Ceruloplasmin As a Marker of Occupational Copper Exposure

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Ceruloplasmin as a marker of occupational copper exposure

ASIM SAHAa, ANIL KARNIKb, NATUBHAI SATHAWARAc, PRADIP KULKARNId AND VEDPRAKASH SINGHe aNational Institute of Occupational Health, Meghani Nagar, Ahmedabad, Gujarat, India bNational Institute of Occupational Health, Meghani Nagar, Ahmedabad, Gujarat, India cNational Institute of Occupational Health, Meghani Nagar, Ahmedabad, Gujarat, India dNational Institute of Occupational Health, Meghani Nagar, Ahmedabad, Gujarat, India eNational Institute of Occupational Health, Meghani Nagar, Ahmedabad, Gujarat, India

Estimation of serum copper to indicate copper status in the human system in the context of moderate chronic occupational copper exposure requires a sophisticated and expensive method. Hence, a search for a suitable marker has been made and few studies have found potential in serum ceruloplasmin. In this context, the present study was initiated to explore whether ceruloplasmin could serve as a predictor of occupational copper exposure. An interviewer- administered questionnaire survey (personal, occupational and health-related information) was undertaken involving 185 employees of a copper handling industry. Serum alkaline phosphatase, serum glutamic pyruvic transaminase (SGPT), serum ceruloplasmin and serum copper were estimated in all the subjects. Multivariate analysis was undertaken using a linear regression model to understand the contribution of serum copper on serum ceruloplasmin values adjusting for the role of other confounders. Serum copper and serum ceruloplasmin values were found to have a statistically signiﬁcant positive correlation (R ¼ 0.169, adjusted R2 ¼ 0.024) after adjustment for other predictors like age, nature of job (department), job duration, smoking, serum alkaline phosphatase and SGPT. This study concludes that the serum ceruloplasmin level can act as a reliable indicator of copper status in the human body following copper exposure in cases of chronic moderate occupational exposure to copper. Journal of Exposure Science and Environmental Epidemiology (2008) 18, 332–337; doi:10.1038/jes.2008.2; published online 20 February 2008

Keywords: ceruloplasmin, occupational copper-exposure, exposure marker.

Introduction brassing, welding, cutting orpolishing of copperarealso sources of exposure (Armstrong et al., 1983). Copper, a reddish metal that occurs naturally not only in soil, Although copper is necessary for good health, exposure to water, sediment and air, but also in plants and animals, is an large doses of copper causes harm to human health. Long- essential element forall known living organisms, including term exposure to copper dust can cause irritation of the nose, human beings. Copper is involved in a variety of critical mouth and eyes and may also result in headache, dizziness, metabolic processes. A large number of enzymatic and nausea and diarrhea (Lenntech water treatment and air structural proteins incorporate copper. Copper can function puriﬁcation holding B.V., 2007) Occurrence of metal fume as an electron transfer intermediate, hence acting in feverhas been observedafterexposureto copperfumes oxidation/reduction of enzymes. In certain cases, copper is (Suciu et al., 1981). Otherstudies have reported hepato- required as a cofactor and in some occasions it acts as an megaly, digestive disorders and a range of respiratory signs allosteric component of enzymes (Hartmann and Evenson, and symptoms (McLaughlin et al., 1992). The seriousness of 1992; Linderand Hazegh-Azam, 1996). effects of copperexposureis expected to increasewith both Copperhas proved to be one of the most useful metals, level and length of exposure (Agency for Toxic Substances mined and worked by men since long past, because of its and Disease Registry, 1990). essential use in various spheres of life. A wide range of To indicate the exposure as well as toxicity of copper in industrial activities may expose workers to dust from rocks an exposed population, determination of serum copper level and ore containing different concentrations of copper has been used as marker. But because clearance of copper (Weant, 1985). Workers exposed to smelting operations, is different from person to person, serum copper alone, although it can be a potential biomarker of potential effect, 1. Address all correspondence to: Dr. A. Saha, Occupational Medicine may not act as an efﬁcient biomarkerof copperexposure. Division, National Institute of Occupational Health, Meghani Nagar, Moreover, atomic absorption spectrophotometry used for Ahmedabad, 380016, India. such analysis is an expensive method, available in only Tel: 91 07922686351. Fax: 91 07922686110. þ þ specialized laboratories. For these reasons, copper-related E-mail: [email protected] Received 9 July 2007; accepted 10 November2007; published online 20 carrier proteins, like ceruloplasmin, have been used for February 2008 monitoring copper exposure (Araya et al., 2003a). Ceruloplasmin and occupational copper exposure Saha et al.

