- Email: [email protected]
Metabolic studies of C3 in man
Journal of Immunological Methods, 30 (1979) 69--75
69
© Elsevier/North-Holland Biomedical Press
METABOLIC STUDIES OF C3 IN MAN
L. BRINCH, P. TEISBERG and I. AAKESSON
Medical Department 7, Ullevaal Hospital and Medical Department A, Rikshospitalet, Oslo, Norway (Received 5 February 1979, accepted 10 May 1979)
The results of metabolic studies with radioactively labelled C3 in 14 healthy individuals are presented. Plasma volume determined by labelled C3 was generally higher than when determined by labelled albumin, and different batches of C3 behaved differently during the early part of the experiments. These phenomena are probably caused by C3 inhomogenities which are difficult to detect by usual biochemical and immunological methods. We have, therefore, excluded the first 24 h of the experiments from the mathematical analysis. In this way, rather narrow limits for the metabolic parameters have been obtained.
INTRODUCTION
Several groups have performed metabolic studies of labelled C3 to investigate the involvement of the complement system in different diseases (Alper and Rosen, 1967; Petz et al., 1968; Carpenter et al., 1969; Sliwinski and Zvaifler, 1972; Charlesworth et al., 1974a, b, c; Potter et al., 1976). These reports show large variations in the calculated fractional catabolic rate (FCR), synthesis rate (SR) and extravascular/intravascular (EV/IV) ratio even in normals. The differences may only to some extent be explained by different methods of analysing the experimental data. The aims of the present study were to elucidate the possible explanations for interlaboratory variations, and to standardize the procedure as far as possible. M A T E R I A L AND METHODS
The material consisted of 14 healthy medical students and medical staff between the ages of 24 and 39 years, 3 females and 11 males. None of the participants had, according to information given by themselves, any disease of a chronic nature, and all were feeling well at the time of the study. No clinical or laboratory studies were performed to verify absence of disease. Informed consent was obtained from all participants. C3 was isolated from HB antigen-free serum by a modification of the m e t h o d described by Lachmann et al. (1973).
70 EDTA was added to the serum to a final concentration of 0.01 M, and the pH adjusted to 6.0. The globulins were precipitated with 20% (w/v) Na2SO4. From then on all procedures were performed at +4~C. The precipitate was dialysed against phosphate buffer, pH 5.6, with 0.002 M EDTA, and dissolved in 0.01 M phosphate buffer, pH 7.0. Chromatography was performed on a DEAE Sephadex A-50 column (Pharmacia Fine Chemicals AB, Uppsala, Sweden) with phosphate buffer, pH 7.0, and a continuous NaC1 gradient. The fractions containing C3 were then chromatographed on a Bio-Gel HT column (Bio-Rad Laboratories, Richmond, CA, U.S.A.). The elution was performed stepwise using disodium hydrogen phosphate/potassium dihydrogen phosphate buffer, pH 7.9, of ionic strengths 7.5, 10, 12.5, and 15 mS measured at +4°C. Native C3 was mainly eluted in the 12.5 mS fraction. The purity of the preparations was tested by high voltage agarose gel electrophoresis, polyacrylamide gel electrophoresis, immunoelectrophoresis against rabbit anti human C3 and rabbit antiserum against human serum proteins (Behring Werke, Marburg/Lahn, F.R.G.) and crossed immunoelectrophoresis in agarose against anti C3. The preparations were sterile filtered with Acrodisc filters, 0.22 grn (Gelman Instrument Company, Ann Arbor, MI, U.S.A.). Pyrogen testing was performed in rabbits. Each batch was immediately frozen at --75°C in sterile vials and stored until labelling was performed with [ 12sI] (Isotope Laboratories, Institutt for Atomenergi, Kjeller, Norway). The chloramine T method, as described by McConahey and Dixon (1966), was employed. Free radioactivity was removed by dialysis at 4°C against sterile Ringer acetate. The labelled preparations were examined for activation products by the previously mentioned methods and by radio-autography after immunoelectrophoresis against anti C3 and antiserum against