Macular degeneration and elevated serum ceruloplasmin

December 1986 Vol. 27/12

Investigative Ophthalmology& Visual Science

A Journal of Dosic and Clinical Research

Articles

Macular Degeneration and ElevatedSerum Ceruloplasmin

Dovid A. Newsome, Mono Swortz,* Nicholas C. Leone,t A. Tyl Hewirr, Frances Wolford,t and Earl D. Miller

Macular degeneration associated with age and drusen, an important cause of visual loss, is associatedclinically with alterations in the retinal pigmented epithelium. Because the pigmented epithelium is acopper-rich tissue with antioxidant properties, the copper economy in patients and controls were studiedby measuring ceruloplasmin. Ceruloplasmin, a multifunctional, copper-binding a-globulin, was signifi-cantly elevated in non-related patients as compared with controls (691 ± 1 5 3 mg/L vs 312 ± 64; P< .001), both by the p-phenylenediamine oxidation technique and radial immunodiffusion assay. When53 members of a large family were divided clinically into persons with and without macular degeneration,the ceruloplasmin concentrations were not significantly different from each other, but were elevated ascompared with non-related controls (P < .001). These differences were not due to an intragroup agemismatch. A group of patients with retinitis pigmentosa had normal serum ceruloplasmin concentrations.This study suggests a relationship between serum ceruloplasmin, trace metals, and the tissue alterationsassociated with macular degeneration that deserves further investigation. Invest Ophthalmol Vis Sci27:1675-1680, 1986

Macular degeneration associated with age and dru-sen is a leading cause of visual loss among aged indi-viduals in the United States and western Europe. Theetiology of macular degeneration is unknown, althoughphototoxicity and oxidative free radicals may be of im-portance.1 Based on clinical observations, the retinalpigmented epithelium (RPE) is significantly involvedin the disease process.2 The RPE has a variety of spe-cialized functions, including antioxidant activity, andmany of its enzymes are copper-dependent. In addition,the RPE has one of the highest copper concentrationsin the body.3 Thus, a better knowledge of factors re-lating to copper balance could be important to fur-thering our understanding of macular degeneration.

From the Michael M. Wynn Center for the Study of Retinal De-generations, The Wilmer Ophthalmological Institute, The JohnsHopkins Hospital and University, Baltimore, Maryland; the *De-partment of Ophthalmology, University of Utah Medical Center,Salt Lake City, Utah; and the fDepartment of Veterinary Science,Utah State University, Logan, Utah.

Supported in part by the Research Fund, Department of VeterinaryScience, Utah State University, in cooperation with and under thesupervision of James L. Shupe, D.V.M.

Submitted for publication: September 30, 1985.Reprint Requests: David A. Newsome, MD, LSU Eye Center, 2020

Gravier Street, New Orleans, LA 70112.

Ceruloplasmin is a multifunctional, copper-bindinga-globulin that appears to be involved in the regulationof copper utilization by enzymatically mediating thetransfer of copper to copper-containing enzymes invarious parts of the body.4"7 Ceruloplasmin also hasantioxidant capabilities, due to the discharge of elec-trons, especially during the course of its catalytic ac-tivity in converting Fe(II) to Fe(III). These electronscan be used to reduce molecular oxygen directly toH2O.8 Blood concentrations of ceruloplasmin can beinfluenced by nutritional and pathological conditions,resulting either in hypoceruloplasminemia (e.g., mal-nutrition, Wilson's disease) or hyperceruloplasminemia(e.g., leukemia, rheumatoid arthritis).4

The relationship between blood and RPE concen-trations of copper and between antioxidant activity inthe blood and similar activity in the RPE is not clear.Consequently, alterations in circulating ceruloplasmincould influence both the transport of copper and thefunctioning of the RPE. For this reason, we comparedthe serum ceruloplasmin concentrations of normal in-dividuals and those with macular degeneration in apopulation from northern Utah, as well as those of agroup of patients with retinitis pigmentosa and otherocular conditions. Our results show that individualswith macular degeneration have elevated cerulo-plasmin.

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1676 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / December 1986 Vol. 27

Fig. 1. If, on examinationof fundus photographs, astudy subject showed drusenand pigment mottling equalto or less than what is shownin this standard photograph,their maculas were graded asnormal.

Materials and Methods

Study subjects were enrolled after obtaining in-formed consent using documents and procedures thathad been approved by the intramural review board.

