Introduction

Ceruloplasmin is a serum ferroxidase responsible for 95% copper transport in the blood. It is well known for its role in the pathogenesis of Wilson disease and regulating iron metabolism.[1] Also, ceruloplasmin is a positive acute-phase reactant, meaning its levels will increase in inflammatory conditions or cell injury.[2] Copper is a trace element that acts as a cofactor for multiple enzymes in critical cellular reactions, for instance, cytochromes which participate in the electron transport chain and redox catalysis.[3] Recent studies have shown that alterations of ceruloplasmin protein and copper homeostasis are associated with Alzheimer's disease.[4][5]

Fundamentals

Ceruloplasmin production occurs in the liver and is a ferroxidase enzyme encoded by the CP gene in humans.[6] Liver hepatocytes predominantly synthesize ceruloplasmin. In the liver, P-type ATPase (ATPase 7B) enzymes are necessary to incorporate copper into apo-ceruloplasmin.[7] Ceruloplasmin is then released into the bloodstream to transport properly to distal sites and to complete its functions in other metabolic processes, namely iron homeostasis and metabolism. A solution of ceruloplasmin is blue, and excess serum levels may give the serum a greenish hue. Each ceruloplasmin molecule can bind to 6 to 8 copper atoms; usually, 50% are in cupric (+2) and 50% are in cuprous (+1) states.[8] 

Issues of Concern

As an acute phase reactant, ceruloplasmin may elevate in multiple conditions such as trauma, tissue damage, infection, or inflammation. It bears mention that ceruloplasmin may increase in states of high estrogen and progesterone; for instance, pregnant women, patients receiving estrogen therapy, and women on oral contraceptives may have high levels of ceruloplasmin.[2] In addition, many medications, notably anti-seizure drugs (e.g., valproic acid), are known to increase ceruloplasmin levels.[9] Measurements of the ceruloplasmin level are not common in standard laboratory testing, but certain symptoms may raise clinical suspicion and prompt the ordering of ceruloplasmin levels.[10] These include the following:

• Anemia
• Ataxia
• Nausea and abdominal pain
• Neuropathy
• Jaundice
• Fatigue
• Behavioral changes
• Tremors
• Difficulty walking or swallowing
• Dystonia
• Sleep disorders

Cellular Level

Ceruloplasmin is an enzyme from the multi-copper oxidase family. It is synthesized primarily in the liver parenchymal cells, with small amounts produced from immune cells, including macrophages and lymphocytes.[11] The peptide chain is synthesized then copper atoms are added from an intracellular ATPase.[12] Although copper availability is not necessary for ceruloplasmin translation, copper binding is essential for the adequate folding of ceruloplasmin protein and the normal attachment of side-chain oligosaccharides. The apo-ceruloplasmin, which does not contain copper or the ATPase, is less structurally stable, and most undergo intracellular degradation. However, a small portion of apo-enzyme will reach circulation but has a short half-life of 5 to 6 hours compared to the holoenzyme (ceruloplasmin plus copper), which has a longer half-life of a few days.[13]

Molecular Level

Ceruloplasmin has a molecular weight of 132kDa, and its crystal structure was identified in 1997. It is a polypeptide chain of 1046 amino acids with three asparagine-linked oligosaccharide side chains, and it contains a total carbohydrate content of 7 to 8%.[14] The size and charge of the molecule depend on the glycosylation of the molecule, the number of copper atoms, peptide chain variations, and polymerization.[15] Ceruloplasmin is highly prone to proteolysis; recent studies have utilized electron paramagnetic resonance spectroscopy techniques to evaluate its structure and concentration.[16]

Function

In general, the ceruloplasmin molecule is a catalyst for redox reactions in plasma. It can oxidize iron from ferrous (2+) to ferric iron (3+), which assists in iron binding to transferrin. Neurological disorders in hereditary ceruloplasmin deficiency may be caused by disordered iron transport in the brain. It is also thought to be involved in controlling membrane lipid oxidation. In the presence of superoxide, ceruloplasmin promotes LDL oxidation in vitro and is colocalized with oxidized LDL in atherosclerotic lesions.[17]