human serum proteins. Protein content was measured with the micro-biuret method. The labelled and unlabelled preparations were tested for biological activity in a haemolytic system with cellular intermediates (Cordis Laboratories, Miami, FL, U.S.A.). Albumin (AB Kabi, Stockholm, Sweden) in a final concentration of 0.5% was added to protect the labelled protein. The batches were divided in 1 ml aliquots for each study. Aliquots of 0.5--1.0 mg protein with activities of 3--5 t~Ci [12sI] were injected intravenously in each subject. Most of the subjects were simultaneously given 5--10 ~Ci [ 131]albumin (Isotope Laboratories, Institutt for Atomenergi, Kjeller, Norway) in order to compare plasma volumes (PV) determined by two different labelled proteins. Plasma volumes were defined as the distribution volumes of labelled proteins 10 min after injection. In the calculated metabolic data we have used the C3 distribution volume. Before and during each study the thyroid gland was blocked with potassium iodide. Blood samples were collected in EDTA Vacutainers 10 min and 5 h after injection and then daily for 7 days. Plasma was immediately separated from the blood cells. Urine was collected in 24 h aliquots for 8 days. Serum
71
samples were taken at the beginning, during and at the end of each study for determination of C3 concentrations by the radial immunodiffusion technique (Mancini et al., 1965). Plasma samples were stored at --75°C until the termination of each study and then counted. If counted earlier, corrections for half-lives of the isotopes were made. Plasma and urine samples were counted in 2 ml aliquots in a Hewlett Packard Tri-Carb Scintillator Spectrometer, model 3002, with appropriate correction for ['3~I] influence on the ['2sI] channel. FCR was determined by the metabolic clearance method as described by Berson and Yalow (1957) and by exponential analysis of the plasma disappearance curve as described by Matthews (1957). The EV/IV ratio was determined from the rate constants (K's), calculated as described by Matthews (1957). K,:
1 -
C,C:(b2 K13
C, + C~ b, bE
Ka, =
C1b2 + C 2 b l
=
EV IV
-- b,):
C,b: + C:b, _ K, a
Ka,
SR was calculated from the formula: SR =
Serum concentration of C3 × PV × FCR Body weight
RESULTS
Starting with approximately 400 ml EDTA-serum the isolation procedure yielded 20--60 mg of C3. The preparations contained only one protein band, on high voltage agarose gel electrophoresis. Immunoelectrophoresis identified the band as C3. Polyacrylamide gel electrophoresis gave one main band with one to three faint contaminant bands. Sodium dodecyl sulphate polyacrylamide gel electrophoresis with fl-mercaptoethanol showed t w o main bands, presumably the a and/3 chains of C3. Crossed immunoelectrophoresis against anti human C3 showed that C3 was in the native form. The prepara-
TABLE 1 DISTRIBUTION V O L U M E S F O R [12si]c3 (BATCH 2) A N D [131I]ALBUMIN Subject
[ 12 s I ]C3 (ml)
[ 1311 ]Albumin (ml)
R a t i o [ 12 s I ]C3/ [ 13 l I ] A l b u m i n
J.L. J.S.
3496 3374
3230 3126
1.08 1.08
T w o subjects studied with this batch did not receive [ lal I]albumin.
72 TABLE 2 DISTRIBUTION VOLUMES F O R [12si]c3 (BATCH 3) AND [ l a l I ] A L B U M I N Subject
[12SI]C3 (ml)
[131I]Albumin (ml)
Ratio[ 12si]C3/ [ ] 311 ]Albumin
M.R. B.R. S.T. P.H. B.F. T. 0. K.K. T.D.
3259 3569 3240 3597 3860 3490 4565 2604
2447 2826 2787 3217 2744 2817 3199 3309
1.33 1.26 1.16 1.12 1.41 1.24 1.43 0.79
tions were haemolytically active before and after labelling. Pyrogen testing was negative. Three batches of C3 were prepared for the present study. For batches 2 and 3 the metabolic data have been c o m p u t e d , only the plasma radioactivity decay curve is presented for batch 1. There was a discrepancy between the distribution volumes obtained at 10 min with [12si]C3 and [13~I]albumin (Tables 1 and 2). The mean plasma radioactivity decay curve of [ 12sI]C3 showed differences between batches in the initial part of each study (Fig. 1). After one to two days, however, the slopes were quite similar for batches 2 and 3. Because of this and for reasons
150 0 0 ~ 4 2
10'
- . , . n 7, n . 2
[
cg
.o_ "O
fg
k.
o:
E
o)
e:
K
!
