Solid phase immunoradiometric assay for porcine serum ferritin

Comp. Biochem. Physiol. Vol. 89B, No. 2, pp. 355-358, 1988

0305-0491/88 $3.00+0.00 © 1988PergamonJournals Ltd

Printed in Great Britain

SOLID PHASE I M M U N O R A D I O M E T R I C ASSAY FOR PORCINE SERUM FERRITIN PAUL C. ADAMS, LAWRIE W. POWELL, JUNE W. HALLIDAY* The Liver Unit, Department of Medicine, University of Queensland, Royal Brisbane Hospital, Brisbane, Qld. 4000, Australia (Tel: 07 253-5127)

(Received 13 April 1987)

Al~traet--1. A solid phase immunoradiometric assay using anti-serum coated polystyrene tubes, is described for the assay of porcine serum ferritin. 2. The mean concentration of ferritin in the serum of both male and female pigs (Sus scrofa) was 12.1/tg/l + 8.7 #g (range < 1-35 #g/l) and no sex differences were observed in 40 pigs from 1 day to 4 years old. 3. Serum ferritin increased with increasing body iron stores in iron loaded pigs as assessed by hepatic iron concentration. 4. The assay is sensitive (detecting less than 1/~g/l), reproducible, specific and it does not cross-react with human or rat ferritin.

MATERIALS AND METHODS

INTRODUCTION Ferritin is an iron storage protein responsible for the storage of 50% of the body's iron. Previous studies have detected ferritin in the serum and tissues of a wide range of species. Despite these studies, the origin of serum ferritin and its physiological role in body iron metabolism are unknown. Studies have shown differences in the iron metabolism of the pig and the rat which indicate that the pig may provide a more appropriate model for the study of human ferritin metabolism. The rat depends on dietary iron rather than iron stores to meet increased requirements. It has a much greater capacity to absorb iron than man and it has been suggested that iron absorption in the rat is not closely related to iron stores (Cook et al., 1973). In addition, daily iron loss is much greater than in the human (Finch et aL, 1978) and biliary excretion of iron in the rat provides a significant pathway for excretion of excess iron (Figueroa and Thompson, 1968) while human studies have shown that the contribution of the biliary system to daily losses of iron is minimal (Green et al., 1968). Ferrokinetic studies in the pig have shown similar results to the human (Furugouri, 1974; Jensen et al., 1956) and neonatal iron metabolism in the pig is also directly comparable to the human (Furugouri, 1973). Like the human, the endogenous loss of iron in the pig is extremely small (Braude et al., 1962) and the biliary excretion of iron is minimal even in an iron loaded state (personal observation). This technique for the measurement of porcine serum ferritin is based on the principles of previously described immunoradiometric assays (Halliday et al., 1975) and it is sensitive to 1 #g/1 ferritin. *Correspondence to Dr June W, Halliday.

Purification of porcine liver ferritin Porcine liver ferritin was isolated and purified by a modification of the method of May and Fish (1977). Fresh pig liver was added to twice its weight in 0.1 M Tris-acetate, 0.01 M 2-mercaptoethanol buffer (TAME) and homogenized in a blender. The homogenate was centrifuged at 10,000g for 20min and the supernatant retained. The supernatant was heated to 75°C for 5 rain and then centrifuged at 10,000g for 20rain and the precipitate discarded. Solid ammonium sulfate was added (31.5g/dl). After settling for 15 min the suspension was centrifued at I0,000 g for 20 min and the supematant discarded. The precipitate was dissolved in a minimal volume of TAME buffer. The solution was concentrated using Aquacide (Calbiocbem, CA) and dialysis tubing. Five millilitre aliquots were applied to a Sepharose 6BCI chromatography column (2.5 x 60 cm) equilibrated with TAME buffer. Ferritin fractions were eluted from the column with TAME buffer and recognized by colour. Pooled fractions were concentrated by Aquacide and applied to a Sephaeryl $200 chromatography column (l.6cm x 100cm). Ferritin fractions were eluted with TAME buffer and recognized by colour. Pooled fractions were again concentrated by Aquacide. The final product was checked for purity by gel electrophoresis. It should be noted that 2-mercaptoethanol is required as a reducing agent to ensure solubility of the ferritin during the purification. In this regard, porcine ferritin differs from human and rat ferritin.