The liver is the crucial organ in copper homeostasis, and more than 90% of the copper exported from the liver to the plasma is in the form of ceruloplasmin. However, ceruloplasmin's role in copper transport from the peripheral tissues is limited. Albumin is the main protein responsible for ionic copper transport from the tissues (such as absorbed copper in the digestive tract). The copper-albumin complex is directed to the liver via the portal system in circulation. Albumin and transcuprein appear to be the other major copper transport proteins, especially immediately post-gastrointestinal absorption.[18]

Testing

Standard Ceruloplasmin measurement is via a blood test, which often occurs by analyzing a serum sample with immunoassays, immunoturbidimetry, or immunonephelometry. The sample must be spun and separated as soon as the laboratory receives it and assays it promptly. The samples may be stored at 4 degrees Celsius for up to three days or longer at -70 degrees Celsius. The serum test is often part of the workup when there is suspicion of Wilson disease and is commonly ordered along with urine copper levels. Several factors, including diet, hormone levels, and other genetic disorders, may influence the resulting ceruloplasmin levels in the urine and serum.[19][20]

Deficiency States 

Low ceruloplasmin levels may indicate the following pathological conditions: Wilson disease, Menke disease, copper deficiency, aceruloplasminemia, or in states of low protein energy intake (e.g., malnutrition.)[21][22]

Excess 

High ceruloplasmin levels may suggest or be present in physiological and pathological circumstances, including pregnancy, oral contraceptive use, copper toxicity/zinc deficiency, lymphoma, lung cancer, acute and chronic inflammation, rheumatoid arthritis, angina, Alzheimer's disease, schizophrenia, and obsessive-compulsive disorder.[23][24][25]

Reference intervals:(adapted from Tietz Textbook of Clinical Chemistry and Molecular Diagnostics by Carl A Burtis et al.)

Age/Interval(mg/L)

• Cord (term): 50 to 330
• Birth to 4 months: 150 to 560
• 5 to 6 months: 260 to 830
• 7 to 36 months: 310 to 900
• 6 to 12 years: 250 to 450

Age/Male/Female

• 13 to 19 years: 150 to 370/220 to 500
• Adult: 220 to 400/250 to 600 (no oral contraceptives)
• Adult: 270 to 660 (on oral contraceptives, estrogens)
• Adult: 300 to 1200 (pregnant)

Wilson disease is diagnosed based on clinical presentation, biochemical tests, and ATP7B mutation analysis. Regarding serum ceruloplasmin testing, only those with low serum ceruloplasmin, low serum copper, and high urine copper meet the criteria for Wilson disease. However, some patients diagnosed with Wilson disease exhibit normal ceruloplasmin levels. When urine and serum concentrations of copper and ceruloplasmin are low, the patient suffers from a copper deficiency. Factors interfering with the body's ability to metabolize copper, such as chelator medications, diet, and iron restriction, may also affect serum ceruloplasmin levels.[26][27] 

Pathophysiology

Ceruloplasmin is undetectable before 20 weeks of gestation. Concentrations gradually rise to 25 to 40% of average adult concentrations and, by six months, are close to adult concentrations.[28]

Special Situations

• There have been reports of inherited aceruloplasminemia in several families, and it is a genetic cause of ceruloplasmin deficiency; these patients have neurodegeneration and iron deposition in the brain.[29] Concerning aceruloplasminemia, a diagnosis of aceruloplasminemia could be suspected based upon a biochemical profile showing very low to absent ceruloplasmin in serum, high ferritin, low copper, low iron, microcytic anemia refractory to iron supplementation, and characteristic imaging features. Homozygous cases have distinctive imaging features with marked hypointensity of the liver, basal ganglia nuclei, thalami, dentate nuclei, and cerebral/cerebellar cortices on T2 MRI. MRI of heterozygous cases may reveal cerebellar atrophy without hypointensity of the basal ganglia.[30] 
• Menke's disease is an X-linked recessive disorder caused by a mutation in the ATP7A protein. With this mutation, copper cannot be transported out of the GI tract; therefore, copper is unavailable to the liver, which causes low ceruloplasmin levels. The patients present in infancy have sparse, brittle, and kinky hair, growth retardation and neurologic degradation, and death if untreated in the first few years of life.[31]
• Wilson disease results from an autosomal recessive mutation in the ATP7B protein. Mutations in ATP7B (located on chromosome 13) lead to Wilson disease. ATP7B is an efflux transporter in the liver, but it is also essential in the Trans-Golgi Network for transferring copper for the metalation of ceruloplasmin and biliary copper excretion.[32] The point mutation H1069Q is the most common ATP7B mutation in patients from Central, Eastern, and Northern Europe, and 50 to 80% of Wilson disease patients from these countries carry at least one H1069Q allele.[33] Mutations resulting in completely absent or non-functional ATP7B protein activity are associated with early-onset, typically hepatic, severe Wilson disease; these mutations are comparatively rare.[34] It is characterized by hepatolenticular degeneration due to copper deposition in organs, including the basal ganglia, cornea, and liver. In Wilson disease, ceruloplasmin levels will usually be low, but urinary excretion of copper will be high. Liver biopsy results will show high copper content. Clinically, a dark ring around the iris suggests a diagnosis of Wilson disease. This sign is known as a Kayser-Fleischer ring.[35]