16o 0 Timeafterinjection (hours). o
,so
Fig. 1. Plasma decay curves of three batches of [12si]c3. The curves represent mean radioactivity for each batch.
73 TABLE 3 SUMMARY OF METABOLIC DATA (BATCH 2) Subject
C3-serum
FCR (%h)
Synthesis rate (mg/kg/h)
(g/l)
I.A. K.S. J.L. J.S.
1.40 1.40 1.80 2.00
EV/ IV ratio
Metabolic clearance
Exponential analysis
Metabolic clearance
Exponential analysis
1.26 0.90 0.97 1.33
1.03 0.77 0.99 0.99
0.95 0.86 0.75 1.08
0.78 0.73 0.76 0.81
0.35 -0.28 0.28
Mean
1.65
1.12
0.95
0.91
0.77
0.30
S.D.
0.30
0.21
0.12
0.14
0.03
0.04
EV/IV ratio for K. S. could not be calculated, as there was only one exponential.
given later it was decided to calculate FCR from the mean metabolic clearance for days 2 : 7 for each subject. With the exponential analysis the plasma decay curve for days 2--7 was resolved into two exponentials, and K,2, K3, and K, 3 calculated. The metabolic data are presented in Fig. 1 and Tables 3 and 4. The subjects were in a steady state according to the serum C3 concentrations. DISCUSSION
The isolation procedure yielded C3 preparations which contained very small amounts of contaminating proteins and C3 fragments. We t o o k great TABLE 4 SUMMARY OF METABOLIC DATA (BATCH 3) Subject
C3-serum
FCR (%/h)
Synthesis rate (rng/kg/h)
(g/l)
M.R. B.R. S.T. P.H. B.F. T.O. K.K. T.D.
2.63 2.17 2.80 2.40 1.99 1.38 1.61 1.50
EV]
IV Metabolic clearance
Exponential analysis
Metabolic clearance
Exponential analysis
ratio
1.16 0.96 1.10 1.25 0.97 1.39 0.98 0.84
0.75 0.64 0.88 1.17 0.80 0.98 0.92 0.85
1.65 1.28 1.33 1.49 1.03 1.03 1.03 0.64
1.07 0.85 1.06 1.40 0.85 0.73 0.97 0.65
0.30 0.27 0.15 0.12 0.25 0.22 0.19 0.19
Mean
2.06
1.08
0.87
1.19
0.95
0.21
S.D.
0.53
0.18
0.16
0.32
0.24
0.06
care to discard all fractions from the Bio-Gel column containing traces of C3 degradation products. Nevertheless the preparations had a lower haemolytic activity than fresh serum containing the same a m o u n t of C3. The same p h e n o m e n o n has been described by R u d d y et al. {1975) and remains unexplained The distribution volume at 10 min was generally higher with [t2sI]C3 than with [131I]albumin. This has also been found by others (Alper and Rosen, 1967; Petz et al., 1968; R u d d y et al., 1975; Potter et al., 1976) and has been ascribed to the use of inhomogeneous C3. An alternative hypothesis is that the labelled C3 binds rapidly to C3 receptors. The difference between batches observed in the present study makes the latter explanation less likely. Differences in the slopes of the first part of the plasma radioactivity decay curves were observed {Fig. 1). After 24 h retained plasma radioactivity varied from 30 to 50%. There were marked between-batch variations but much smaller within-batch differences. The batch showing the most rapid initial decay also showed the highest distribution volume of [ ~25I] C3 at 10 min. We have to make the reservation that our data are limited in quantity, and it is important to point out that similar variations in behaviour of different C3 preparations have been observed by others (Alper and Rosen, 1967). From the second day our curves generally agree with previous studies. Most probably the differences between batches are due to differences in the preparations, and n o t to biological differences in the experimental subjects. Despite the fact that the C3 preparations seem biochemically and immunologically to contain mainly pure C3 in its native form, subtle molecular changes must have taken place. Some of the molecules are therefore broken down at a more rapid rate than native C3, the extreme example being batch 1. Thus we find it incorrect to present metabolic data obtained with a preparation of this quality. We consider that the first 24 h of the experiment should not be used in the calculations, except that the initial distribution volume of [ ~25I] C3 may be used as the plasma volume. Data from days 2 to 7 are thus analysed according to the metabolic clearance method, and in the exponential analysis we