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Assessment of copper status in pregnancy by means of determining the specific oxidase activity of ceruloplasmin
Clinica Chimica Acta 312 Ž2001. 123–127 www.elsevier.comrlocaterclinchim
Assessment of copper status in pregnancy by means of determining the specific oxidase activity of ceruloplasmin M. Olga Louro a , J. Angel Cocho b, J. Carlos Tutor a,) a
b
Laboratorio Central, Hospital Clınico UniÕersitario, Santiago de Compostela, Spain ´ Laboratorio de Alteraciones Metabolicas y Nutricionales, Hospital Clınico UniÕersitario, Santiago de Compostela, Spain ´ ´ Received 21 February 2001; received in revised form 18 June 2001; accepted 22 June 2001
Abstract Background: Conditions not directly related to copper nutriture, such as pregnancy, infections and inflammation, which increase serum copper concentration even during copper deprivation, may be expected to conceal changes in copper status. It has been suggested that the specific enzymatic activity of ceruloplasmin Žactivity per unit mass of enzyme protein. may be a sensitive indicator of copper status and is not affected by factors such as hormones or sex. In this study, we investigated the behaviour of specific oxidase activity of ceruloplasmin and the copperrceruloplasmin ratio in pregnant women. Methods: Copper, immunoreactive ceruloplasmin and its oxidase activity were determined in serum from 52 women in the last trimester of normal pregnancy, and in 50 control women of similar age living in the same area and who were not taking oral contraceptives. The results are expressed as mean " S.E.M. Results: In the group of pregnant women, significantly higher serum levels of copper, ceruloplasmin and its oxidase activity were found than in the control group Ž p - 0.001.. In both groups, a high correlation was found between these biochemical variables Ž r G 0.905, p - 0.001.. However, in the group of pregnant women the specific oxidase activity for ceruloplasmin Ž364.4 " 3.3 vs. 407.5 " 3.8 Urg. and the copperrceruloplasmin ratio Ž2.82 " 0.03 vs. 3.19 " 0.04 mgrmg. were significantly lower than in the control group Ž p - 0.001.. Conclusions: Although pregnancy accelerates the rate of ceruloplasmin protein synthesis and release with an increase of serum copper, the decrease in specific oxidase activity of circulating ceruloplasmin would be an indicator of the degree of depletion of the mother’s copper deposits in order to deal with the foetus’ needs. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Ceruloplasmin; Specific oxidase activity; Pregnant women; Copper status
1. Introduction The significant increase in the concentration of circulating copper due to hormonal impregnation in women who are pregnant or taking oral contraceptives is a well-documented issue w1–5x. This ef) Corresponding author. Fax: q34-981-950-403r34-981-950852. E-mail address: [email protected] ŽJ.C. Tutor..
fect is due to an increase in the hepatic synthesis and secretion into the extracellular environment of ceruloplasmin Žferroxidase I, EC 1.16.3.1., a multifunctional protein w6x which integrates within its molecule the major fraction of copper present in the blood plasma w1,2,5x. Similarly, other conditions also not directly related to copper nutriture, such as infection and inflammatory reactions, may increase serum copper w7x. Accordingly, conditions which tend to elevate serum copper concentration may obscure the
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 6 0 7 - 6
124
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127
changes in copper status even during copper deprivation w2,7x. Recent studies in humans suggest that the specific enzymatic activity of ceruloplasmin Žactivity per unit mass of enzyme protein. is a more sensitive indicator of copper status than either serum copper and ceruloplasmin or erythrocyte superoxide dismutase w2x, and is not influenced by factors such as age, gender, hormones w8x or the use of plasma or serum w9x. In this study, we indicate the results obtained for the specific oxidase activity of ceruloplasmin in a group of women in the last 3-month period of normal pregnancy, when supply of copper to the foetus is at its greatest level w10x.
determined by atomic absorption spectrometry using a previously described method w12x. The within- and between-run precision results yielded CVs of less than 3.0% and the recovery is 97.0–99.7%. When the data presented a Gaussian distribution, parametric tests were used ŽStudent’s t-test for differentiating averages and Pearson’s correlation coefficient.. When the data did not have a Gaussian distribution, non-parametric tests were used ŽWilcoxon’s test for comparing populations and Spearman’s correlation coefficient.. For normality, Shapiro– Wilk’s test was used or the skewness and kurtosis coefficients according to the size of the sample.