Moderate exposure to copper takes place on a long-term Methods basis in case workers are employed in many copper-handling industries (e.g., smelting industries). Evidence is accumulat- The present study was carried out in a unit of the copper ing about different health effects taking place after a long handling industry where copper ore is used as raw material period of such moderate level exposures (Agency for Toxic and copper rods/wires are produced over a serial process of Substances and Disease Registry, 2004). In such situations, smelting, refining and casting of rods/wires. Acid (sulfuric where effects may take a long time to appear, monitoring of acid and phosphoric acid) as well as fertilizer is also produced exposure is important, and, accordingly, availability of a (as byproducts) by this industry. All the employees working biomarker of such exposure is extremely useful. in the unit (185) were included in this study. All the workers Ceruloplasmin, an a-2-glycoprotein that contains more employed in the industries were male. Necessary approval than 95% of copperin plasma, not only acts as a copper was obtained from the Institutional Ethics Committee of transport protein but also a multicopper oxidase protein with National Institute of Occupational Health, India. Informed six atoms of copper incorporated within it plays an important consent was taken from all the participants. All the role in the mobilization and oxidation of iron into participants were interviewed with the help of an inter- transferring (Osaki and Johnson, 1966). Its ferroxidase viewer-administered questionnaire to obtain their personal activity has led to the understanding that there is a molecular (age, habit, education status etc.), occupational (nature of link between copperand ironmetabolism. Ceruloplasmin job, experience etc.) and health-related information. mobilizes iron into plasma from iron storage cells in liver. Blood samples were drawn from the cubital vein and An equally important role ceruloplasmin serves is that, after transferred into separate heparinized tubes. Serum was its biosynthesis in liver, it acts as a major copper transport obtained by centrifuging. Serum glutamic pyruvic transami- vehicle, comparable to transferrin (Frieden and Hsieh, nase (SGPT) and alkaline phosphatase was measured using 1976a). Evidence is accumulating that copperatoms of clinical analyzer. Quantitative determination of cerulo- ceruloplasmin are a prerequisite for copper utilization in the plasmin was carried out by turbidimetric immunoassay using biosynthesis of cytochrome oxidase and other copper a dialab kit. Copper concentration was measured by flame proteins (Frieden and Hsieh, 1976b). atomic absorption spectrophotometry using atomic absorp- So far as biomarkers of copper exposure are concerned, tion spectrophotometer (AAS) make Perkin-Elmer model besides blood copperlevel, otherserum proteinsexposed to 3100. Detection limit and sensitivity was (0–1000) IU/l for copper in the circulation may also serve as an important alkaline phosphatase (Wilkinson and Winsten, 1969), biomarker for copper. The biomarkers of effect available are (0–450) mg/dl and 0.1 mg/dl forSGPT (Bradley et al., efficient and helpful in diagnosing coppertoxicity at high 1972), (0–90) and 0.1 mg/dl forceruloplasmin (Poulik and levels of exposure over a short time period because there are Weiss, 1982). important clinical manifestations and typical changes in Data analysis was carried out using EPInfo 6 and SPSS blood due to oxidative stress induced by copper. However, 6.1.4 software. Initially, a descriptive analysis was carried traditional effect markers like homocysteine, liver glutamic out. Afterwards, multivariate analysis was undertaken using oxaloacetic transaminase, glutamic pyruvic transaminase a linear regression model. Ceruloplasmin values were entered (GPT) and g-glutamyl transpeptidase (GGT), Zn–Cu- as a dependent variable and age, department (grading of superoxide dismutase (SOD) activity in erythrocytes, departments was carried out from 1 to 6 in increasing order glutathione and IL-2 production, and urinary copper of environmental copper exposure), job duration, smoking excretion after 2,3-dimercapto-1-propane sulfonic acid chal- status (grade 1 was allotted to never smoker group and grade lenge (mainly blood biochemical and urinary indicators) are 2 was offeredto smokeras well as past smokergroup), serum not sensitive enough to reveal small changes in copper status, coppervalues, alkaline phosphatase values and SGPT values which may be relevant for long-term health (Kehoe et al., were entered as predictors. While making gradation for 2000; Araya et al., 2003b). Moreover, they are not specific to departments, environmental copper exposure data were copper toxicity alone. Studies have already made efforts to obtained from environmental monitoring records of the find the potential of ceruloplasmin as a biomarkerforcopper industry and were used for gradation. Grade 1 was offered exposure (Araya et al., 2003b). Different researchers have for lowest exposure and grade 6 was offered for highest observed positive correlation between serum copper and exposure. Grade 1 to grade 6 was offered to office job, ceruloplasmin (Davidoff et al., 1978, Magalova et al., 1997; fertilizer production, acid production, casting of rods/wires, Boz et al., 2005). However, the role of ceruloplasmin is yet to job at refinery and job at smelter, respectively. Serum copper, be established as a biomarkerforcopperexposureand serum alkaline phosphatase, SGPT and serum ceruloplasmin consequently more evidence is needed to support the values being normally distributed, no data transformation hypothesis. In this backdrop, this present study was initiated was necessary and these variables were entered as such in a to explore whether ceruloplasmin could serve as a predictor linear regression model. Analysis was carried out initially by of copperexposure. accommodating all variables at a time in the modal (Enter

