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The introduction of electrophoresis for the separation of biologic molecules earned Arne Tiselius the Nobel Prize in chemistry and ushered in a new era of investigation resulting in the characterization of the proteins of human plasma. In the course of such studies, Holmberg and Laurell(1) used ammonium sulfate to obtain a bluish green fraction of plasma from which they isolated a protein containing most of the copper found in serum. Owing to the sky blue color of the purified protein they named their discovery caeruloplasmin(1). In this same year, Wilson's disease was found to result from hepatic copper accumulation and soon thereafter Scheinberg and Gitlin(2) demonstrated a deficiency of ceruloplasmin in the sera of patients with this disorder. Despite intense investigation the biologic role of ceruloplasmin has only recently been revealed by identification of patients with aceruloplasminemia.

PHYSIOLOGIC FUNCTION

Ceruloplasmin is a multicopper oxidase, using the facile electron chemistry of copper to oxidize selected substrates via a single-step, four-electron reduction of oxygen. These enzymes have been highly conserved in evolution and are characterized by the presence of three types of spectroscopically distinct copper ions(3). In ceruloplasmin, three of these copper ions form a trinuclear cluster responsible for oxygen binding and activation during enzymatic catalysis(4). Recent determination of the crystal structure of ceruloplasmin provides a model for the process of electron transfer from the oxidized substrate to molecular oxygen and permits delineation of the amino acids which directly contribute to the trinuclear copper center(5).

Ceruloplasmin is an abundant serum protein that contains greater than 95% of the copper present in the blood plasma(6). This protein is synthesized in hepatocytes and secreted into the serum with six atoms of copper incorporated during biosynthesis(7). Ceruloplasmin is an acute-phase reactant, and the serum concentration increases during infection, inflammation, and trauma largely due to effects on hepatic ceruloplasmin gene expression(8). The intracellular copper concentration does not affect the rate of synthesis or secretion of apoceruloplasmin; however, a failure to incorporate copper during biosynthesis results in the secretion into the plasma of an unstable apoprotein which is devoid of oxidase activity and rapidly turned over(9,10). In patients with Wilson's disease an inability to transfer copper into the secretory pathway for holoceruloplasmin biosynthesis results in a decrease in serum ceruloplasmin concentration secondary to the rapid degradation of the secreted apoprotein. Consistent with this observation, the Wilson's disease gene encodes a copper-transporting ATPase which has been shown to reside in the trans-Golgi network of hepatocytes(11–13) (Fig. 1).

Figure 1
figure 1

Localization of the Wilson's disease protein in HepG2 cells. Immunofluorescence using a polyclonal antibody reveals abundant Wilson's disease protein in the trans-Golgi network of the secretory pathway. This protein transports copper into this pathway for incorporation into apoceruloplasmin before secretion of the holoprotein into the plasma.

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Although ceruloplasmin can oxidize several substrates in vitro, Freiden and colleagues demonstrated that this protein plays a direct role in the mobilization of iron from parenchymal tissues and the subsequent oxidation and incorporation of ferric iron into circulating apotransferrin(14,15). In a series of elegant metabolic studies, Cartwright and colleagues demonstrated that pigs made copper deficient by dietary restriction developed iron overload in conjunction with a diminution of circulating ceruloplasmin. These copper-deficient animals had impairment in tissue iron release into the plasma that was restored after administration of ceruloplasmin(16,17). Support for the role of ceruloplasmin as a ferroxidase also comes from genetic experiments in Saccharomyces cerevisiae, which reveal that high affinity iron uptake in this organism is dependent upon the presence of the homologous multicopper oxidase Fet3(18). Fet3 functions as a ferroxidase at the plasma membrane, oxidizing iron in the extracellular milieu and acting in concert with a membrane permease to promote iron uptake(19). The Wilson's disease protein homolog has also been identified in this organism and shown to be essential for delivery of copper to Fet3 in the yeast secretory pathway, revealing a remarkable evolutionary conservation of the function of these copper proteins in iron homeostasis(20,21).

