Is ceruloplasmin a physiological ferroxidase?

Clin. Biochem. 5, 115-120 (1972) IS C E R U L O P L A S M I N A P l t Y S I O L O G I C A L F E R R O X I D A S E ? M. H. K. SHOKEIR* Section of Medical Genetics Department of Pediatrivs University of Saskatchewan Saskatoo~t, Canada

SUMMARY 1. The hypothesis t h a t the physiological role of ceruloplasmin is as a ferroxidase enzyme which effects the binding of ferric iron to t r a n s f e r r i n was investigated by m e a s u r i n g serum ceruloplasmin activity, total transf e r r i n and iron saturated t r a n s f e r r i n levels as well as e s t i m a t i n g percent s a t u r a t i o n in sera f r o m newborns, their mothers and unrelated controls. The results indicate t h a t the group with the lowest ceruloplasmin activity displays the highest ferric s a t u r a t e d t r a n s f e r r i n as well as the highest percent saturation. Conversely, mothers have the highest ceruloplasmin activity and the lowest i r o n - t r a n s f e r r i n serum levels. 2. The f i n d i n g s do not support the above hypothesis.

CERULOPLASMIN, A COPPER-CONTAINING ~2 SERUM GLOBULIN was discovered by Holmberg and Laurell (1). The copper, which confers intense blue coloration on the protein, constitutes the prosthetic group in the oxidase activity displayed by ceruloplasmin(2,3,4). It has been suggested t h a t ceruloplasmin carries out its physiological function by virtue of this oxidase p r o p e r t y (5). A n u m b e r of compounds have been invoked as potential substrates, e.g. d i h y d r o x y p h e n y l a l a n i n e (DOPA), epinephrine, serotonin and ascorbic acid. To date, the evidence to t h a t effect has been conflicting and on the whole unconvincing (6), for although ceruloplasmin exhibits m a r k e d oxidase activity t o w a r d s aromatic diamines in vitro (7,8,9), this does not appear to be a functionally crucial a t t r i b u t e of the protein in vivo. *The author is Queen Elizabeth II Scientist and the research was supported by M.R.C. of Canada. Correspondence: Dr. M. H. K. Shokeir, Section of Medical Genetics, Ellis Hall, University Hospital, Saskatoon, Saskatchewan, Canada

116

SHOKEIR

F u r t h e r m o r e , it has been suggested t h a t ceruloplasmin m i g h t be involved in regulating copper absorption from the gut (10), mainly by curbing excess diffusion. The capacity o f ceruloplasmin to dissociate reversibly half or more of its copper content was t h o u g h t to be pertinent to this proposed role. tIowever, the circumstances required to achieve this dissociation do not obtain in the intestinal mucosa where copper absorption occurs. A possible copper t r a n s p o r t role for ceruloplasmin has also been raised

(11). In 1969, we advanced the hypothesis t h a t ceruloplasmin enzymatically mediates copper t r a n s f e r to copper-containing enzymes notably cytochrome C oxidase and tyrosinase. Data were presented which disclosed diminished cytochrome C oxidase activity in extracts f r o m leucocytes of patients with Wilson's disease - - where serum ceruloplasmin concentration is grossly deficient. Subsequently corroborating evidence was submitted to show t h a t ~Cu labelled ceruloplasmin could t r a n s f e r its copper to cytochrome C oxidase whereas °'Cu bound to albumin could not gain access to the enzyme (13). Despite the above work it has to be stated t h a t the biological function normally subserved by ceruloplasmin is yet unknown. More recently the idea has been put f o r w a r d t h a t ceruloplasmin m a y physiologically function as ferroxidase (14), responsible for mobilization of iron from the cells to the plasma and converting ferrous to ferric iron; thereby causing the s a t u r a t i o n of the t r a n s f e r r i n with the metal. Evidence in support of the hypothesis has been adduced f r o m previous work (15,16). We decided to investigate this novel suggestion by studying the concentrations of both saturated and total t r a n s f e r r i n s in the sera of newborns. Correlation between the levels of i r o n - s a t u r a t e d - t r a n s f e r r i n and ceruloplasmin activity was attempted. The findings do not support the hypothesis t h a t ceruloplasmin primarily functions as a ferroxidase. MATERIALS AND METHODS