3. Results 2. Subjects and methods Fifty-two women in the last trimester of normal pregnancy were studied, with a mean Ž"S.E.M.. age of 27.5 " 2.0 years Žrange 15–43 years.. The control group comprised of 50 healthy women living in the same area with a mean Ž"S.E.M.. age of 31.3 " 1.9 years Žrange 18–45 years. and who were not taking oral contraceptives. Serum samples were taken between 8:00 and 10:00 a.m. Immunoreactive ceruloplasmin was determined by radial immunodiffusion using agar plates Nor-Partigen with monospecific antiserum ŽDade-Behring, Liederbach, Germany.. Oxidase activity of the ceruloplasmin was determined at 30 8C in a Cobas Bio analyzer ŽRoche Diagnostics, Basel, Switzerland. using o-dianisidine as a substrate w11x, which permits the expression of the enzyme activity in terms of International Units. The within- and between-run precision results of this technique yielded CVs of less than 1.3%. The total serum copper was
Table 1 shows the results obtained both in the group of pregnant women and the control group studied. The levels of serum copper, immunoreactive ceruloplasmin and oxidase activity are significantly higher in pregnant women Ž p - 0.001.. In turn, specific oxidase activity and the copperrceruloplasmin ratio are significantly higher in the control female subjects Ž p - 0.001. with the differences being clinically significant according to Harris’ criteria w13x. We did not find a statistical or clinically significant difference for the specific oxidase activity between the healthy female controls Ž n s 50, mean 407.5 " 3.8 Urg, medians 405.2 Urg. and male controls Ž n s 51, mean 398.2 " 4.0 Urg, media n s 398.3 Urg.. In a pool of sera from pregnant women, it was not possible to find any inhibitors of the oxidase activity or disturbing agent of the immunochemical determination of the ceruloplasmin. In the groups of pregnant and control women, high correlation coefficients were found between the
Table 1 Serum ceruloplasmin levels and related variables in the control and pregnant women Controls Ž n s 50.
Ceruloplasmin Žmgrl. Oxidase activity ŽUrl. Specific oxidase activity ŽUrg. Copper Žmgrl. CopperrCeruloplasmin Žmgrmg.
Pregnant women Ž n s 52.
Mean " S.E.M.
Median
Mean " S.E.M.
Median
335.7 " 8.0 136.1 " 2.8 407.5 " 3.8 1069.3 " 27.6 3.19 " 0.04
317 132 405 1045 3.23
758.8 " 18.7 276.1 " 6.9 364.4 " 3.3 2171.2 " 49.3 2.82 " 0.03
742 270 366 2155 2.87
Significance Ž p .
- 0.001 - 0.001 - 0.001 - 0.001 - 0.001
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127
levels of serum copper, ceruloplasmin and its oxidase activity Ž r G 0.905, p - 0.001.. In the female control subjects, a highly significant correlation was found between specific oxidase activity and the copperrceruloplasmin ratio Ž r s 0.603, p - 0.001.; however, for pregnant women no statistical significance was found by correlating these variables Ž r s 0.208.. Neither group produced a significant correlation between specific oxidase activity and the ceruloplasmin concentration or its oxidase activity.
4. Discussion Ceruloplasmin is a multifunctional protein, performing catalytic, antioxidant and copper transport functions w6x. Its enzymatic character is unique in being able to oxidise both organic and inorganic substrates. The oxidase activity of ceruloplasmin towards aromatic amines is very well correlated with its ferroxidase activity w14–18x, and is derived from the reduction of select copper atoms in the oxidase sites of the holoprotein. The significantly increased levels of copper, ceruloplasmin and its oxidase activity, as well as the high degree of correlation between these variables, all found in the group of pregnant women, are widely confirmed in the literature w1–5,19–22x. However, at least to our knowledge, the fact that specific oxidase activity and the copperrceruloplasmin ratio may decrease significantly during normal pregnancy has not been previously dealt with. Milne’s studies w2,7,8x, suggest that in adults the specific oxidase activity of ceruloplasmin is a better indicator of copper status than serum concentrations of copper or ceruloplasmin. Specific oxidase activity would not be influenced by non-dietary factors such as age, gender or hormone use w7,8x. Ruelland et al. w22x indicated that during pregnancy and in experimental oestrogenic impregnation, immunochemical determination of ceruloplasmin would give falsely decreased results, and the best choice for the quantification of serum ceruloplasmin would be to determine its oxidase activity. Obviously, if this assertion is correct, specific oxidase activity would be increased in pregnant women, which is not in line with our results. Similarly, Milne’s results indicate that the use of oral contraceptives, while significantly