Participants with macular degeneration and theirunaffected controls were born, reared, and dwell in thenorthern Utah area. All subjects were Caucasian. Fifty-three of our subjects were blood relatives distributedamong three generations of one family, called hereFamily A. Detailed genealogic histories were wellknown for all participants. Thus, we know that ourstudy group does not represent a sample of an inbredpopulation. This study was not population-based;therefore, we do not know what proportion of the area'stotal macular degeneration population our samplerepresents. Retinitis pigmentosa (RP) subjects werefrom various regions of the continental U.S. After in-formed consent was obtained, a detailed history ofocular and general medical status, medications, familylineage, and diseases were elicited by a combination ofinterview and questionnaire techniques. All partici-pants received thorough ocular examinations. Cor-rected Snellen acuity at 20 feet, and near acuity withJaeger type, were determined. Anterior-segment ex-amination, Schiotz intraocular-pressure determina-tions, and dilated direct and indirect funduscopy werealso performed. Fundus appearance was recorded viaa portable (Kowa Optimed, Torrance, CA) or station-ary (Carl Zeiss Co., Thornwood, NY) fundus cameraon Kodacolor (Eastman Kodak, Rochester, NY) ASA64 or 25 film, respectively. Subjects with findings re-quiring immediate attention were so informed, and re-ferred to their personal ophthalmologists.

Affected Groups

The diagnosis of age-related maculopathy was madeif drusen and pigmentary changes were present and thebest corrected visual acuity was 20/25 or worse withno other apparent explanation. The presence of drusenand pigmentary changes greater than those in a stan-dard photograph (Fig. 1) was confirmed by an inde-pendent experienced reader who reviewed the subjects'photographs in a masked fashion.

Eye diseases other than macular degeneration in-cluded macular holes, macular pucker, retinal detach-ment, progressive myopia, and RP. RP was diagnosedon the basis of a history of night blindness, findings oftypical pigmentary changes, including intraretinal pig-ment migration, depigmentation of the RPE withmacular sparing, arteriolar narrowing, vitreous abnor-malities, preservation of relatively good central acuityuntil late in the course of the disease, and the presenceof peripheral visual field defects, as assessed by Gold-mann perimetry. Both simplex (no affected siblings)and multiplex (one or more affected siblings) cases wereincluded.

Unaffected Control Group

These subjects had no macular degeneration andwere not blood relatives of affected subjects. This groupincluded some spouses of affected subjects.

Blood relatives of affected subjects formed the "un-affected relatives" control group. Macular findings inthis group were less than the standard photograph. Theremainder of the examination, with the exception oflens changes in some subjects, was normal.


No. 12 MACUIAR DEGENERATION AND CERULOPLA5MIN / Newsome er ol. 1677

Table 1. Comparison of mean-grouped serum ceruloplasmin concentrations

Group

Unaffected; controls non-Family AUnaffected; Family AAffected, non-Family AAffected, Family AOther eye diseasesRP

Numberof Persons

211834341326

Age

Mean ± S.D.

62.6 ±8.858.2 ±7.070.2 ±8.161.7 zt 9.161.3 ±7.139.7 ±8.2

Ceruloplasmin (mg/l)

Mean ± S.D.

312±64655 ±87691 ±153641±143318±55295 ± 50

Range

188-446560-866432-988*424-996*220-408206-394

P-Value ofComparison With

Unaffected,Non-Family A

__<.00l<.00l<.00l>.05>.05

* Statistically significant relative to other two groups, as well as to non-Family A controls.

Serum Collection and Handling

Peripheral venous blood samples were withdrawn,using sterile precautions, into evacuated tubes speciallyprepared to avoid trace-metal contamination (TerumoMedical Corp., Elkton, MD). Blood was allowed toclot at room temperature for 45-75 min. Serum wasseparated by centrifugation and divided among severalsiliconized vials, to minimize freeze-thawing. Aftertemporary storage at -20° , serum was transported ondry ice for permanent storage at —70° until assayed.Ceruloplasmin assays were usually done within 60 daysof collection, although repeat determinations on serafrom ten randomly selected individuals showed littlevariation after an additional 45 days at —70°, in agree-ment with the findings of other laboratories.9"10

Ceruloplasmin Assay

Serum ceruloplasmin concentrations were deter-mined by a colorimetric assay for its oxidase activityusing p-phenylenediamine (PPD) (Eastman (EastmanKodak, Rochester, NY) #103 1988) as a substrate.9 Allglassware used in this procedure was nitric acid-washed.Standard curves, using known concentrations of pu-rified human ceruloplasmin (0.1-0.5 mg/ml, Sigma(Sigma Chemical, St. Louis, MO) Cat. #13307), weredetermined for each group assayed. Sera were dilutedwith acetate buffer (see below), as necessary, to obtainOD readings in the linear range of the standard curve.Lipemic or hemolyzed serum samples were includedin the study, since these characteristics do not signifi-cantly affect ceruloplasmin determinations.10 Twosamples with a visible precipitate were not used, sincethe precipitate may have contained ceruloplasmin.