resolve the decay curve from the same days into two exponentials and calculate the metabolic parameters from slopes and intercepts. With these procedures rather narrow limits are obtained for FCR, SR and EV/IV ratio in normals, and the results with the two methods agree fairly well (Tables 1--4). Our values for FCR are generally lower than previously reported by others, which partly may be related to exclusion of data from the first 24 h. R u d d y et al. (1975) also excluded the very first part of their experiments, and their 'corrected' FCR compares better with our results than the values reported from other laboratories. There are obvious other reservations that must be made in the interpretation of the data obtained in studies of this kind. As each batch of labelled protein must be used within 4--6 weeks, the number of studies which can be
75
performed must be limited. Because of the duration of the experiments and the high degree of cooperation required from the participants, we have n o t involved the same subjects in studies with different batches. We have therefore presented the experimental data with calculated means and standard deviations only, without statistical comparisons between groups. The integrated analysis m e t h o d (Nosslin, 1973) is generally useful for protein turnover studies and has also been employed in some reports on C3 metabolism. In our hands, probably due to the difficulties encountered in the initial part of the studies, this method has not been found suitable. The presented procedure has standardized the method fairly well. It is nevertheless advisable to include normal controls for each batch of labelled C3 used in patient studies. REFERENCES Alper, C.A. and F. Rosen, 1967, J. Clin. Invest. 46, 2021. i~erson, S.A., R.S. Yalow, S.S. Schreiber and J. Post, 1953, J. Clin. Invest. 32,746. Berson, S.A. and R.S. Yalow, 1957, Fed. Proc. 16, 13S. Carpenter, C.B., S. Ruddy, I.H. Shehadeh, H.J. Miiller-Eberhard, J.P. Merrill and K. Frank Austen, 1969, J. Clin. Invest. 48, 1495. Charlesworth, J.A., D. Gwyn Williams, E. Sherington, P.J. Lachmann and D.K. Peters, 1974a, J. Clin. Invest. 53, 1578. Charlesworth, J.A., D. Gwyn Williams, E. Sherington and D.K. Peters, 1974b, Clin. Sci. Molec. Med. (Oxf.) 4 6 , 2 2 3 , Charlesworth, J.A., D.G. Williams, P. Naish, P.J. Lachmann and D.K. Peters, 1974c, Clin. Exp. Immunol. 16,445. Lachmann, P.J., M.J. Hobart and W.P. Aston, 1973, In: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 5.1. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2, 235. Matthews, C.M.E., 1957, Phys. Med. Biol. 2, 36. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29,185. Nosslin, B., 1973, In: Protein Turnover, CIBA Foundation Symposium 9. (Elsevier, Amsterdam) p. 113. Petz, L.D., D.S. Fink, E.A. Letsky, H.J. Fudenberg and H.J. Miiller-Eberhard, 1968, J. Clin. Invest. 47, 2469. Potter, B.J., E. Elias and E. Anthony Jones, 1976, J. Lab. Clin. Med. 88,427. Ruddy, S., C.B. Carpenter, K.W. Chin, J.N. Knostman, N.A. Soter, O. GStze, H.J. MiillerEberhard and K.F. Austen, 1975, Medicine 54,165. Sliwinski, A.J. and N.J. Zvaifler, 1972, Clin. Exp. Immunol. 11, 21.
69
© Elsevier/North-Holland Biomedical Press
METABOLIC STUDIES OF C3 IN MAN
L. BRINCH, P. TEISBERG and I. AAKESSON
Medical Department 7, Ullevaal Hospital and Medical Department A, Rikshospitalet, Oslo, Norway (Received 5 February 1979, accepted 10 May 1979)
The results of metabolic studies with radioactively labelled C3 in 14 healthy individuals are presented. Plasma volume determined by labelled C3 was generally higher than when determined by labelled albumin, and different batches of C3 behaved differently during the early part of the experiments. These phenomena are probably caused by C3 inhomogenities which are difficult to detect by usual biochemical and immunological methods. We have, therefore, excluded the first 24 h of the experiments from the mathematical analysis. In this way, rather narrow limits for the metabolic parameters have been obtained.