125
increasing serum levels of copper and ceruloplasmin, does not affect specific oxidase activity w8x, and we did not find a statistical or clinically significant difference for the specific oxidase activity between the healthy female and male control groups. During pregnancy, there is a transference of copper from the mother’s tissue reserves specially in the liver to the foetus, who stores it in order to meet future extrauterine requirements w3x; however, the exact mechanism of this placental transport has not been explained w3,23,24x. Transfer across the placenta of ceruloplasmin is unlikely due to this protein’s high molecular mass Ž132 kDa., and its presence in foetal blood is considered to represent foetal synthesis w3,23,24x. Although transport of copper to extrahepatic tissues by ceruloplasmin has been established w6,25x, the identity of the specific copper atoms in the protein, which takes part in this transport, has not yet been discovered w6x. However, it appears that ceruloplasmin retains full oxidase activity following a reaction with cells, and this gives us grounds to consider that copper in oxidase sites is not used for transport w6x. Subsequently, if this hypothesis is correct, then the decrease in specific oxidase activity of ceruloplasmin in pregnancy is not due to an impoverishment in copper at the placental level, even supposing that this were to occur. Nevertheless, it has been shown that the free copper and copper bound to amino acids is probably the best form transferred to the foetus w23,26x, and that there is no essential role for maternal ceruloplasmin in placental or foetal copper transport or metabolism w27x. Copper availability helps to determine the ratio of aporholoceruloplasmin secreted from the liver w28x, and it is well established that 75% of all copper is transferred through the placenta from mother to foetus in the last 10–12 weeks of pregnancy w10x. The decrease of the mother’s tissue reserves of copper in this period of gestation would lead to an increase in the relative proportion of the serum apoprotein, andror other molecular forms, which have low copper content in the oxidase sites, and consequently to a decrease in the specific oxidase activity of the circulating ceruloplasmin. The fact that the copperr ceruloplasmin ratio is significantly lower in the group of pregnant women rather than the control group Ž p - 0.001. may not be explained entirely due to a
126
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127
lower mean proportion of free copper in the first group Ž0.9% vs. 1.4%. w26x, and also suggests the presence of ceruloplasmin forms with a low copper content during pregnancy. Similar results for the specific oxidase activity of ceruloplasmin were obtained by DiSilvestro w21x during lactation, and this could represent, at least partly, an incomplete reversal of the effects of pregnancy. On the contrary, the increase of circulating ceruloplasmin in the inflammatory response is accompanied by a decrease in the aporholoprotein relationship w29x. The highly significant correlation between specific oxidase activity and the copperrceruloplasmin ratio in the female control group Ž p - 0.001. is to be expected, as ceruloplasminic copper represents approximately 93% of the total in this population group w12x. However, the lack of a significant correlation between these two variables in the group of pregnant women is more difficult to explain, particularly when we consider that they showed a decrease similar in relation to the control group Žapproximately 11%. and its interindividual variability ŽCV ( 7%. is very similar in both groups of women studied. The presence of ceruloplasmin forms with fewer copper atoms in the oxidase sites of its molecule, may lead to a disproportion between the specific oxidase activity and the copperrceruloplasmin ratio. It is unlikely that the lower specific oxidase activity in pregnant women would be also due to an inactivation of circulating ceruloplasmin by the oxidative stress, as Pacht and Davies w30x have postulated to explain their findings among smokers. Winyard et al. w31x have reported that the ferroxidase activity of ceruloplasmin is reduced by exposure in vitro to superoxide radical or hydrogen peroxide. However, Gutteridge’s w32x data do not confirm this results. Moreover, it would be rash to extrapolate the results of Winyard et al. to in vivo conditions. In patients with rheumatoid arthritis w29x or Duchenne muscular dystrophy w33x, we have not observed a significant negative correlation between the specific oxidase activity of ceruloplasmin and the serum concentration of thiobarbituric acid-reacting substances a marker of the oxidative stress. In conclusion, serum copper or ceruloplasmin concentration may not be sensitive indices of shortterm or marginal copper depletion because of their sensitivity to factors not related to dietary copper