Sera: Newborn sera: Cord blood was obtained by free flow without pressing from the placental stump of the umbilical cord immediately a f t e r severance. All 220 newborns from whose cords blood specimens were obtained were healthy, full-term, m a t u r e i n f a n t s with birth-weight r a n g i n g from 2700-4100 g. All the mothers studied were normal, healthy women between 19 and 39 years of age. Blood specimens were obtained from both mothers and controls by venipuncture and collected in vacuum tubes. Healthy women between 20 and 40 years of age who were neither pregn a n t nor receiving oral contraceptives (17), and healthy men within a similar age range represent the. control group. Blood samples were d r a w n on mothers and independent controls while fasting. Antisera: Commercial specific rabbit a n t i - h u m a n t r a n s f e r r i n antiserum (Behringwerke, A.G., M a r b u r g - L a h n , West G e r m a n y ) was used in this study.

C E R U L O P L A S M I N AS F E R R O X I D A S E

117

Ceruloplasmin enzymatic assay: This was determined spectrophotometrically by several modifications of the method of Ravin (18,19,20) using paraphenylene diamine dihydrochloride as a substrate. Quantitative immunodiffusion assay: The level of total t r a n s f e r r i n in the serum was assayed by quantitative double i m m u n o d i f f u s i o n in tubes; 6 x 50 mm culture tubes were coated with a dried agar film, using 1'~ I o n a g a r No. 2 A n t i - t r a n s f e r r i n antiserum diluted ~ in normal saline was added to an equal volume of 1% I o n a g a r to produce a final 1/~ dilution of antiserum. This m i x t u r e was kept at a t e m p e r a t u r e of 50 ° to prevent solidification of agar. 50 k of the antiserum a g a r m i x t u r e was pipetted in the bottom of each tube and allowed to gel. A layer of 0.5'/i I o n a g a r in saline, 300 k in volume, was next placed on top of the gelled a n t i s e r u m with a micropipette. On top of this 10 h of h u m a n serum to be tested was layered. F o r every determination, full concentration and 1/_, dilution of serum were used. The procedure was performed in two replicas. The tubes were finally sealed with p a r a f f i n and incubated for 3 days at 37 °. The position of the precipitin band was ascertained at 72 h by m e a s u r i n g the distance f r o m the top of the agar column to the precipitin band and f r o m the top to the interface between the antiserum and the agar. The ratio between the two distances ("f) is a function of the initial concentration of the antigen (transferrin). In the preparation of the calibration curves, against which the amounts of t r a n s f e r r i n in unknown samples were read, 1/~2, 1/,, 1/8 dilutions of standard serum (of known t r a n s f e r r i n level) as well as the full concentration were used. The "f values thus obtained were plotted versus t r a n s f e r r i n level on semi log paper and t r a n s f e r r i n levels in unknown sera were then read off according to the ~f obtained. Total iron binding capacity was determined on all samples by a method adapted f r o m R a m s a y (21). Satm'ated transferrin levels were determined on all samples by dialysing transferrin-bound-iron by addition of dilute hydrochloric acid following initial t r e a t m e n t with ascorbic acid, 2,4,6 T r i p y r i d y l - 1,3,5 triazine (TPTZ) which readily complexes with f e r r o u s ion was used as a recipient. The complex has a m a x i m u m absorbancy at 593 mu, and can, thus, be assayed spectrophotometrically (22). By combining the previous two approaches a percent - saturation of t r a n s f e r r i n could be obtained. RESULTS

Table 1 summarizes ceruloplasmin activities obtained in sera f r o m newborns, their mothers and unrelated male and female controls. Mean serum ceruloplasmin activity in neonates was about 3 ~ t h a t of their mothers and 5 ~ t h a t of controls. Table 1 also shows the f i n d i n g s on measurem e n t of total as well as iron-saturated t r a n s f e r r i n in sera f r o m the above

118

SHOKEIR

TABLE

1

CERULOPLASMINACTIVITIES, TOTAL AND IRON-SATURATEDTRANSFERRIN AND PERCENTSATURATION IN SERA FROM NEWBORNS, THEIR MOTHERS AND UNRELATED MALE AND FEMALE CONTROLS

Group Newborns

Mothers Female Controls Male Controls

Mean serum Mean serum Mean serum ironceruloplasmin total transferrin saturated transNo. activity (mg per (mg per 100 ml) ferrin (mg per i00 Investigated 100 ml) :t: S.D. 4- S.D. ml) 4- S.D.