INTRODUCTION
Several groups have performed metabolic studies of labelled C3 to investigate the involvement of the complement system in different diseases (Alper and Rosen, 1967; Petz et al., 1968; Carpenter et al., 1969; Sliwinski and Zvaifler, 1972; Charlesworth et al., 1974a, b, c; Potter et al., 1976). These reports show large variations in the calculated fractional catabolic rate (FCR), synthesis rate (SR) and extravascular/intravascular (EV/IV) ratio even in normals. The differences may only to some extent be explained by different methods of analysing the experimental data. The aims of the present study were to elucidate the possible explanations for interlaboratory variations, and to standardize the procedure as far as possible. M A T E R I A L AND METHODS
The material consisted of 14 healthy medical students and medical staff between the ages of 24 and 39 years, 3 females and 11 males. None of the participants had, according to information given by themselves, any disease of a chronic nature, and all were feeling well at the time of the study. No clinical or laboratory studies were performed to verify absence of disease. Informed consent was obtained from all participants. C3 was isolated from HB antigen-free serum by a modification of the m e t h o d described by Lachmann et al. (1973).
70 EDTA was added to the serum to a final concentration of 0.01 M, and the pH adjusted to 6.0. The globulins were precipitated with 20% (w/v) Na2SO4. From then on all procedures were performed at +4~C. The precipitate was dialysed against phosphate buffer, pH 5.6, with 0.002 M EDTA, and dissolved in 0.01 M phosphate buffer, pH 7.0. Chromatography was performed on a DEAE Sephadex A-50 column (Pharmacia Fine Chemicals AB, Uppsala, Sweden) with phosphate buffer, pH 7.0, and a continuous NaC1 gradient. The fractions containing C3 were then chromatographed on a Bio-Gel HT column (Bio-Rad Laboratories, Richmond, CA, U.S.A.). The elution was performed stepwise using disodium hydrogen phosphate/potassium dihydrogen phosphate buffer, pH 7.9, of ionic strengths 7.5, 10, 12.5, and 15 mS measured at +4°C. Native C3 was mainly eluted in the 12.5 mS fraction. The purity of the preparations was tested by high voltage agarose gel electrophoresis, polyacrylamide gel electrophoresis, immunoelectrophoresis against rabbit anti human C3 and rabbit antiserum against human serum proteins (Behring Werke, Marburg/Lahn, F.R.G.) and crossed immunoelectrophoresis in agarose against anti C3. The preparations were sterile filtered with Acrodisc filters, 0.22 grn (Gelman Instrument Company, Ann Arbor, MI, U.S.A.). Pyrogen testing was performed in rabbits. Each batch was immediately frozen at --75°C in sterile vials and stored until labelling was performed with [ 12sI] (Isotope Laboratories, Institutt for Atomenergi, Kjeller, Norway). The chloramine T method, as described by McConahey and Dixon (1966), was employed. Free radioactivity was removed by dialysis at 4°C against sterile Ringer acetate. The labelled preparations were examined for activation products by the previously mentioned methods and by radio-autography after immunoelectrophoresis against anti C3 and antiserum against human serum proteins. Protein content was measured with the micro-biuret method. The labelled and unlabelled preparations were tested for biological activity in a haemolytic system with cellular intermediates (Cordis Laboratories, Miami, FL, U.S.A.). Albumin (AB Kabi, Stockholm, Sweden) in a final concentration of 0.5% was added to protect the labelled protein. The batches were divided in 1 ml aliquots for each study. Aliquots of 0.5--1.0 mg protein with activities of 3--5 t~Ci [12sI] were injected intravenously in each subject. Most of the subjects were simultaneously given 5--10 ~Ci [ 131]albumin (Isotope Laboratories, Institutt for Atomenergi, Kjeller, Norway) in order to compare plasma volumes (PV) determined by two different labelled proteins. Plasma volumes were defined as the distribution volumes of labelled proteins 10 min after injection. In the calculated metabolic data we have used the C3 distribution volume. Before and during each study the thyroid gland was blocked with potassium iodide. Blood samples were collected in EDTA Vacutainers 10 min and 5 h after injection and then daily for 7 days. Plasma was immediately separated from the blood cells. Urine was collected in 24 h aliquots for 8 days. Serum