intakes. The assessment of copper status is difficult and controversial, but the specific enzymatic activity of ceruloplasmin seems to be a sensitive index. Although pregnancy accelerates the rate of ceruloplasmin protein synthesis and release, the decrease in its specific oxidase activity would be an indicator of the degree of depletion of the mother’s copper reserves. In women with dietary copper restriction, the arterial blood pressure after sustained handgrip exercise of moderate intensity was negatively correlated with the specific oxidase activity of ceruloplasmin w34x. This suggests an impaired human blood pressure response in association with altered copper status, and supports the study of ceruloplasmin specific activity in pregnancy-induced hypertension. In connection with this, the recent results obtained for the serum ceruloplasmin and its ferroxidase activity by Vitoratos et al. w35x in women with pre-eclampsia are highly promising. Acknowledgements The authors wish express their appreciation to J. Pedreira for his technical assistance. References w1x Houot O, Tarallo P. Le cuivre. In: Chappuis P, editor. Les Oligoelements en Medecine et Biologıe. ´ ´ Paris: Lavoisier Tec and Doc 1991:459–98. w2x Milne DB. Trace elements. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd edn. Philadelphia: Saunders 1999:1029–55. w3x Speich M. Le cuivre dans la grossesse normale, diabetique et en perinatologıe. ´ Rev Fr Gynecol Obstet 1990;85:34–9. w4x Berg G, Kohlmeier L, Brenner H. Effect of oral contraceptive progestins on serum copper concentration. Eur J Clin Nutr 1998;52:711–5. w5x Fernandez MD. Cobre. In: Cocho JA, Escanero JF, Gonzalez-Buitrago JM, editors. Elementos Traza: Aspectos Bioquımicos, Analıticos y Clınicos. Barcelona: Comite´ de ´ ´ ´ Publicaciones de la Sociedad Espanola Clınica ˜ de Bioquımica ´ ´ y Patologıa ´ Molecular 1998:223–59. w6x Saenko EL, Yaropolov AI, Harris ED. Biological functions of ceruloplasmin expressed through copper binding sites and cellular receptor. J Trace Elem Exp Med 1994;7:69–88. w7x Milne DB. Assessment of copper nutritional status. Clin Chem 1994;40:1479–84. w8x Milne DB, Johnson PE. Assessment of copper status: effect of age and gender on reference ranges in healthy adults. Clin Chem 1993;39:883–7.
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127 w9x Louro MO, Tutor JC. Serum and plasma ceruloplasmin in humans. J Clin Chem Clin Biochem 1989;27:511–3. w10x Wachnik A. The physiological role of copper and the problems of copper nutritional deficiency. Nahrung 1988;32: 755–65. w11x Louro MO, Tutor JC. Determinacion ´ de la actividad oxidasica ´ de la ceruloplasmina en los analizadores Cobas Bio y Cobas Fara. Acta Bioquim Clin Latinoam 1991;25:119–25. w12x Cocho JA, Louro MO, Tutor JC. Determinacion ´ del cobre serico no unidoi a ferroxidasa I Žceruloplasmina.. Quim Clin ´ 1992;11:142–6. w13x Harris EK. Proposed goals for analytical precision and accuracy in single-point diagnostic testing. Arch Pathol Lab Med 1988;112:416–20. w14x Johnson DA, Osaki S, Frieden E. A micromethod for the determination of ferroxidase Žceruloplasmin. in human serum. Clin Chem 1967;13:142–50. w15x Takayanagi M, Yashiro T. Determination of human serum ceruloplasmin based on its ferroxidase activity. Jpn J Clin Chem 1985;14:233–8. w16x Takayanagi M. Determination of human serum ceruloplasmin by ion chromatography based on its ferrosidase activity. J Chromatogr, Biomed Appl 1986;374:378–82. w17x Takayanagi M, Goto S, Fukuda T, Yashiro T. Determination of human serum ceruloplasmin by measurement of its X X 3,3 ,5,5 -tetramethylbenzidine oxidase activity. J Pharm Biomed Anal 1987;5:403–7. w18x Erel O. Automated measurement of serum ferroxidase activity. Clin Chem 1998;44:2313–9. ¨ ¨ w19x Ozgunes H, Beksac MS, Duru S, Kayakirilmaz K. Instant effect of induced abortion on serum ceruloplasmin activity, copper and zinc levels. Arch Gynecol 1987;240:21–5. ¨ ¨ w20x Ozgunes H, Beksac MS, Atac FB, et al. Ceruloplasmin activity, copper and zinc determinations in predicting the prognosis of threatened abortion. Trace Elem Electrolytes 1994;11:139–42. w21x Disilvestro RA. Plasma levels of immunoreactive ceruloplasmin and other acute phase proteins during lactation. Proc Soc Exp Biol Med 1986;183:257–61. w22x Ruelland A, Gobron S, Cloarec L. Du choix d’une technique de dosage de la ceruloplasmine dans le syndrome inflamma´ toire et dans le cours du troisieme trimestre de la grossesse. Ann Biol Clin 1982;40:11–5.