Preparation of anti-ferritin antiserum Antiserum to porcine liver ferritin was produced in rabbits by three intramuscular injections at weekly intervals of 1 nag purified porcine liver ferritin mixed with Freund's complete adjuvant.

Preparation of purified anti-ferritin antibody A globulin fraction of rabbit anti-serum was passed through a Sepharose 4B column (1 x 5era) containing purified porcine liver ferritin and the antibody was eluted

355

PAUL C. ADAMSet al.

356

from the column with 5-10 ml of 3.0 M thiocyanate pH 6.5 at 4°C. The fractions were pooled and 10 mi of sample was applied to a Sepharuse G50 column (2.5 x 60cm) equilibrated in 0.1 M borate in 0.5 M NaC1, pH 8.0 at 4°C. After a column volume of buffer was passed through the column, 3 mi fractions were collected into 10 test tubes. Protein concentration was estimated by absorbance at 280 urn. The highest fractions were pooled and the presence of antibody was confirmed by agarose gel diffusion immunoprecipitation against purified porcine ferritin.

lodination of anti-ferritin antibody Fifty micrograms of purified anti-ferritin antibody was iodinated with I mCi ~2sIaccording to the method of Bolton and Hunter (1973). Following evaporation of the solvent with a gentle stream of dry nitrogen the purified antibody (approx. 100 pl) was added to the vial and stirred overnight. Free iodine was removed by passage through a Sephadex G-25 column equilibrated with phosphate buffered saline (PBS), 20 mM Na2HPO 4, 20 mM NaH2PO 4, in 0.1 M NaC1, pH 7.4. Ten fractions of 1 mi were taken and the radioactivity of 20/d aliquots of each fraction was determined. The fraction with the highest counts was stored at 4°C. Assay of ferritin Flat bottomed polystyrene tubes (NC 1, Disposable Products, Adelaide, Australia) were coated by the addition of 500/zl of rabbit antiserum to porcine liver ferritin in a dilution of 1: 5000 in PBS and incubated for 24 hr at 20°C. The tubes were washed 5 times with 0.1 M saline by an automated procedure and aspirated dry. The coated tubes remained stable for a period of at least two months. To prepare a standard curve, a stock solution of 1000/zg/1 of porcine ferritin was prepared in PBS. From this solution 30/~1 was added to 1.47ml of veronal buffer (0.05 M veronal, 0.1 M NaCI, pH 8.0) containing 4% bovine serum albumin (VBS) to make a solution of 20#g/1. A standard curve from 0-10pg/l was made up in VBS. One hundred microlitres of each standard was added to tubes. Blank tubes contained 100pl of VBS. Triplicate standard tubes were used for each point of the curve. A standard curve was derived from 0-I0/~g/1 (Fig. 1A) and from 0-100/~g/1 (Fig. 1B). Estimation of ferritin concentration of serum samples Porcine serum (100/zl) was diluted I/2 and 1/4 in VBS and added to duplicate tubes. The tubes were incubated in an upright position for 24 hr at 20°C. The tubes were then washed 5 times with 0.1 M NaC1 and aspirated dry. One

15-

u

tOE

I

I

I

I

I

I

I

50 Serum ferritin (,u.g/L)

I

I

I

tO0

Fig. l B. Porcine serum ferritin assay standard curve from 0-100 #g/1. hundred microfitres of l~sI labelled anti-ferritin antibody containing approximately 200,000 counts/rain (Specific activity = 105 counts//zg of protein) was added carefully to the bottom of each tube. The tubes were incubated for a further 24 hr at 20°C, washed again with 0.1 M NaCI, aspirated dry and counted in a gamma counter.

Determination of normal range Blood was obtained by jugular vein puncture in 18 male and 22 female pigs ranging in age from 1 day to 4 years. Serum samples were assayed at multiple dilutions. Iron loading of pigs Pigs of approximately 10 weeks of age were fed on a diet of regular pig chow (Purina Pig Chow) mixed with 2.5% carbonyl iron (GAF Australia) and water and fed as mush. Animals were fed for up to 6 weeks on this diet. The bioavailability of the iron enriched diet was assessed by measuring the iron absorption of three pigs before and after iron loading. Iron absorption was measured using oral SgFe ferric citrate and a total body counter as previously described (Cook et al., 1972). Measurement of hepatic iron content A small sample of liver (1-2 rag) was dried to constant weight in foil for 48 hr. The dried liver was carefully weighed into acid washed glassware and digested as previously described (Bassett et al., 1986). The iron content was determined by atomic absorption spectrophotometry.