Clinical Significance

Treatment for Aceruloplasmin and Wilson Disease

Treatment for aceruloplasminemia mainly falls to chelation therapy and increasing serum ceruloplasmin. Fresh frozen plasma (FFP), which contains ceruloplasmin combined with IV desferrioxamine, effectively decreases iron content in the liver. Repetitive FFP treatment can improve neurologic signs/symptoms.

Treatment options for Wilson disease include chelation therapy (D-penicillamine, trientine, and tetrathiomolybdate) or zinc salts. Medical therapy is continued lifelong as copper accumulation is not manageable with dietary restriction alone. It is important to note that there may be an associated paradoxical worsening of neurological status after initiation of chelation therapy. While this is not fully understood, studies suggest that increased mobilization of free copper by chelator therapy may lead to acute worsening upon initiation of treatment.[34]

Complications

Diabetes mellitus, ataxia, dystonia, Parkinsonism, psychiatric problems (mostly mood disorders), cardiac disease, thyroid dyscrasia, anemia, and liver damage are common complications shared by both Wilson disease and aceruloplasminemia. Amenorrhea and frequent abortions may be complications of copper toxicity that may affect fertility. Antipsychotic drugs are applied in severe mania and to treat psychotic symptoms. In Wilson disease, antipsychotics increase the risk of neurological deterioration and hepatic injury. As such, antipsychotics with low EPS risk, such as clozapine or quetiapine, should be used.[36]

Enhancing Healthcare Team Outcomes

Surveillance should include yearly glucose tolerance tests starting at the age of 15 or the age of diagnosis if, after 15 years old, to monitor for the inception of diabetes mellitus. Regular eye exams should also be a consideration, as retinopathy is a prominent feature in aceruloplasminemia and Wilson disease. An Annual Echocardiogram (ECG), evaluation of thyroid and liver function, and a complete blood count (CBC) starting at the time of diagnosis should be performed to monitor for other potential complications. Blood transfusions should be approached cautiously as the increased iron load may be challenging for patients with these disorders.

In patients with Wilson disease, it is strongly recommended to perform familial screening. The American Association for Study of Liver Diseases and the European Association for the Study of Liver both separately recommend screening first-degree relatives of the affected individuals, suggesting siblings or offspring only.[37]

While most patients with Wilson disease who become pregnant have successful pregnancies, a discussion is necessary about the potential side effects of anti-copper therapy, as all available anti-copper drugs can pass into the breast.                                                      

Ceruloplasmin Interpretation Adapted from Labtestsonline [See exhibit 2]

Wilson Disease

Wilson disease presents between ages 5 to 35.[38]

The clinical presentation may include the following symptoms:

• Hepatolenticular degeneration
• Kayser-Fleischer rings
• Parkinsonism
• Ataxia
• Personality changes
• Depression
• Psychosis
• Acute liver failure
• Chronic liver failure
• Cirrhosis 
• Renal issues (renal tubular acidosis with aminoaciduria)
• Secondary Hemolysis is less common
• Most patients with Wilson disease have low ceruloplasmin levels

Treatment may include: 

• Penicillamine
• Trientine
• Zinc