The complete amino acid sequence of ceruloplasmin was determined by Putnam and colleagues using protein chemistry(22). These studies demonstrated the single chain structure of this protein and revealed an internal triplicate homology within the sequence. Subsequently, human ceruloplasmin cDNA clones were isolated and characterized which confirmed the amino acid sequence determined by earlier protein analysis(23–25). Studies using these clones localized the ceruloplasmin gene to chromosome 3q23-25 and revealed abundant, tissue-specific expression of this gene in the liver. The human ceruloplasmin gene consists of 19 exons spanning greater than 45 kb(26). A processed pseudogene for ceruloplasmin has been detected on chromosome 8 by in situ hybridization(27). Although not expressed, the occurrence of this sequence in the human genome has methodologic implications in the detection of ceruloplasmin gene mutations in patients with aceruloplasminemia(vide infra).

METABOLISM

Evidence of ceruloplasmin synthesis can be detected in the human fetal liver and yolk sac as early as the 5th wk of gestation(28). The serum concentration rises steadily throughout gestation, and half-life analysis at birth indicates that nearly all the ceruloplasmin present in newborn serum is derived from endogenous synthesis(28). The serum concentration of ceruloplasmin remains low for the first several postnatal months, and adult levels are not achieved until the 1st y of life. This rise in serum ceruloplasmin concentration parallels the onset of biliary copper excretion and represents the slow postnatal increase in holoceruloplasmin synthesis. Metabolic studies using131 I indicate a t1/2 of 5.5 d for human ceruloplasmin(29,30). Kinetic studies using radioactive copper yield a similar half-life, indicating little or no exchange of ceruloplasmin copper in tissues(31). This observation suggests that, although ceruloplasmin contains almost all the copper found in the serum, this protein plays no direct role in copper transport.

About 10% of circulating ceruloplasmin is apoprotein, which is synthesized and secreted from the liver without copper and turns over rapidly in the circulation with a t1/2 of 5 h. Biosynthetic studies reveal that copper availability serves only to determine the ratio of apo- and holoceruloplasmin secreted from the cell and thus at steady state the serum ceruloplasmin concentration represents the sum of the these two forms, determined by intracellular copper availability and the observed differences in their turnover(32,33). Given the differences in half-lives, these data indicate that under normal circumstances the rate of synthesis and secretion of the apo-and holoprotein is roughly equivalent. As anticipated from such a model, an increase in the hepatic copper pool of normal individuals results in a sustained increase in the concentration of holoceruloplasmin in the serum(34).

ACERULOPLASMINEMIA

In addition to the decreased serum ceruloplasmin in patients with Wilson's disease, earlier studies revealed that approximately 15% of obligate heterozygotes have a slightly decreased serum ceruloplasmin and a 3-fold increase in hepatic copper concentration(35,36). In the course of these investigations, individuals were identified with inherited differences in serum ceruloplasmin concentration where the incidence and inheritance pattern suggested this was not due to the heterozygote state of Wilson's disease(37,38). Analysis of one such family led to the designation of hereditary hypoceruloplasminemia and suggested that a decrease in serum ceruloplasmin concentration to 50% of normal is not associated with any clinical abnormality(39). Despite these earlier findings, two reports appeared in the literature which described individuals with dementia, diabetes, retinal degeneration, and basal ganglia symptoms in association with a complete absence of circulating serum ceruloplasmin(40,41). Analysis of the serum ceruloplasmin concentrations in families of affected patients revealed an autosomal recessive inheritance pattern, and molecular genetic studies have demonstrated the presence of mutations in the ceruloplasmin gene(42–48).

A typical pedigree in one such family from Belfast, Ireland, is shown in Figure 2 along with the DNA sequence from exon 13, indicating a single nucleotide deletion in affected family members. As would be anticipated from the incidence and inheritance pattern of this disease, consanguinity has been observed in most cases. Thus far, six distinct mutations have been characterized in the ceruloplasmin gene, each of which would result in an alteration of the open reading frame such that the amino acid ligands in the carboxyl-terminus determinating the essential trinuclear copper cluster would be eliminated (Table 1). As ceruloplasmin synthesized without this region would be incapable of binding copper, these results are consistent with the observed lack of detectable ceruloplasmin oxidase activity in the serum of affected patients.

Figure 2
figure 2

Pedigree and nucleotide sequence analysis in a kindred with aceruloplasminemia. (A) A pedigree of an affected family is shown with serum ceruloplasmin concentrations as indicated(mg/dL). Solid squares indicate proband and his younger brother.(B) Sequence ladder of normal and mutant allele in heterozygote V-6 as well as the affected individuals. (Reproduced from Harris et al.(46): Quarterly Journal of Medicine, 89:355-359, 1996, with permission from Oxford University Press.)