Journal of Exposure Science and Environmental Epidemiology (2008) 18(3) 333 Sahaetal. Ceruloplasmin and occupational copper exposure method) and then by accommodating the predictors in a smokers. Mean family size of workers was 3.87(±1.15) stage wise manner (stepwise forward LR method). Correla- persons and mean income was 9998 (±4759) rupees (Indian tion between serum ceruloplasmin and different other currency). The industry in which this study was undertaken variables was also calculated. While calculating the correla- had been operating for only 10 years and therefore maximum tion coefﬁcient between serum ceruloplasmin and variable, job duration observed was 10 years (mean job duration was adjustment for other variables was carried out. 5.6±9.5 years). The majority of workers (36.2%) were working in smelting activity, whereas 14.1%, 10.8% and 17.8% were working in reﬁnery, casting of rods/wires and Results acid production, respectively. The mean serum copper level observed in the subjects was 101.74 (±21.45) mg/dl, whereas Table 1 describes the characteristics of the study subjects. mean SGPT, mean serum alkaline phosphatase and mean Mean age of the study subjects was 31.2 years (±5.9). The serum ceruloplasmin levels were 32.38 (±19.85) IU/l, 50.75 majority of subjects were below 40 years of age (90.8%). (±20.79) IU/l and 27.94 (±8.04) mg/dl, respectively. Almost 15% of study subjects wereeithersmokersorpast Table 2 shows the distribution of serum ceruloplasmin values according to different characteristics of the study Ta bl e 1 . Characteristics of the study participants. population. The ceruloplasmin value was a little higher in subjects of 40 or more years of age. Similarly, it was a bit Parameters (characteristics) Values (N ¼ 185) higher in subjects having more than 5 years of job experience

Age (years) o30 82 (44.3%) 30–39 86 (46.5%) Table 2 . Distribution of serum ceruloplasmin values according to Z40 17 (9.2%) different characteristics of the study population. Mean age (years)±SD 31.2±5.9 Characteristics Ceruloplasmin values ANOVA results Education (Mean±SD) (mg/dl) Secondary 124 (66.9%) Above secondary 61 (33.1%) Age (years) o30 27.01±7.53 Marital status 30–39 28.17±8.34 F ¼ 2.057, d.f. ¼ 2, Married 155 (83.8%) P ¼ 0.128 Unmarried 30 (16.2%) Z40 31.27±8.39