Each reaction mixture and corresponding blankcontained 50 til serum (whole or diluted, as appropri-ate), 2.05 ml 0.1 M acetate buffer (pH 5.45 at 37°),and 1.0 ml 27.6 mM acetate-buffered PPD solution(pH 5.45 at 37°). After a 5 min lag phase, due to theoxidation of the serum ascorbic acid, the blanks wereterminated by the addition of 50 /xl 1.5 M Na azidesolution. Reaction mixtures were terminated with azideafter an additional 30 min incubation at 37°. The ab-

sorbance of the reaction mixtures and correspondingblanks was determined immediately at a wavelengthof 530 nm in a Beckman (Beckman Instruments, Inc.,Irvine, CA) DU-8 recording spectrophotometer usinga cuvette of 1 cm optical-path length. Serum cerulo-plasmin concentration was determined by comparisonof the blank-corrected absorbance values to the stan-dard curve, and the data expressed as mg/l.

Ceruloplasmin concentrations also were determinedfor all control subjects (normals, other macular andretinal, and RP), and some of the affected subjects bya commercial radial immunodiffusion technique (Bio-science Laboratories, Columbia, MD). This assay doesnot differentiate between the holo- and apo-cerulo-plasmin, whereas the oxidase assay detects only holo-ceruloplasmin.

For comparison, determinations were made of theconcentration of transferrin, a plasma protein capableof binding iron, copper, and zinc. These determinationswere made by a commercial laboratory (BioscienceLaboratories) using a single radial immunodiffusiontechnique with a 200-400 mg/dl reference range.

Liver-function studies were also performed on ali-quot portions of the sera used for ceruloplasmin de-terminations. Both alanine and aspartate aminotrans-ferase (SGPT and SGOT) activities were determinedby a commercial laboratory (Bioscience) using a stan-dard spectrophotometric technique to measure theconversion of NADH to NAD at 320 nm. Three per-sons with values above 35 IU (at 37°) out of a total of149 were not included in this study, because elevatedlevels of these enzymes may indicate conditions asso-ciated with altered levels of ceruloplasmin.4

Results

Clinical Observations

Macular changes in affected individuals ranged fromsparse drusen and pigmentary changes to old disciformscars. We encountered drusen that varied both in sizeand apparent consistency from soft to crystalline. Ofthe legally blind eyes with macular degeneration, 71%



lOOO

900

800

^ 700c

- 600zI 500<r

£ 400

a. 300UJ

o200

100

All Mac.Degen.

AllUnaffected

Family AOnly:Mac

Degen.

Family AOnly:

Unaffected

Non-Family A

Only:Unaffected

Non-Family A

Only:Mac.Degen.

OtherEye

Diseases

RP

Fig. 2. Comparison of thedistributions of serum ceru-loplasmin concentrationsamong the various groupsstudied shows the generallyelevated amounts detected inpersons with macular degen-eration and normal amountsin persons without disease, orwith retinitis pigmentosa orother macular diseases. Theexception is the group ofFamily A members, who ei-ther had macular changes toomild to meet the criteria forthe diagnosis of macular de-generation, or showed nochanges.

(26/36) had exudative lesions, and 29% had areolaratrophy.

The non-Family A affected group was generally olderthan the non-Family A unaffected group. Of the formergroup, 65% were age 60 yr or older, while only 33% ofthe latter were that old (Table 1). Affected and unaf-fected members of Family A had similar age distri-butions (Table 1).

Associated Diseases

None of the individuals in the study had conditionsknown to be associated with reduced ceruloplasmin(e.g., malnutrition, Wilson's disease). Small, similarnumbers in affected and unaffected groups had con-ditions that might be associated with ceruloplasminelevation (see Discussion), such as osteoarthritis (12 of68 affected; 8 of 39 unaffected) or history of thyroiddisease (5 of 68 affected; 2 of 39 unaffected).