Percent saturation

220 220

1.8 d= 1.1 53.8 =~ 5.1

179.4 -4- 25.2 338.7 ± 42.1

131.2 ~ 21.4 69.7 4- 19.6

73% 2O%

100

35.8 +

245.9 ± 29.4

88.6 ~= 24.1

36%

100

34.4 ± 3.9

252.1 ~ 31.6

95.8 ~ 17.6

38%

3.8

f o u r g r o u p s . M e a n p e r c e n t s a t u r a t i o n of s e r u m t r a n s f e r r i n is also i n d i c a t ed f o r t h e r e s p e c t i v e g r o u p s . B y i n s p e c t i n g t h e T a b l e it is r e a d i l y a p p a r e n t t h a t no p o s i t i v e c o r r e l a tion could be e s t a b l i s h e d b e t w e e n c e r u l o p l a s m i n a c t i v i t y a n d t h e level of i r o n - s a t u r a t e d t r a n s f e r r i n . I n f a c t in t e r m s of b o t h r e l a t i v e a n d a b s o l u t e values the group with the lowest serum ceruloplasmin activity (Newborns) a p p e a r s to h a v e t h e h i g h e s t p e r c e n t - s a t u r a t i o n of t r a n s f e r r i n as well as t h e h i g h e s t s e r u m c o n c e n t r a t i o n of the i r o n - s a t u r a t e d p r o t e i n . T h e o b v e r s e holds true; the m o t h e r s ' sera exhibit the highest ceruloplasmin activity b u t t h e l o w e s t level of s a t u r a t e d t r a n s f e r r i n . T h e m e a n p e r c e n t - s a t u r a t i o n is e v e n l o w e r t h a n m a y be a n t i c i p a t e d f r o m t h e level of i r o n - s a t u r a t e d t r a n s f e r r i n . T h i s is due to t h e e l e v a t e d t o t a l t r a n s f e r r i n c o n c e n t r a t i o n in t h e i r s e r a - - a f e a t u r e of l a t e p r e g n a n c y a n d e a r l y p u e r p e r i u m . DISCUSSION T h e a b o v e d a t a c l e a r l y d e m o n s t r a t e t h a t o v e r 9 5 ~ r e d u c t i o n of s e r u m c e r u l o p l a s m i n a c t i v i t y as e n c o u n t e r e d in c o r d blood, is a s s o c i a t e d n o t w i t h a d i m i n u t i o n b u t w i t h a n i n c r e a s e in t r a n s f e r r i n i r o n s a t u r a t i o n . A n u m b e r of s e r a of n e w b o r n i n f a n t s h a d no m e a s u r a b l e c e r u l o p l a s m i n a c t i v i t y , h o w e v e r , t h e i r i r o n s a t u r a t e d t r a n s f e r r i n levels t o g e t h e r w i t h p e r c e n t s a t u r a t i o n w e r e e l e v a t e d w h e n c o m p a r e d to t h e i r m o t h e r s or n o r m a l controls. C o n v e r s e l y , m o t h e r s of n e w b o r n s , w i t h s u b s t a n t i a l l y e l e v a t e d s e r u m c e r u l o p l a s m i n a c t i v i t i e s , d i s p l a y e d c o n s i s t e n t d i m i n u t i o n in b o t h t h e level of i r o n s a t u r a t e d t r a n s f e r r i n as well as p e r c e n t i r o n s a t u r a t i o n of s e r u m t r a n s f e r r i n . No p o s i t i v e c o r r e l a t i o n could be e s t a b l i s h e d b e t w e e n cerulop l a s m i n a c t i v i t y in s e r a of n e o n a t e s a n d t h e i r s e r u m t r a n s f e r r i n i r o n s a t u r a t i o n . I n d e e d , n e g a t i v e c o r r e l a t i o n c a n be s h o w n to e x i s t in t h i s s i t u a t i o n .

CERULOPLASMIN AS F E R R O X I D A S E

119

The findings herein presented may be regarded as evidence against the suggestion that ceruloplasmin's principal physiological function is a ferroxidase enzyme, responsible for achieving transferrin saturation with ferric iron (14). It may be argued, however, that only a very small fraction (perhaps 5 ~ or less) of normal ceruloplasmin activity is required to effect normal saturation of transferrin. Nevertheless, it is hard to envisage a physiological system in which the redundancy exceeds 9 5 ~ and the essential component is 5% or less. Although the two recently proposed functions for ceruloplasmin (12,14) are not necessarily mutually exclusive, the balance of evfdence hitherto available supports a role for the protein as an enzymatic mediator of copper transfer to other copper containing enzymes notably cytochrome C oxidase and possibly tyrosinase.

