Experimental chronic copper poisoning in sheep
Copper poisoning was produced in sheep by feeding them copper sulfate (CuS04·5H20). In sheep given daily oral doses of 1.5 g CuS04'SH20 as 1 percent solution, severe signs of hemolysis appeared in 24 to 26 days. Signs of hemoglobinuria, hemoglobinemia and jaundice were accompanied by a rapid drop in hematocrit to a low of 6 percent in four days. This disease was subsequently called subacute copper poisoning. Sheep fed copper sulfate as a powder added to the ration hemolyzed after day 42. The hemolytic crises were mild and some sheep went through two crises before they were killed. One sheep was fed copper for 131 days without signs of a crisis. This regimen was referred to as chronic copper poisoning. Aspects of chronic copper poisoning studied included plasma assays for liver and kidney function, total plasma copper concentration and ceruloplasmin activity. Histological changes in the liver and kidney were examined at postmortem. Erythrocyte (RBC) pentose phosphate shunt pathway e~zymcs and other Jlematologic parameters were recorded. RBC morphology was studied using scanning electron microscopy. Subcellular liver copper storage was determined by differential centrifugation of post-mortem liver homogenate and examination of glutaraldehyde fixed liver samples using transmission electron microscopy. Increased activities of plasma glutamate oxaloacetate transaminase (GOT), sorbitol dehydrogenase (SDH) , lactate dehydrogenase (LDH) and sulfobromophthalein (BSP) clearance time during the hemolytic crisis implied destruction of liver tissue which was confirmed by histology. Increase in liver function tests prior to hemolytic crisis was noted in the subacutely poisoned sheep and in one of the five sheep that were chronically poisoned. A similar trend was noted in plasma copper concentration. Plasma ceruloplasmin activity did not change. Liver function tests and plasma copper for early diagnosis of chronic copper poisoning may be of use in animals which would later undergo severe crises. Increases in plasma blood urea nitrogen (BUN) and creatinine have a prognostic importance in hemolytic crisis. Erythrocyte glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) remained unaffected until after the hemolytic crisis when their activities increased due to an increase in circulating immature erythrocytes. Erythrocyte reduced glutathione (GSH) decreased by nearly SO percent during crisis. Decrease of GSH was not attributable to in vivo inhibition of pentose phosphate shunt enzymes. Scanning electron microscopy revealed that erythrocytes retained a normal biconcave shape up to 48 hOUTS before hemolytic crisis. Changes observed on the day preceeding the crisis incJ.uded irregularly distributed swellings attributed to Heinz bodies, fragmentation of the erythrocyte membrane and spicule formation in a few cells. Hemolytic crisis was associated with marked distortion and rounding due to increased fragmentation. Many cells contained pits where Heinz bodies had been located previously. Sheep with normal liver copper concentrations stored nearly 40 percent in the 14,000 g pellet. In chronically poisoned sheep, over 40 percent of liver copper appeared in the nuclear fraction (600 g pellet), while subacutely poisoned sheep accumulated copper mostly in the cytosol. Liver cytosol of sheep with normal or slightly elevated copper concentr~tion had most copper in a high molecular weight (MW) fraction (75,000) while a low molecular weight fraction (12,000) showed increased binding in copper poisoned sheep. A fourth copper fraction (MW approximately 5000) was recorded in copper po~soned sheep. This fraction bound the most copper in subacutely poisoned sheep and may be related to appearance and severity of hemolytic crisis signs. D-penicillamine, 0.5 g as a 2 percent solution given daily as an intravenous (rV) injection for four days, did not significantly lower hepatic copper content in housed South- Jown sheep. A mean of 3.8 mg copper was mobilized through the urine in four days. The IV treatment did not affect the health of the sheep.