71
samples were taken at the beginning, during and at the end of each study for determination of C3 concentrations by the radial immunodiffusion technique (Mancini et al., 1965). Plasma samples were stored at --75°C until the termination of each study and then counted. If counted earlier, corrections for half-lives of the isotopes were made. Plasma and urine samples were counted in 2 ml aliquots in a Hewlett Packard Tri-Carb Scintillator Spectrometer, model 3002, with appropriate correction for ['3~I] influence on the ['2sI] channel. FCR was determined by the metabolic clearance method as described by Berson and Yalow (1957) and by exponential analysis of the plasma disappearance curve as described by Matthews (1957). The EV/IV ratio was determined from the rate constants (K's), calculated as described by Matthews (1957). K,:
1 -
C,C:(b2 K13
C, + C~ b, bE
Ka, =
C1b2 + C 2 b l
=
EV IV
-- b,):
C,b: + C:b, _ K, a
Ka,
SR was calculated from the formula: SR =
Serum concentration of C3 × PV × FCR Body weight
RESULTS
Starting with approximately 400 ml EDTA-serum the isolation procedure yielded 20--60 mg of C3. The preparations contained only one protein band, on high voltage agarose gel electrophoresis. Immunoelectrophoresis identified the band as C3. Polyacrylamide gel electrophoresis gave one main band with one to three faint contaminant bands. Sodium dodecyl sulphate polyacrylamide gel electrophoresis with fl-mercaptoethanol showed t w o main bands, presumably the a and/3 chains of C3. Crossed immunoelectrophoresis against anti human C3 showed that C3 was in the native form. The prepara-
TABLE 1 DISTRIBUTION V O L U M E S F O R [12si]c3 (BATCH 2) A N D [131I]ALBUMIN Subject
[ 12 s I ]C3 (ml)
[ 1311 ]Albumin (ml)
R a t i o [ 12 s I ]C3/ [ 13 l I ] A l b u m i n
J.L. J.S.
3496 3374
3230 3126
1.08 1.08
T w o subjects studied with this batch did not receive [ lal I]albumin.
72 TABLE 2 DISTRIBUTION VOLUMES F O R [12si]c3 (BATCH 3) AND [ l a l I ] A L B U M I N Subject
[12SI]C3 (ml)
[131I]Albumin (ml)
Ratio[ 12si]C3/ [ ] 311 ]Albumin
M.R. B.R. S.T. P.H. B.F. T. 0. K.K. T.D.
3259 3569 3240 3597 3860 3490 4565 2604
2447 2826 2787 3217 2744 2817 3199 3309
1.33 1.26 1.16 1.12 1.41 1.24 1.43 0.79
tions were haemolytically active before and after labelling. Pyrogen testing was negative. Three batches of C3 were prepared for the present study. For batches 2 and 3 the metabolic data have been c o m p u t e d , only the plasma radioactivity decay curve is presented for batch 1. There was a discrepancy between the distribution volumes obtained at 10 min with [12si]C3 and [13~I]albumin (Tables 1 and 2). The mean plasma radioactivity decay curve of [ 12sI]C3 showed differences between batches in the initial part of each study (Fig. 1). After one to two days, however, the slopes were quite similar for batches 2 and 3. Because of this and for reasons
150 0 0 ~ 4 2
10'
- . , . n 7, n . 2
[
cg
.o_ "O
fg
k.
o:
E
o)
e:
K
!
16o 0 Timeafterinjection (hours). o
,so
Fig. 1. Plasma decay curves of three batches of [12si]c3. The curves represent mean radioactivity for each batch.
73 TABLE 3 SUMMARY OF METABOLIC DATA (BATCH 2) Subject
C3-serum
FCR (%h)
Synthesis rate (mg/kg/h)
(g/l)
I.A. K.S. J.L. J.S.
1.40 1.40 1.80 2.00
EV/ IV ratio
Metabolic clearance
Exponential analysis
Metabolic clearance
Exponential analysis
1.26 0.90 0.97 1.33
1.03 0.77 0.99 0.99
0.95 0.86 0.75 1.08
0.78 0.73 0.76 0.81
0.35 -0.28 0.28
Mean
1.65
1.12
0.95
0.91
0.77
0.30
S.D.