2000 --

RESULTS

iO00 ~

Figures 1A and B show the standard curve obtained from 0-10/tg/1 and 0-100 /z g/1, respectively. Individual points were the means o f triplicate observations.

0

I

I

I

I

I

2

3

4

i

I

I

I

I

I

5

6

7

8

9

I0

Ferritin (/-EJ/ L)

Fig. IA. Porcine serum ferritin assay standard curve from

0-10~,g/1.

Non-specific binding Tubes containing only VBS were used to determine the non-specific binding of protein to the antibody and these values have been subtracted. The counts bound in the absence of ferritin represented 0.6% of the total radioactivity bound.

Solid phase IRA for serum ferritin

357

Accuracy

Serum results

The recovery of added ferritin was determined by the addition of 0 . 1 # g - l . 0 # g / l standard, 0.5#g5.0#g/l standard, 1.0#g-10gg/l standard. Mean recoveries of ferritin were 94___4.5% at the 0.1 #g level, 100 _+ 5.8% at the 0.5 #g level and 96 +_ 7.5% at the 1/tg level.

Serum from 40 pigs was assayed for serum ferritin. The ferritin levels ranged from less than 1-283/~g/l. The range in normal pigs from 1 day to 4 years old was from < 1 to 35 #g/I, with an arithmetic mean of 12.1 _+ 8.7#g/1 and a geometric mean of 13.5#g/1. Levels up to 55gg/l were seen in pigs fed the 2.5% carbonyl iron diet. The highest value of 283 #g/l was seen in a post operative pig with severe infection.

Sensitivity Sensitivity defined as the smallest single value that can be distinguished from zero was calculated at the zero point of the curve from 10 curves and a mean sensitivity of 0.31 + 0.18 #g/l was obtained.

Specificity Samples were assayed at three dilutions and were linear when plotted. The addition of non-radioactive antibody prior to 12sI anti-ferritin reduced the final radioactivity to the level of the blanks, while the addition of bovine serum albumin had no effect. Human and rat ferritin were undetectable in this assay.

Reproducibility (a) lntra-assay variation. Five sera were run within the same batch (Table 1).

Iron loading of pigs Having determined a normal range for hepatic iron of 5-10 pmol/g of dry liver in seven pigs of approximately 12 weeks of age, hepatic iron was determined in pigs which had been fed 2.5% oral carbonyl iron for 6 weeks. Hepatic iron levels were increased 4-7 times over those of normal pigs and light microscopy (stained by Prussian blue) revealed parenchymal iron deposits in a portal distribution with no fibrosis or inflammation. Iron absorption initially ranged from 10-18% and decreased to less than 1% after 6 weeks of iron loading in three pigs.

Correlation with hepatic iron Figure 2 shows the relationship between serum ferritin and hepatic iron in seven pigs.

(b) Inter-assay variation. Two sera were run in different batches on different days (Table 2). Precision Precision was determined according to the method of Virasoro et al. (1971). Standard deviations (SD) and coefficients of variation (CV) for different serum concentrations were calculated according to Snedecor from the differences between duplicate determination. (SD = ~x/"~-~/2N where d is the difference between duplicates and N is the number of estimations) (Table 3).

DISCUSSION An assay for porcine ferritin has been developed based on the principles of a solid phase immunoradiometric assay for human ferritin (Halliday et al., 1975). The assay is simple to perform and the sensitivity, precision, reproducibility, recovery and specificity compare favourably with ferritin assays for other species. Previously described assays for the measurement of human and rat ferritin are unsatisfactory for the measurement of porcine ferritin

Table i. Intra-assayvariation Serum N Ferritin (#g/l) A 20 12.8+ 1.2 B 10 8.4 + 0.83 C 10 6.4 + 0.60 D 10 8.3 + 0.47 E 12 9.1 + 0.92 N = number.

50-

40-

Table 2. Inter-assayvariation Serum N Ferritin (#g/l) A 10 10.2+ 0.88 A 10 10.0_+0.92 B 10 8.4 + 0.83 B 10 7.8 _+0.24 N = number.

~3o-

E

20"

03

Table 3. Precisionof ferritin determinations Mean ferritin Numberof (#g/l) pairs SD CV 3.44 6.42 10.1 23.5

14 7 10 4

283 2 SD = Standard deviation; CV ffiCoefficientof variation.