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w23x Speich M, Murat A, Auget JL, Bousquet B, Arnaud P. Magnesium, total calcium, phosphorus, copper and zinc plasma and erythrocytes of venous cord blood from infants of diabetic mothers: comparison with a reference group by logistic discriminant analysis. Clin Chem 1992;38:2002–7. w24x Alevic-Juretic A, Frkovic A, Orlic Z. Umbilical cord copper content in newborns from the northern Adriatic area ŽCroatia.. Fresenius Envir Bull 1999;8:53–8. w25x Harris ED. Copper transport: an overview. Proc Soc Exp Biol Med 1991;196:130–40. w26x Noubah AM, Al-Awqati MA. Ultrafiltrable copper and related analytes in maternal and cord blood. Clin Chem 1990; 36:860–4. w27x Harris ZL, Klomp LWJ, Gitlin JD. Aceruloplasminemia: an inherited neurodegenerative disease with impairment of iron homeostasis. Am J Clin Nutr 1998;76:972S–7S. Žsuppl.. w28x Gitlin JD. A ceruloplasminemia. Pediatr Res 1998;44:271–6. w29x Louro MO, Cocho JA, Mera A, Tutor JC. Immunochemical and enzymatic study of ceruloplasmin in rheumatoid arthritis. J Trace Elem Med Biol 2000;14:174–8. w30x Pacht ER, Davis WB. Decreased ceruloplasmin ferroxidase activity in cigarette smokers. J Lab Clin Med 1988;111: 661–8. w31x Winyard P, Lunec J, Brailsford S, Blake D. Action of free radical generating systems upon the biological and immunological properties of ceruloplasmin. Int J Biochem 1984;16: 1273–8. w32x Gutteridge JMC. Antioxidant properties of proteins, ceruloplasmin, albumin and transferrin. A study of their activity in serum and synovial fluid from patients with rheumatoid arthritis. Biochem Biophys Acta 1986;869:119–27. w33x Bernabeu F, Louro MO, Castro M, Fernandez MP, Paz JM, Tutor JC. Immunoreactive ceruloplasmin and its oxidase activity in Duchenne muscular distrophy. Clin Chem Enzymol Commun 1995;6:291–5. w34x Lukaski HC, Kleavy LM, Milne DB. Effect of dietary copper on human autonomic cardiovascular function. Eur J Appl Physiol 1988;58:74–80. w35x Vitoratos N, Salamakelis E, Dalamaga N, Kassanos D, Creatsas G. Defective antioxidant mechanisms via changes in serum ceruloplasmin and total iron binding capacity of serum in women with pre-eclampsia. Eur J Obstet Gynecol 1999; 84:63–7.
Assessment of copper status in pregnancy by means of determining the specific oxidase activity of ceruloplasmin M. Olga Louro a , J. Angel Cocho b, J. Carlos Tutor a,) a
b
Laboratorio Central, Hospital Clınico UniÕersitario, Santiago de Compostela, Spain ´ Laboratorio de Alteraciones Metabolicas y Nutricionales, Hospital Clınico UniÕersitario, Santiago de Compostela, Spain ´ ´ Received 21 February 2001; received in revised form 18 June 2001; accepted 22 June 2001
Abstract Background: Conditions not directly related to copper nutriture, such as pregnancy, infections and inflammation, which increase serum copper concentration even during copper deprivation, may be expected to conceal changes in copper status. It has been suggested that the specific enzymatic activity of ceruloplasmin Žactivity per unit mass of enzyme protein. may be a sensitive indicator of copper status and is not affected by factors such as hormones or sex. In this study, we investigated the behaviour of specific oxidase activity of ceruloplasmin and the copperrceruloplasmin ratio in pregnant women. Methods: Copper, immunoreactive ceruloplasmin and its oxidase activity were determined in serum from 52 women in the last trimester of normal pregnancy, and in 50 control women of similar age living in the same area and who were not taking oral contraceptives. The results are expressed as mean " S.E.M. Results: In the group of pregnant women, significantly higher serum levels of copper, ceruloplasmin and its oxidase activity were found than in the control group Ž p - 0.001.. In both groups, a high correlation was found between these biochemical variables Ž r G 0.905, p - 0.001.. However, in the group of pregnant women the specific oxidase activity for ceruloplasmin Ž364.4 " 3.3 vs. 407.5 " 3.8 Urg. and the copperrceruloplasmin ratio Ž2.82 " 0.03 vs. 3.19 " 0.04 mgrmg. were significantly lower than in the control group Ž p - 0.001.. Conclusions: Although pregnancy accelerates the rate of ceruloplasmin protein synthesis and release with an increase of serum copper, the decrease in specific oxidase activity of circulating ceruloplasmin would be an indicator of the degree of depletion of the mother’s copper deposits in order to deal with the foetus’ needs. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Ceruloplasmin; Specific oxidase activity; Pregnant women; Copper status
1. Introduction The significant increase in the concentration of circulating copper due to hormonal impregnation in women who are pregnant or taking oral contraceptives is a well-documented issue w1–5x. This ef) Corresponding author. Fax: q34-981-950-403r34-981-950852. E-mail address: [email protected] ŽJ.C. Tutor..