Smoking Smoking Neversmoker 158 (85.4%) Neversmoker 27.97 ±8.28 F ¼ 0.023, d.f. ¼ 2, Eversmoker 27 (14.6%) P ¼ 0.873 Eversmoker 27.72 ±6.58 Job experience (years) Upto 5 years 86 (46.5%) Nature of job (Department) Z6 years 99 (53.5%) Office job 28.09±6.01 Mean job experience (±SD) (years) 5.6 ± 9.5 Fertilizer production 28.74±6.72 Acid production 24.55±6.98 F ¼ 1.762, d.f. ¼ 1, Nature of job (Department) P ¼ 0.122 Office job 28 (15.1%) Casting of rods/wires 30.2±9.81 Fertilizer production 11 (5.9%) Refinery 29.54±8.69 Acid production 33 (17.8%) Smelter28.12 ±8.38 Casting of rods/wires 20 (10.8%) Refinery 26 (14.1%) Job Experience Smelter67 (36.2%) Upto 5 years 27.79±8.19 F ¼ 0.73, d.f. ¼ 1, P ¼ 0.806 Serum copper (mg/dl) Z6 years 28.07±7.48 r100 91 (49.2%) 4100 94 (50.8%) Serum copper (mg/dl) Mean serum copper (mg/dl) 101.74 ± 21.45 r100 26.82±8.19 F ¼ 3.509, d.f. ¼ 1, P ¼ 0.059 SGPT (IU/l) 4100 29.02±7.79 r40 141 (76.2%) 440 44 (23.8%) SGPT (IU/l) Mean SGPT (IU/l) 32.38±19.85 r40 27.66±7.66 F ¼ 0.73, d.f. ¼ 1, Mean serum alkaline phosphatase (IU/l) 50.75±20.79 P ¼ 0.601 Mean serum ceruloplasmin (mg/dl) 27.94±8.04 440 28.84±9.18

334 Journal of Exposure Science and Environmental Epidemiology (2008) 18(3) Ceruloplasmin and occupational copper exposure Saha et al. and among subjects who had SGPT levels more than 40 IU/l. Discussion The value of ceruloplasmin was also higher in subjects who had serumcopperlevels higherthan 100 mg/dl. The median Relevant biological effects associated with short-term mild to normal value for serum copper being 100 mg/dl (IPCS, moderate copper excess are unknown. It is difficult to identify WHO, 1998) and upperlimit of SGPT value being 40 IU/l markers of such early effects because limits of homeostatic (Bradley et al., 1972), these variables were dichotomized at range are still unidentified, and early changes may represent these levels while examining the distribution of serum adaptive responses that do not necessarily imply risk of ceruloplasmin values. However, no such difference observed damage. However, evidence is accumulating about the health between different groups was statistically significant. effects of moderate level copper exposure occurring on a So far as correlation between serum ceruloplasmin and long-term basis (as occurs in different occupational settings) different other variables is concerned, a statistically signifi- (Agency forToxic Substances and Disease Registry,1990). cant positive correlation was observed between serum Monitoring of exposure is of importance in such situations, ceruloplasmin and serum copper levels as well as between and, accordingly, availability of exposure biomarker may be serum ceruloplasmin level and age of workers (Table 3). of use. Efforts have been made by different researchers to However, the correlation coefficient, although statistically identify a suitable marker for such exposure (mainly short- significantly different from zero, was quite small. term exposure). In a study involving adult healthy volunteers, Table 4 shows the effect of different variables on serum exposure of additional 50–60 mg of copper/kg/day for 3 ceruloplasmin values (after multivariate analysis through months orup to 159 mg/kg/day for2 months (a tentatively linear regression model). When all the variables were entered safe and adequate dose of intake of copperbeing 2–3 mg/day simultaneously (Entermethod) in the linearregression for adults (National Research Council, 1980)) resulted in no model, age and serum copper showed significant contribution significant changes of SOD activity in erythrocytes, of copper on serum ceruloplasmin. On stepwise entry of predictors (Forward LR method) in the model, all the variables except serum copper were excluded from the model, while serum 60 coppershowed statistically significant contribution on serum ceruloplasmin. Serum copper and serum ceruloplasmin 50 values showed significant positive correlation (R ¼ 0.169, adjusted R2 ¼ 0.024) (Figure 1). 40

30 Ta bl e 3 . Correlation between serum ceruloplasmin and different other variables. 20

a Serum Ceruloplasmin Variables Correlation coefﬁcient Signiﬁcance (P-value) 10

Serum copper 0.164 0.02 SGPT 0.024 0.737 0 Serum ALP À0.004 0.956 0 50 100 150 200 Age 0.157 0.026 Serum Copper Job duration À0.118 0.095 Figure 1. Relationship between serum copper and serum ceruloplas- aAdjusted foreach other. min values (uploaded separately as an image).