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Table 1 Ceruloplasmin gene mutations identified in patients with aceruloplasminemia
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PATHOLOGY

Patients with aceruloplasminemia have normal liver histology on biopsy with no evidence of hepatic copper accumulation but a marked increase in iron in both hepatocytes and reticuloendothelial cells(49). The serum ferritin concentration is elevated, the serum iron concentration is decreased, and most patients have a mild anemia. Autopsy findings reveal iron in the pancreas with accumulation in the islets of Langerhans in association with a selective loss of insulin-producing cells(49). All patients evidence abnormal glucose metabolism and most develop insulin-dependent diabetes.

The disruption of iron homeostasis in aceruloplasminemia is understood by considering the role of ceruloplasmin as a plasma ferroxidase. Within the iron cycle ceruloplasmin functions to oxidize ferrous iron for subsequent transfer to plasma apotransferrin. This transferrin-bound iron is then predominantly used for Hb biosynthesis during erythropoiesis. Once in the circulation the Hb-bound iron remains within the erythrocyte until these cells are turned over in the reticuloendothelial system, and the iron is then subsequently released and recycled to transferrin (Fig. 3). Ceruloplasmin is essential for the release of iron from these reticuloendothelial storage sites. The low serum iron in patients with aceruloplasminemia reflects this impairment in iron release from the reticuloendothelial system; however, the mild anemia indicates that alternative mechanisms of iron oxidation are available to provide sufficient transferrin-bound ferric iron for erythropoiesis. In addition, the absence of ceruloplasmin leads to an accumulation of ferrous iron in the plasma, which is rapidly removed from the circulation by the liver and other tissues, analogous to what is observed in atransferrinemia, hereditary hemochromatosis, and transfusion-dependent iron overload(50). In the healthy individual 5% of the normal serum ceruloplasmin concentration is sufficient to sustain plasma iron turnover rates, explaining why abnormalities of iron homeostasis are not observed in patients with Wilson's disease(17). Although all patients with aceruloplasminemia have low plasma copper owing to the absence of this protein in the serum, copper metabolism in such patients is entirely normal, indicating that there is no essential role for ceruloplasmin in copper homeostasis.

Figure 3
figure 3

Role of ceruloplasmin as a plasma ferroxidase. The systemic iron cycle is shown illustrating the function of ceruloplasmin (Cp) in mediating iron release from the reticuloendothelial system and subsequent oxidation and incorporation into transferrin (Tf). (Reproduced with permission from Harris et al(6): American Journal of Clinical Nutrition 67:972S-977S.)

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NEUROPATHOLOGY

The predominant clinical features in aceruloplasminemia are neurologic and result from progressive degeneration of the basal ganglia. T2-weighted magnetic resonance brain imaging reveals decreased signal intensity in the basal ganglia, consistent with iron deposition in these regions. Furthermore, examination of brain tissue at autopsy demonstrates abundant iron deposition in neurons and glia within the basal ganglia and thalamus with clear evidence of neuronal loss in these same tissues(49). Patients exhibit clinical symptoms consistent with these findings, including subcortical dementia, dystonia, dysarthria, and movement disorders. Although visual symptoms are not prominent clinically, ophthalmologic examination does reveal iron deposition and photoreceptor loss in the peripheral regions of the retina. Involvement of the CNS distinguishes aceruloplasminemia from other known disorders of iron homeostasis and indicates that ceruloplasmin plays an essential role in brain iron metabolism. Interestingly, these pathologic findings of iron accumulation are similar to those observed in Hallervorden-Spatz disease, but no information is currently available on the mechanisms of iron accumulation in this rare disease(51).