Table 2. Comparison of mean serumtransferrin concentrations

Group

Unaffected,non-Fam-ily A

All affectedAffected

Family Aonly

UnaffectedFamily Aonly

Other eyediseases

RP

Numberof Persons

2468

35

18

1016

Transferrin (mg/dl)

Mean±S.D.

276 ± 30278 ±60

270 ± 44

296 ± 30

278 ±65288 ± 30

Range

180-344151-368

180-358

180-356

176-351230-388

P- Value ofDifference

With Controls

>.O5

>.O5

>.O5

>.O5>.O5

Ceruloplasmin and Transferrin

A wide range of serum ceruloplasmin concentrationswas found for both macular degeneration and controlgroups when all study participants were analyzed to-gether (Fig. 2). In general, ceruloplasmin concentra-tions were higher for macular degeneration (n = 690mg/1; a = 154 mg/1) than for the unaffected controls(n = 628 mg/1; a = 103 mg/1). This difference is mar-ginally significant (t = 1.99; df = 96; P < .05). Sinceceruloplasmin levels do not correlate with age for themacular degeneration group (r = 0.19), the controlgroup (r = .0002), or for the two groups combined (r= 0.21), we conclude that the observed difference inceruloplasmin concentrations between groups was notdue to the age mismatch.

An interesting observation relating to the unaffectedgroup was the apparent bimodal distribution of ceru-loplasmin levels. It is possible that there could be agenetic component to this variation, since approxi-mately one-half of the unaffected group (n = 18) andone-half of the macular degeneration group (n = 34)were blood relatives (Family A). When data for FamilyA members and non-Family A members were analyzedseparately, several points were apparent (Fig. 2). First,the bimodal distribution seen when all unaffected con-trols were analyzed is now segregated into two groups,with Family A unaffected members having high ce-ruloplasmin and non-Family A unaffected controlshaving ceruloplasmin within normal limits. Second,the differences in ceruloplasmin levels between non-Family A macular degeneration and control groups isquite striking (641 ± 153 mg/1 for macular degenera-tion vs 312 ± 64 mg/1 for control) (Fig. 1, Table 1).Third, ceruloplasmin levels in both macular degener-ation groups were high, and did not split into twogroups, as did unaffected individuals. Fourth, there is


No. 12 MACULAR DEGENERATION AND CERULOPLASMIN / Newsome er ol. 1679

no apparent difference in ceruloplasmin levels betweenthe Family A macular degeneration group (641 ± 143mg/1) and the Family A unaffected group (655 ± 87mg/1) (Table 2), of which approximately 35% had dru-sen, but not to the degree that would allow for classi-fication as macular degeneration (Fig. 2, Table 1).However, it should be noted that, in comparing thetwo groups of Family A subjects to the two groups ofnon-Family A subjects, we find, from an analysis ofvariance, that the concentration differences are signif-icant (F[3, 94] = 4.2, P< 0.01).

Although some individual variability with time wasseen, persons with an elevated ceruloplasmin concen-tration initially, or a normal concentration initially,tended to maintain that level (Fig. 3). Elevated ceru-loplasmin in macular degeneration may be specific forthis condition, since patients with a variety of otherocular conditions, including RP, had normal cerulo-plasmin levels (Fig. 2, Table 1). All controls (includingRP subjects) and about 10% of the macular degener-ation subjects also had serum ceruloplasmin concen-tration determined by quantitative radial immunodif-fusion assay. With rare exception, the immune-deter-mined values were within 10% of the quantities basedon oxidase activity (not shown).

To identify the source of significance, multiple t-tests were performed. Since six comparisons weremade, the t values must occur at P < .01 to be regardedas significant. Using this criterion, both Family A andnon-Family A macular disease patients have signifi-cantly elevated group ceruloplasmin concentrations ascompared with non-Family A unaffected subjects.

No significant differences in serum transferrin levelswere found between the same groups of subjects (Ta-ble 2).