fect is due to an increase in the hepatic synthesis and secretion into the extracellular environment of ceruloplasmin Žferroxidase I, EC 1.16.3.1., a multifunctional protein w6x which integrates within its molecule the major fraction of copper present in the blood plasma w1,2,5x. Similarly, other conditions also not directly related to copper nutriture, such as infection and inflammatory reactions, may increase serum copper w7x. Accordingly, conditions which tend to elevate serum copper concentration may obscure the
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 6 0 7 - 6
124
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127
changes in copper status even during copper deprivation w2,7x. Recent studies in humans suggest that the specific enzymatic activity of ceruloplasmin Žactivity per unit mass of enzyme protein. is a more sensitive indicator of copper status than either serum copper and ceruloplasmin or erythrocyte superoxide dismutase w2x, and is not influenced by factors such as age, gender, hormones w8x or the use of plasma or serum w9x. In this study, we indicate the results obtained for the specific oxidase activity of ceruloplasmin in a group of women in the last 3-month period of normal pregnancy, when supply of copper to the foetus is at its greatest level w10x.
determined by atomic absorption spectrometry using a previously described method w12x. The within- and between-run precision results yielded CVs of less than 3.0% and the recovery is 97.0–99.7%. When the data presented a Gaussian distribution, parametric tests were used ŽStudent’s t-test for differentiating averages and Pearson’s correlation coefficient.. When the data did not have a Gaussian distribution, non-parametric tests were used ŽWilcoxon’s test for comparing populations and Spearman’s correlation coefficient.. For normality, Shapiro– Wilk’s test was used or the skewness and kurtosis coefficients according to the size of the sample.
3. Results 2. Subjects and methods Fifty-two women in the last trimester of normal pregnancy were studied, with a mean Ž"S.E.M.. age of 27.5 " 2.0 years Žrange 15–43 years.. The control group comprised of 50 healthy women living in the same area with a mean Ž"S.E.M.. age of 31.3 " 1.9 years Žrange 18–45 years. and who were not taking oral contraceptives. Serum samples were taken between 8:00 and 10:00 a.m. Immunoreactive ceruloplasmin was determined by radial immunodiffusion using agar plates Nor-Partigen with monospecific antiserum ŽDade-Behring, Liederbach, Germany.. Oxidase activity of the ceruloplasmin was determined at 30 8C in a Cobas Bio analyzer ŽRoche Diagnostics, Basel, Switzerland. using o-dianisidine as a substrate w11x, which permits the expression of the enzyme activity in terms of International Units. The within- and between-run precision results of this technique yielded CVs of less than 1.3%. The total serum copper was
Table 1 shows the results obtained both in the group of pregnant women and the control group studied. The levels of serum copper, immunoreactive ceruloplasmin and oxidase activity are significantly higher in pregnant women Ž p - 0.001.. In turn, specific oxidase activity and the copperrceruloplasmin ratio are significantly higher in the control female subjects Ž p - 0.001. with the differences being clinically significant according to Harris’ criteria w13x. We did not find a statistical or clinically significant difference for the specific oxidase activity between the healthy female controls Ž n s 50, mean 407.5 " 3.8 Urg, medians 405.2 Urg. and male controls Ž n s 51, mean 398.2 " 4.0 Urg, media n s 398.3 Urg.. In a pool of sera from pregnant women, it was not possible to find any inhibitors of the oxidase activity or disturbing agent of the immunochemical determination of the ceruloplasmin. In the groups of pregnant and control women, high correlation coefficients were found between the
Table 1 Serum ceruloplasmin levels and related variables in the control and pregnant women Controls Ž n s 50.
Ceruloplasmin Žmgrl. Oxidase activity ŽUrl. Specific oxidase activity ŽUrg. Copper Žmgrl. CopperrCeruloplasmin Žmgrmg.
Pregnant women Ž n s 52.