0.31 0.60 0.16 2.1

10

9.0 9.3 1.6 8.9

3.5

I0-

I I0

I 20

I 30 Hepotic

I 40

I 50

I 60

I 7'0

iron ( / ~ r n o l / g )

Fig. 2. Hepatic iron vs serum fcrritin.

I 80

358

PAUL C. ADAMSet al.

because of the lack of cross-reactivity between the species. Serum ferritin values were low in these pigs compared to other species such as the human or the rat with a mean of 12.1 #g/l and a range from less than 1-35#g/1. Our experiments in which the diet was supplemented with 2.5% carbonyl iron show that even when the body iron stores as assessed by hepatic iron concentration were elevated the serum ferritin was only 55/~g/1. This value would be within the normal range in a human and an iron overloaded human may have serum ferritin levels over 1000 #g/1. Therefore, even though iron deficiency has been described in young pigs it is unlikely to explain the low circulating levels of serum ferritin. A recently described porcine hepatic ferritin receptor could contribute to the regulation of a low circulating ferritin level in the pig (unpublished observations). Although there was a positive correlation between hepatic iron and serum ferritin, there appears to be a wide range of normal values for serum ferritin and the value of serum ferritin as a method of assessment of body iron stores remains to be determined. Iron deficiency appears to be so common in piglets that iron supplementation of the diet is routine. This assay permits studies of ferritin metabolism in the pig, an animal model that more closely resembles the human than does the rat. Radioisotopes preclude many of the studies of ferritin metabolism in the human and the pig model bears many similarities to the human. Studies on the receptor-mediated uptake, clearance and excretion of ferritin in normal and iron overloaded pigs should add to our knowledge of normal ferritin metabolism in the human and to a further understanding of the role of ferritin in the pathophysiology of iron overload states. Acknowledgements--This study was supported in part by

the National Health and Medical Research Council of Australia. Dr Paul C. Adams is supported by a Canadian Liver Foundation Fellowship The authors are indebted to L. Duplock and G. Ramm for their technical assistance.

REFERENCES

Bassott M., Halliday J. W. and PoweU L. W. (1986) Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron levelassociated with fibrosis. Hepatology 6, 24-29. BoRon A. and Hunter W. (1973) The labelling of proteins to high specific radioactivities by conjugation to an ~25I containing acylating agent. Biochem. J. 133, 529-539. Braude R., Chamberlain A., Kotarbinska M. and Mitchell K. (1962) The metabolism of iron in piglets given labelled iron either orally or by injection. Br. J. Nutr. 16, 427-449. Cook J., Hershko C. and Finch C. (1973) Storage iron kinetics--V. Iron exchange in the rat. Br. J. Haematol. 25, 695-706. Cook J., Layrisse M., Martinez-Torres C., Walker R., Monsen E. and Finch C. (1972) Food iron absorption measured by an extrinsic tag. J. Clin. Invest. 51, 805-815. Figueroa W., Thompson J. (1968) Biliary iron excretion in normal and iron loaded rats after desferrioxamine and CaDTPA. Am. J. Physiol. 215, 807-810. Finch C., Ragan H., Dyer I. and Cook J. (1978) Body iron loss in animals (40343). Proc. Soc. Exp. Biol. Med. 159, 335-338. Furugouri K., (1973) Developmental changes in the nonheine iron composition of the liver and spleen in piglets. J. Anim. Sci. 36, 265-270. Furugouri K. (1974) Kinetics in iron metabolism in piglets. J. Anim. Sci. 38, 1249-1255. Green R., Charlton R., Seftel H., Bothwell T., Mayet F., Adams B., Finch C. and Layrisse M. (1968) Body iron excretion in man. A collaborative study. Am. J. Med. 45, 336-353. Halliday J. W., (3era K. and Powell L. W. (1975) Solid phase radioimmunoassay for serum ferritin. Clin. Chim. Acta 58, 207-214. Jensen W., Bush J., Ashenbrucker H., Cartwright (3. and Wintrobe M. (1956) The kinetics of iron metabolism in normal growing swine. J. Exp. Med. 103, 145-159. May M. and Fish W. (1977) The isolation and properties of porcine ferritin and apoferritin. Archs Biochem. Biophysiol. 182, 396-403. Virasoro E., Copinschi G., Bruno O. and Leclercq R. (1971) Radioimmunoassay of human growth hormone using a charcoal dextran separation procedure. Clin. Chim. Acta 31, 294-297.