REFERENCES 1. HOLMBERG, C. G. and LAURELL, C. B. Investigations in serum copper. II - Isolation of the c o p p e r - - c o n t a i n i n g protein and a description of some of its properties. Acta. Chem. Scand. 2, 550-556 (1948). 2. BROMAN, L., MALMSTROM, B. G., AASER, R. and VANNGARD, T. The role of copper in the catalytic action of laccase and ceruloplasmin. Biochim. Biophys. Acta. 75, 365-376 (1962). 3. MORELL, A. G. and SCHE~NBERG, I. H. Preparation of an apoprotein from ceruloplasmin by reversible dissociation of copper, Science, 127, 588-590 (1958). ~;. KASPER, C. B. and DEUTSCH, H. F. Physiochemical properties of human ceruloplasmin. J. Biol. Chem. 238, 2325-2337 (1963). 5. LAURELL, C. B. (1960). Metal binding proteins; isolation and function. Chapter 10 in Plasma Proteins, PUTNAM, F. ed. New York Academic Press, Vol. 1, pp. 360-369. 6. MORELL, A. G., AISEN, P. and SCHEINBERG, I. H. Is ceruloplasmin an ascorbic acid oxidase? J. Biol. Chem. 237, 3455-3457 (1962). 7. HOLMBERG, C. G. and LAURELL, C. B. Oxidase reactions in human plasma caused by ceruloplasmin. Scandinav. J. Clin. and Lab. Invest. 3, 103-107 (1951). 8. AKERFELDT, S. Oxidation of N N - - d i m e t h y l p-phenylene diamine by serum from patients with mental disease. Science, 125, 117-119 (1957). 9. McCASHER, P. J. Paraphenylene diamine oxidase activity and copper levels in mammalian plasmas. Nature, 190, 887-889 (1961). 10. SOHEINBERG, J. H. and MORE~LL, A. G. Exchange of ceruloplasmin copper with ionic Cu64 with reference to Wilson's disease. J. Clin. Invest. 36, 1193-1201 {1957). 11. HOLMBERG, C. in Wilson's Disease: Some Current Concepts. (Springfield, Ill.: Thomas p. 96 (1961). 12. SHOKEIR, M. H. K. and SHREFFLER, D. C. Cytochrome oxidase deficiency in Wilson's disease: A suggested ceruloplasmin function. Proc. Nat. Acad. Sci. (Wash.) 62, 867-872 (1969). 13. MARCEAU, N., ASPIN, N. and SASS-KORTSAK, A. The in vivo transfer of ceruloplasmin copper to cytochrome oxidase. Canad. Fed. Biol. Soc. Abst. 13, p. 127 (1970). 14. ROESER, H. P., LEE, G. R., NACHT, S. and CARTWRIGHT, G. E. The role of eeruloplasmin in iron metabolism. J. Clin. Invest. 49, 2408-2417 (1970).

120

SHOKEIR

15. RAGAN, H. A., NACHT, S., LEE, BISHOP, C. R. and CARTWRIGHT, G. E. Effect

of ceruloplasmin on plasma iron in copper-deficient swine. Amer. J. Physiol. 217, 1320-1323 (1969). 16. LEE, G. R., NACHT, S., LUKENS, J. N. and CARTWRIGHT, G. E. Iron metabolism in copper deficient swine. J. Clin. Invest. 47, 2058-2069 (1968). 17. SHOKEIR, M. H. K. Oral contraceptives and ceruloplasmin activity. Lancet, 2, 1192 (1968). 18. RAVIN, H. A. An improved colarimetric enzymatic assay of ceruloplasmin. J. Lab. Clin. Med., 58, 161-168 (1961). 19. COX, D. W. Factors influencing serum ceruloplasmin levels in normal individuals. J. Lab. Clin. Med. 68, 893-904 (1966). zO. SHOKEIR, M. H. K. Quantitative variation in serum ceruloplasmin among ethnic groups. Clin. Genet. 1, 166-170 (1970). 21. RAMSAY, W. N. M. The determination of the total iron-binding capacity of serum Clin. Chim. Acta 2, 221-226 (1957). £~2. YOUNG, D. S. and HICKS, J. M. Method for the automatic determination of serum iron. J. Clin. Path. 18, 98-102 (1965).