27.3: Serum ceruloplasmin (24b.2)

Ceruloplasmin is the major copper-containing protein in the α2-globulin fraction of human serum. More than 60% to 95% of serum copper is associated with cerulo­plasmin (Wirth and Linder, 1985), so changes in serum copper generally parallel the level of cerulo­plasmin in the blood.

Cerulo­plasmin (EC 1.16.3.1) is a copper transport protein synthesized primarily in the liver. It is a single-chain glyco­protein with six copper atoms per molecule. A ferr­oxidase, it assists in iron transport by oxidizing intra­cellular Fe²⁺ to Fe³⁺, which can then combine with trans­ferrin. Cerulo­plasmin is also among those enzymes involved in the acute-phase reaction of inflam­mation and in the scavenging of oxygen radicals to protect cells against oxidative damage (Linder and Hazegh-Azam, 1996).

Cerulo­plasmin levels in serum, like copper, are significantly reduced in cases of severe copper deficiency that can occur with the hereditary diseases associated with disturb­ances in copper meta­bolism: Menkes kinky hair syndrome and Wilson's disease. However, the response of ceruloplasmin to marginal copper deficiency or short-term copper depletion is variable (Milne and Nielsen, 1993; 1996). In most short-term studies of copper depletion (e.g., Milne and Nielsen, 1996) or sup­plement­ation (e.g., Kehoe et al., 2000), there has been little or no response by cerulo­plasmin (Figure 24b.2).

Many of the factors that influence serum copper also affect serum cerulo­plasmin and limit its usefulness as a marker of copper status, except in cases of severe copper deficiency. Furthermore, the compli­cations and variations in measuring cerulo­plasmin and copper in disorders such as Wilson's disease mean that their value as biomarkers need to be carefully assessed (Walshe, 2010).

• Factors affecting ceruloplasmin

Age and gender influence serum cerulo­plasmin activity in the same manner as serum copper as shown in a Table 24b.3 (Fischer et al., 1990).

Estrogen-containing preparations tend to increase the serum cerulo­plasmin activity, as shown in Table 24b.4 (Fischer et al., 1990). Cerulo­plasmin synthesis and secretion by the liver is regulated in the long term by estrogen, probably through stabil­ization of mRNA (Middleton and Linder, 1993).

Pregnancy can increase serum cerulo­plasmin activity (Fischer et al., 1990) again via the effect of estrogen on cerulo­plasmin synthesis. This effect may be related to the copper needs of the growing fetus during pregnancy (Lee et al., 1993).

Infection and inflammatory stress can lead to markedly elevated cerulo­plasmin activity levels in serum (Brown, 1998). Cerulo­plasmin is an acute-phase protein and the inflammatory hormones (interleukin-1 [IL-1], IL-6, and tissue necrosis factor) regulate its acute synthesis and secretion by the liver (Linder and Hazegh-Azam, 1996). Hence, it is not surprising that serum cerulopla­smin activity is also elevated in rheum­atoid arthritis (DiSilvestro et al.,1992).

• Interpretive criteria

Reference data for serum ceruloplasmin are limited. Salmenperä et al. (1986) have published reference values for exclusively breastfed infants followed during the first year of life, and they are shown in Figure 24b.3. Levels increase steadily during the first year of life, paralleling the trend for serum copper.

Milne (1994). suggests that a ratio of enzyme activity (cerulo­plasmin oxidase activity) to cerulo­plasmin protein concen­tration may be a useful index of copper status. The ratio has several advantages. Effects of age, sex, or hormone use are small (Table 24b.6)

Table 24b.6 Effect of age, gender, and hormone use on mean ceruloplasmin concentrations, measured both enzymatically (ENZ) and by radial immunodiffusion (RID). The ratio of ENZ/RID is also shown. FOC, females taking oral contraceptives; FE,females using estrogen therapy. Significance levels from analysis of variance (ANOVA) are also shown (interactions between gender and decade were nonsignificant in all three cases). Data from Milne and Johnson, Clinical Chemistry: 39, 883–887, 1993

Age (years)	Group	Enzyme activity (mg/L)	RID (mg/L)	ENZ/RID
20–29	M (n = 10) F (n = 10) FOC (n = 10)	408 503 612	266 330 371	1.56 1.53 1.71
30–39	M (n = 10) F (n = 10) FOC (n = 11)	416 516 744	296 314 525	1.49 1.66 1.42
40–49	M (n = 11) F (n = 11)	442 519	337 359	1.31 1.48
50–59	M (n = 9) F (n = 13) FE (n = 4)	441 516 593	310 370 384	1.43 1.44 1.57
60–69	M (n = 8) F (n = 10)	470 559	284 354	1.70 1.60
70+	M (n = 7) F (n = 7)	485 470	332 368	1.51 1.29
ANOVA	Gender Decade	0.0001 NS	0.0002 0.03	NS NS

(Milne and Johnson, 1993), and it appears to be more sensitive to changes in copper status than either the enzymatic activity or immuno­reactive protein alone (Milne et al., 1988). Indeed, in some indiv­iduals, cerulo­plasmin oxidase activity (but not cerulo­plasmin protein concentration) was depressed during experi­mental copper depletion (Milne et al., 1988). Further, in young women, cerulo­plasmin oxidase activity has been reported as inversely related to physio­logical changes due to copper depletion (e.g., auto­nomic cardio­vas­cular function)(Lukaski et al., 1988).

Table 24b.6 presents mean cerulo­plasmin values measured both enzym­atically (ENZ) and by radial immuno­diffusion, and the ratio of the enzyme activity to the immuno­reactive protein for males and females by age and hormone use (Milne and Johnson, 1993). Values for cerulo­plasmin for the two assays were standard­ized to mg/L by using purified human ceruloplasmin as a standard.

• Measurement of serum ceruloplasmin

Serum ceruloplasmin can be assayed enzym­atically by measuring p-phenyl­enedia­mine oxidase activity(Sunderman and Nomoto, 1970). Cerulo­plasmin protein levels can be measured immuno­chemically by radial immuno­diffusion techniques (Mancini et al., 1965; Buffone et al., 1979). Alternatively, nephel­ometry(Buffone et al., 1979) or immuno­electo­phoresis techniques (Gibbs and Walshe, 1979) and ELISA (various commercial kits are available) can be used. Some discrepancies have been reported between radial immuno­diffusion and nephel­ometry, which may be attributed to different sources of anti­bodies used for the assays (Buffone et al., 1979). Analytical variation for serum cerulo­plasmin performed by radial immuno­diffusion ranges up to 6% (Gallagher et al., 1989).