Discussion

Ceruloplasmin, an «-2 glycoprotein of 132,000 mo-lecular weight, capable of binding six copper atoms permolecule, is a normal component of human blood. Ithas been reported to be the major source of serumferrous oxidase activity, as well as serum antioxidantactivity.4'8" and is elevated in a variety of acute infec-tious and chronic disease states,12 malignancy,13 preg-nancy and oral contraception administration,14151617

and myocardial infarction.4 Ceruloplasmin is lower inthe nephrotic syndrome12 and in patients with hepto-lenticular degeneration (Wilson's disease).1218 Ceru-loplasmin oxidase activity is also lower in newbornsthan in adults.19'20 While ceruloplasmin levels do varywith age, they are relatively stable between adolescenceand old age.10'21 Genetic influences producing mod-erately low ceruloplasmin concentrations with no ap-parent disease have also been reported.10

1000

900

800

5 700

~ 600zI 5OO<% 400

ir 300iij

o

200

100

1981 1982 1983 1984 1985

Fig. 3. Serum ceruloplasmin concentrations were obtained on fol-low-up visits from nearly all study patients. This graph shows theusually modest variation over 3'/2 years' time of the ceruloplasminfor four representative individuals from these groups: (1) maculardegeneration, non-Family A—solid circles; (2) unaffected, non-FamilyA—open squares; (3) Family A, macular degeneration—solid tri-angles; and (4) Family A, unaffected—open triangles.

Ceruloplasmin concentrations were variable (coef-ficient of variation ranges from 13-22%) within eachof our groups. The degree of variability is consistentwith other reported series.91015'21 However, the actualconcentrations in most macular degeneration subjectsin this study were higher than reported values for nor-mal. Preliminary results on macular degeneration pa-tients from the mid-Atlantic region have also shownsignificantly elevated ceruloplasmin in comparisonwith controls living in the same region (data notshown). The absence in both studies of elevated trans-ferrin, another liver-synthesized transport protein,supports the notion that ceruloplasmin changes maybe suggestive of a specific relationship to macular de-generation, while not necessarily being diagnostic.

In contrast to the data obtained from unrelated nor-mal and affected individuals, the results of the familystudy showed that the unaffected, as well as the affected,members of the large family had elevated ceruloplas-min levels. The group elevation was significant whencompared to that of unaffected, unrelated controls fromthe same area, as well as to published "normal" values(female 170-360 mg/1; male 160-320 mg/1).91015'21

This is, to our knowledge, the first report of a familialmacular degeneration-related occurrence of elevatedceruloplasmin. The reason for the apparent discrepancybetween the two groups of data is not clear. It is possiblethat there is one form of macular degeneration that isa genetic predisposition related to altered ceruloplas-min, and that those unaffected individuals with ele-



vated ceruloplasmin levels are responding to a chronicstimulus which will ultimately manifest itself as clini-cally recognizable, age-related maculopathy. Thisseems possible, because at least some of these individ-uals (35% of unaffected Family A members) showedsubthreshold evidence of macular change. Alterna-tively, this finding could be a covariant with no causalrelationship. Following these unaffected family mem-bers as they age will be extremely important in deter-mining if they were predisposed to this condition at ayounger age.

Normally, there is a correlation between serum cop-per and ceruloplasmin levels. For example, in individ-uals with rheumatoid arthritis22 and in women takingestrogen,1617 there is a co-elevation of both. However,in preliminary studies on affected individuals, serumcopper concentrations were not elevated, even in thepresence of elevated ceruloplasmin. Copper-free ce-ruloplasmin is not known to have oxidase activity.However, since we measured the amount of cerulo-plasmin based on this activity, these patients presum-ably have increased serum antioxidant capacity. Therelative amounts of ceruloplasmin in affected and un-affected individuals are confirmed by the immunolog-ical assay, which is not dependent on either coppercontent or oxidase activity. Therefore, the elevatedconcentrations of ceruloplasmin are real, and do not1

merely reflect anomalously increased oxidase-specificactivity. However, preliminary data indicating normalserum copper concentrations in affected individualsraise some interesting questions regarding the molec-ular heterogeneity of ceruloplasmin23 and the bindingcapacities for copper and other cations in our subjects.Investigations to study some of these possibilities arecurrently underway.

Key words: macular degeneration, drusen, serum cerulo-plasmin, serum copper, retinal pigment epithelium, bloodantioxidant activity

Acknowledgments

We gratefully acknowledge the invaluable assistance of Mrs.Florence Butler in subject recruitment and scheduling, ofMarge Hoffman, RN, in conducting field clinics, of Dr. JohnW. Carlisle for making facilities available for field clinics, ofDr. Legrand Shupe for continued support and assistance, ofElsira Pina in performing some ceruloplasmin determina-tions, and of Dr. Robert Massof in discussing the manuscript.