Mean " S.E.M.
Median
Mean " S.E.M.
Median
335.7 " 8.0 136.1 " 2.8 407.5 " 3.8 1069.3 " 27.6 3.19 " 0.04
317 132 405 1045 3.23
758.8 " 18.7 276.1 " 6.9 364.4 " 3.3 2171.2 " 49.3 2.82 " 0.03
742 270 366 2155 2.87
Significance Ž p .
- 0.001 - 0.001 - 0.001 - 0.001 - 0.001
M.O. Louro et al.r Clinica Chimica Acta 312 (2001) 123–127
levels of serum copper, ceruloplasmin and its oxidase activity Ž r G 0.905, p - 0.001.. In the female control subjects, a highly significant correlation was found between specific oxidase activity and the copperrceruloplasmin ratio Ž r s 0.603, p - 0.001.; however, for pregnant women no statistical significance was found by correlating these variables Ž r s 0.208.. Neither group produced a significant correlation between specific oxidase activity and the ceruloplasmin concentration or its oxidase activity.
4. Discussion Ceruloplasmin is a multifunctional protein, performing catalytic, antioxidant and copper transport functions w6x. Its enzymatic character is unique in being able to oxidise both organic and inorganic substrates. The oxidase activity of ceruloplasmin towards aromatic amines is very well correlated with its ferroxidase activity w14–18x, and is derived from the reduction of select copper atoms in the oxidase sites of the holoprotein. The significantly increased levels of copper, ceruloplasmin and its oxidase activity, as well as the high degree of correlation between these variables, all found in the group of pregnant women, are widely confirmed in the literature w1–5,19–22x. However, at least to our knowledge, the fact that specific oxidase activity and the copperrceruloplasmin ratio may decrease significantly during normal pregnancy has not been previously dealt with. Milne’s studies w2,7,8x, suggest that in adults the specific oxidase activity of ceruloplasmin is a better indicator of copper status than serum concentrations of copper or ceruloplasmin. Specific oxidase activity would not be influenced by non-dietary factors such as age, gender or hormone use w7,8x. Ruelland et al. w22x indicated that during pregnancy and in experimental oestrogenic impregnation, immunochemical determination of ceruloplasmin would give falsely decreased results, and the best choice for the quantification of serum ceruloplasmin would be to determine its oxidase activity. Obviously, if this assertion is correct, specific oxidase activity would be increased in pregnant women, which is not in line with our results. Similarly, Milne’s results indicate that the use of oral contraceptives, while significantly
125
increasing serum levels of copper and ceruloplasmin, does not affect specific oxidase activity w8x, and we did not find a statistical or clinically significant difference for the specific oxidase activity between the healthy female and male control groups. During pregnancy, there is a transference of copper from the mother’s tissue reserves specially in the liver to the foetus, who stores it in order to meet future extrauterine requirements w3x; however, the exact mechanism of this placental transport has not been explained w3,23,24x. Transfer across the placenta of ceruloplasmin is unlikely due to this protein’s high molecular mass Ž132 kDa., and its presence in foetal blood is considered to represent foetal synthesis w3,23,24x. Although transport of copper to extrahepatic tissues by ceruloplasmin has been established w6,25x, the identity of the specific copper atoms in the protein, which takes part in this transport, has not yet been discovered w6x. However, it appears that ceruloplasmin retains full oxidase activity following a reaction with cells, and this gives us grounds to consider that copper in oxidase sites is not used for transport w6x. Subsequently, if this hypothesis is correct, then the decrease in specific oxidase activity of ceruloplasmin in pregnancy is not due to an impoverishment in copper at the placental level, even supposing that this were to occur. Nevertheless, it has been shown that the free copper and copper bound to amino acids is probably the best form transferred to the foetus w23,26x, and that there is no essential role for maternal ceruloplasmin in placental or foetal copper transport or metabolism w27x. Copper availability helps to determine the ratio of aporholoceruloplasmin secreted from the liver w28x, and it is well established that 75% of all copper is transferred through the placenta from mother to foetus in the last 10–12 weeks of pregnancy w10x. The decrease of the mother’s tissue reserves of copper in this period of gestation would lead to an increase in the relative proportion of the serum apoprotein, andror other molecular forms, which have low copper content in the oxidase sites, and consequently to a decrease in the specific oxidase activity of the circulating ceruloplasmin. The fact that the copperr ceruloplasmin ratio is significantly lower in the group of pregnant women rather than the control group Ž p - 0.001. may not be explained entirely due to a