Ceruloplasmin does not cross the blood-brain barrier, and this implies that any direct role for this protein in CNS iron metabolism must involve expression within this site. Indeed examination of ceruloplasmin gene expression within the CNS reveals astrocyte-specific gene expression throughout the cerebral microvasculature(52,53). Ceruloplasmin is also expressed by astrocytes surrounding specific neurons in the substantia nigra and other basal ganglia as well as by astrocytic Müller glia cells in the retina(52–54) (Fig. 4). Biosynthetic studies using primary cultures of neurons and glia confirm this cell-specific expression and indicate that ceruloplasmin is synthesized and secreted by astrocytes with kinetics identical to that observed in hepatocytes(52). Although a glycophosphotidylinositol-linked membrane form of ceruloplasmin is detected in an astrocyte cell line, pulse-chase studies indicate that all of the ceruloplasmin synthesized by primary astrocyte cultures is secreted into the media, making the relevance of this anchored form unclear at this time(55).

Figure 4
figure 4

Ceruloplasmin gene expression in the substantia nigra. (A and B) In situ hybridization of human substantia nigra pars compacta reveals ceruloplasmin mRNA in astrocytes (arrows) surrounding dopaminergic neurons(mdn). Original magnification, ×1000 (A),×2000 (B). (Reproduced from Klomp and Gitlin(53): Human Molecular Genetics, 5:1898-1996, 1996, with permission from Oxford University Press.)

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The observation that ceruloplasmin is synthesized in the CNS suggests that the absence of this protein at this site in affected patients is directly responsible for the iron accumulation and neuronal degeneration. As such it is envisioned that astrocyte-secreted ceruloplasmin may function in an analogous manner as in the plasma, oxidizing ferrous iron after the release of this metal from the cerebral microvasculature (Fig. 5). In aceruloplasminemia, the inappropriate accumulation of ferrous iron by glial cells may then lead to cell-specific injury with subsequent loss of glia-derived trophic factors essential for neuronal survival. Alternatively it is also possible that the accumulation of ferrous iron may directly result in oxidant-mediated injury to the CNS. This model would be consistent with observations in patients with aceruloplasminemia indicative of free-radical mediated tissue injury including an increase in plasma lipid peroxidation and an interruption of peroxisomal β-oxidation of fatty acids(56,57). It is also possible that neuronal degeneration results from impaired iron delivery to neurons owing to the diminution of transferrin-bound ferric iron. Elucidation of the precise function of ceruloplasmin in the CNS as well as mechanisms of neuronal loss in affected patients must await the development of a suitable animal model in which to examine this process directly.

Figure 5
figure 5

Model of ceruloplasmin function in CNS. Ferrous iron (Fe2+) which accumulates in the absence of ceruloplasmin ferroxidase activity is shown as resulting in tissue injury via free radical generation. (Reproduced with permission from Harris et al.(6): American Journal of Clinical Nutrition 67:972S-977S.)

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Although aceruloplasminemia is a fatal neurodegenerative disease, clinical and laboratory studies indicate that iron accumulation precedes the onset of neurologic symptoms by several years. This observation suggests a potential therapeutic approach, and a preliminary study of the iron-chelator deferoxamine did indicate a reduction in body iron stores as well as amelioration of neurologic symptoms in a severely affected patient(58). If these preliminary observations are supported by clinical trials, this approach may prove useful as a preventative therapy in asymptomatic individuals. As the complete absence of serum ceruloplasmin in diagnostic of this disorder, the development of an effective therapy would make it feasible to consider population screening.

CONCLUSION

The presence of mutations in the ceruloplasmin gene in patients with CNS iron accumulation and neuronal degeneration indicates an essential role for this protein in brain iron homeostasis and neuronal survival. The pathophysiology of aceruloplasminemia suggests the presence of an iron cycle in the CNS analogous to that previously characterized in the systemic circulation. Presumably the presence of such a cycle serves to minimize the effects of systemic iron deficiency on CNS function. This observation has important implications for the cognitive defects observed in children with sustained iron deficiency in the perinatal period where previous studies have suggested a critical window for iron repletion within the CNS(59). In addition, recent studies suggest that iron-mediated free-radical injury is a central mechanism underlying hypoxicischemic brain damage in the newborn infant(60). As holoceruloplasmin biosynthesis is diminished in the fetus and newborn infant it will be worth pursuing the role of this protein in iron homeostasis in the newborn brain. Thus the discovery of aceruloplasminemia provides a starting point for elucidation of the mechanisms of CNS iron homeostasis during development and may permit the creation of novel therapeutic approaches to prevent or ameliorate neurodegeneration in a variety of childhood diseases where abnormalities in brain iron metabolism have been implicated.