References

1. Young RW: A theory of central retinal disease. In Future Di-rections in Ophthalmological Research, Sears ML, editor, NewHaven, Yale University Press, 1981, pp. 237-270.

2. Green WR and Key SN III: Senile macular degeneration: A his-topathologic study. Trans Am Ophthalmol Soc 75:180, 1977.

3. Bowness JM, Morton RA, and Shakir MH: Distribution of copperand zinc in mammalian eyes. Occurrence of metals in melaninfractions from eye tissues. Biochem J 51:521, 1952.

4. Frieden E: Ceruloplasmin: The serum copper transport proteinwith oxidase activity. In Copper in the Environment, Part II,Nriagu JO, editor, New York, John Wiley & Sons, Inc., 1979,pp. 241-284.

5. Shokeir MHK and Shreffler DC: Cytochrome oxidase deficiencyin Wilson's disease: A suggested ceruloplasmin function. ProcNatl Acad Sci USA 62:867, 1969.

6. Marceau N and Aspin N: Distribution of ceruloplasmin-bound67Cu in the rat. Am J Physiol 222:106, 1972.

7. Marceau N and Aspin N: The intracellular distribution of ra-diocopper derived from ceruloplasmin and from albumin.Biochim Biophys Acta 328:338, 1973.

8. Osaki S, Johnson DA, and Frieden E: The possible significanceof the ferrous oxidase activity of ceruloplasmin in normal humanserum. J Biol Chem 241:2746, 1966.

9. Sunderman FW Jr and Nomoto S: Measurement of human serumceruloplasmin by its p-phenylenediamine oxidase activity. ClinChem 16:903, 1970.

10. Cox DW: Factors influencing serum ceruloplasmin levels in nor-mal individuals. J Lab Clin Med 68:893, 1966.

11. White A, Handler P, and Smith EL: Principles of Biochemistry,5th Edition, New York, McGraw-Hill, 1973, pp. 395-396.

12. Markowitz H, Gubler CJ, Mahoney JP, Cartwright GE, andWintrobe MM: Studies on copper metabolism. XIV Copper,ceruloplasmin and oxidase activity in sera of normal humansubjects, pregnant women, and patients with infection, hepato-lenticular degeneration and the nephrotic syndrome. J Clin Invest34:1487, 1955.

13. Hughes NR: Serum transferrin and ceruloplasmin concentrationsin patients with carcinoma, melanoma, sarcoma and cancers ofhematopoietic tissues. Aust J Exp Biol Med Sci 50:97, 1972.

14. Hrgovcic M, Tessmer CF, and Minckler TM: Serum copper levelsin lymphoma and leukemia. Special reference to Hodgkin's dis-ease. Cancer 21:743, 1968.

15. Ray GR, Wolf PH, and Kaplan HS: Value of laboratory indicatorsin Hodgkin's disease: Preliminary results. Natl Cancer Inst Mon-ogr 36:315, 1973.

16. Caruthers ME, Hobbs CB, and Warren RL: Raised serum copperand ceruloplasmin levels in subjects taking oral contraceptives.JClinPathol 19:498, 1966.

17. Halsted JA, Hackley BM, and Smith JC Jr: Plasma-zinc andcopper in pregnancy and after oral contraceptives. Lancet 2:278,1968.

18. Scheinberg IH and Gitlin D: Deficiency of ceruloplasmin in pa-tients with hepatolenticular degeneration (Wilson's disease). Sci-ence 116:484, 1952.

19. Pojerova A and Tovarek J: Ceruloplasmin in early childhood.ActaPaediat49:113, 1960.

20. Shokeir MHK: Investigations of the nature of ceruloplasmin de-ficiency in the newborn. Clin Genet 2:223, 1971.

21. Yunice AA, Lindeman RD, Czerwinski AW, and Clark M: In-fluence of age and sex on serum copper and ceruloplasmin levels.J Gerontol 29:277, 1974.

22. Grennan DM, Knudson JM-L, Dunckley J, MacKinnon MJ,Myers DB, and Palmer DG: Serum copper and zinc in rheu-matoid arthritis and osteoarthritis. New Zealand Med J 91:47,1980.

23. Morell AG and Scheinberg IH: Heterogeneity of human ceru-loplasmin. Science 131:930, 1960.


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Macular degeneration and elevated serum ceruloplasmin