Ta bl e 4 . Effect of different variables on serum ceruloplasmin values.

Variables Standardized coefﬁcient (b) t Signiﬁcance

All variables entered Age 0.190 2.317 0.022 simultaneously in linear Department 0.025 0.339 0.735 regression model Job duration À0.047 À0.578 0.564 Smoking À0.036 À0.476 0.635 Serum copper 0.215 2.911 0.004 Serum alkaline phosphatase À0.087 À1.172 0.243 SGPT 0.002 0.175 0.129 R ¼ 0.293, adjusted R2 ¼ 0.049 Variables entered stepwise Serum copper 0.169 2.455 0.015 R ¼ 0.169, adjusted R2 ¼ 0.024

Journal of Exposure Science and Environmental Epidemiology (2008) 18(3) 335 Sahaetal. Ceruloplasmin and occupational copper exposure concentration (in serum, erythrocytes and mononuclear cells) environmental exposure through personal monitoring and to and of serum ceruloplasmin. Neither was found different by find correlation between serum ceruloplasmin levels and such gendernorage (Araya et al., 2003a). Anothersimilarstudy copper exposure values. Moreover, the observed correlation undertook analysis of 195 participants but showed no coefficient, although statistically significant, was quite small. correlation between copper exposure and copper status Nevertheless, because of the small correlation between serum variables like serum copper, serum ceruloplasmin and SOD copper and ceruloplasmin, ceruloplasmin may find use as a activity (Araya et al., 2003b). On the other hand, a study of biomarkerof copperexposureacrosspopulation orin five groups of individuals observed significant correlation comparing exposed and unexposed populations, providing between serum copper and serum ceruloplasmin in all that the sample size is sufficiently large to achieve strong populations except the smoker group (healthy subjects, oral statistical significance. contraceptive users, non-smoking pretreatment lymphoma patients and treated lymphoma patients) (Davidoff et al., 1978). Anotherstudy of 128 healthy volunteers(57 men and Acknowledgements 71 women) aged 20–74 years, observed a weak correlation between serum levels of copper and ceruloplasmin (Magalova We hereby acknowledge Mr AM Suthar, Mr MM Patel, et al., 1997). A statistically significant correlation between MrU Desai, MrV Rathod, MrS Dodia and Ms S Patel for serum copper and serum ceruloplasmin was also observed in theircooperationand help duringthis study. patients of gastrointestinal tract cancers (Boz et al., 2005). We hereby declare that this article is neither published nor Serum copper and ceruloplasmin levels are elevated in cancer underconsiderationforpublication in any otherjournal. We patients as a part of a biological response to cancer, rather also confirm that necessary consent was given from the than its cause (Willingham and Sorenson, 1986) and many concerned study subjects and the ethical committee as well as studies have looked for correlation between serum copper the scientific advisory committee of the National Institute of and ceruloplasmin levels in cancer patients, although the Occupational Health, India. The study was approved by the relevance of the findings of such studies to the general National Institute of Occupational Health, India. population may be limited. A study designed to understand how ceruloplasmin, an indicator of copper status, reflects Conflictofinterest changes of copperstatus among healthy individuals observed The authors state no conflict of interest. that ceruloplasmin values in serum represent a reliable indicatorof copperstatus among the normalpopulation (Mendez et al., 2004). One more study in which serum References copper and ceruloplasmin values of 338 individuals were Agency forToxic Substances and Disease Registry. Toxicological Profile for retrospectively reviewed observed a linear relationship Copper. United States Public Health Service, 1990. between the two variables [Deming LR equation Agency forToxic Substances and Disease Registry. 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