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lower mean proportion of free copper in the first group Ž0.9% vs. 1.4%. w26x, and also suggests the presence of ceruloplasmin forms with a low copper content during pregnancy. Similar results for the specific oxidase activity of ceruloplasmin were obtained by DiSilvestro w21x during lactation, and this could represent, at least partly, an incomplete reversal of the effects of pregnancy. On the contrary, the increase of circulating ceruloplasmin in the inflammatory response is accompanied by a decrease in the aporholoprotein relationship w29x. The highly significant correlation between specific oxidase activity and the copperrceruloplasmin ratio in the female control group Ž p - 0.001. is to be expected, as ceruloplasminic copper represents approximately 93% of the total in this population group w12x. However, the lack of a significant correlation between these two variables in the group of pregnant women is more difficult to explain, particularly when we consider that they showed a decrease similar in relation to the control group Žapproximately 11%. and its interindividual variability ŽCV ( 7%. is very similar in both groups of women studied. The presence of ceruloplasmin forms with fewer copper atoms in the oxidase sites of its molecule, may lead to a disproportion between the specific oxidase activity and the copperrceruloplasmin ratio. It is unlikely that the lower specific oxidase activity in pregnant women would be also due to an inactivation of circulating ceruloplasmin by the oxidative stress, as Pacht and Davies w30x have postulated to explain their findings among smokers. Winyard et al. w31x have reported that the ferroxidase activity of ceruloplasmin is reduced by exposure in vitro to superoxide radical or hydrogen peroxide. However, Gutteridge’s w32x data do not confirm this results. Moreover, it would be rash to extrapolate the results of Winyard et al. to in vivo conditions. In patients with rheumatoid arthritis w29x or Duchenne muscular dystrophy w33x, we have not observed a significant negative correlation between the specific oxidase activity of ceruloplasmin and the serum concentration of thiobarbituric acid-reacting substances a marker of the oxidative stress. In conclusion, serum copper or ceruloplasmin concentration may not be sensitive indices of shortterm or marginal copper depletion because of their sensitivity to factors not related to dietary copper
intakes. The assessment of copper status is difficult and controversial, but the specific enzymatic activity of ceruloplasmin seems to be a sensitive index. Although pregnancy accelerates the rate of ceruloplasmin protein synthesis and release, the decrease in its specific oxidase activity would be an indicator of the degree of depletion of the mother’s copper reserves. In women with dietary copper restriction, the arterial blood pressure after sustained handgrip exercise of moderate intensity was negatively correlated with the specific oxidase activity of ceruloplasmin w34x. This suggests an impaired human blood pressure response in association with altered copper status, and supports the study of ceruloplasmin specific activity in pregnancy-induced hypertension. In connection with this, the recent results obtained for the serum ceruloplasmin and its ferroxidase activity by Vitoratos et al. w35x in women with pre-eclampsia are highly promising. Acknowledgements The authors wish express their appreciation to J. Pedreira for his technical assistance. References w1x Houot O, Tarallo P. Le cuivre. In: Chappuis P, editor. Les Oligoelements en Medecine et Biologıe. ´ ´ Paris: Lavoisier Tec and Doc 1991:459–98. w2x Milne DB. Trace elements. In: Burtis CA, Ashwood ER, editors. Tietz Textbook of Clinical Chemistry. 3rd edn. Philadelphia: Saunders 1999:1029–55. w3x Speich M. Le cuivre dans la grossesse normale, diabetique et en perinatologıe. ´ Rev Fr Gynecol Obstet 1990;85:34–9. w4x Berg G, Kohlmeier L, Brenner H. Effect of oral contraceptive progestins on serum copper concentration. Eur J Clin Nutr 1998;52:711–5. w5x Fernandez MD. Cobre. In: Cocho JA, Escanero JF, Gonzalez-Buitrago JM, editors. Elementos Traza: Aspectos Bioquımicos, Analıticos y Clınicos. Barcelona: Comite´ de ´ ´ ´ Publicaciones de la Sociedad Espanola Clınica ˜ de Bioquımica ´ ´ y Patologıa ´ Molecular 1998:223–59. w6x Saenko EL, Yaropolov AI, Harris ED. Biological functions of ceruloplasmin expressed through copper binding sites and cellular receptor. J Trace Elem Exp Med 1994;7:69–88. w7x Milne DB. Assessment of copper nutritional status. Clin Chem 1994;40:1479–84. w8x Milne DB, Johnson PE. Assessment of copper status: effect of age and gender on reference ranges in healthy adults. Clin Chem 1993;39:883–7.
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