0.30
0.21
0.12
0.14
0.03
0.04
EV/IV ratio for K. S. could not be calculated, as there was only one exponential.
given later it was decided to calculate FCR from the mean metabolic clearance for days 2 : 7 for each subject. With the exponential analysis the plasma decay curve for days 2--7 was resolved into two exponentials, and K,2, K3, and K, 3 calculated. The metabolic data are presented in Fig. 1 and Tables 3 and 4. The subjects were in a steady state according to the serum C3 concentrations. DISCUSSION
The isolation procedure yielded C3 preparations which contained very small amounts of contaminating proteins and C3 fragments. We t o o k great TABLE 4 SUMMARY OF METABOLIC DATA (BATCH 3) Subject
C3-serum
FCR (%/h)
Synthesis rate (rng/kg/h)
(g/l)
M.R. B.R. S.T. P.H. B.F. T.O. K.K. T.D.
2.63 2.17 2.80 2.40 1.99 1.38 1.61 1.50
EV]
IV Metabolic clearance
Exponential analysis
Metabolic clearance
Exponential analysis
ratio
1.16 0.96 1.10 1.25 0.97 1.39 0.98 0.84
0.75 0.64 0.88 1.17 0.80 0.98 0.92 0.85
1.65 1.28 1.33 1.49 1.03 1.03 1.03 0.64
1.07 0.85 1.06 1.40 0.85 0.73 0.97 0.65
0.30 0.27 0.15 0.12 0.25 0.22 0.19 0.19
Mean
2.06
1.08
0.87
1.19
0.95
0.21
S.D.
0.53
0.18
0.16
0.32
0.24
0.06
care to discard all fractions from the Bio-Gel column containing traces of C3 degradation products. Nevertheless the preparations had a lower haemolytic activity than fresh serum containing the same a m o u n t of C3. The same p h e n o m e n o n has been described by R u d d y et al. {1975) and remains unexplained The distribution volume at 10 min was generally higher with [t2sI]C3 than with [131I]albumin. This has also been found by others (Alper and Rosen, 1967; Petz et al., 1968; R u d d y et al., 1975; Potter et al., 1976) and has been ascribed to the use of inhomogeneous C3. An alternative hypothesis is that the labelled C3 binds rapidly to C3 receptors. The difference between batches observed in the present study makes the latter explanation less likely. Differences in the slopes of the first part of the plasma radioactivity decay curves were observed {Fig. 1). After 24 h retained plasma radioactivity varied from 30 to 50%. There were marked between-batch variations but much smaller within-batch differences. The batch showing the most rapid initial decay also showed the highest distribution volume of [ ~25I] C3 at 10 min. We have to make the reservation that our data are limited in quantity, and it is important to point out that similar variations in behaviour of different C3 preparations have been observed by others (Alper and Rosen, 1967). From the second day our curves generally agree with previous studies. Most probably the differences between batches are due to differences in the preparations, and n o t to biological differences in the experimental subjects. Despite the fact that the C3 preparations seem biochemically and immunologically to contain mainly pure C3 in its native form, subtle molecular changes must have taken place. Some of the molecules are therefore broken down at a more rapid rate than native C3, the extreme example being batch 1. Thus we find it incorrect to present metabolic data obtained with a preparation of this quality. We consider that the first 24 h of the experiment should not be used in the calculations, except that the initial distribution volume of [ ~25I] C3 may be used as the plasma volume. Data from days 2 to 7 are thus analysed according to the metabolic clearance method, and in the exponential analysis we resolve the decay curve from the same days into two exponentials and calculate the metabolic parameters from slopes and intercepts. With these procedures rather narrow limits are obtained for FCR, SR and EV/IV ratio in normals, and the results with the two methods agree fairly well (Tables 1--4). Our values for FCR are generally lower than previously reported by others, which partly may be related to exclusion of data from the first 24 h. R u d d y et al. (1975) also excluded the very first part of their experiments, and their 'corrected' FCR compares better with our results than the values reported from other laboratories. There are obvious other reservations that must be made in the interpretation of the data obtained in studies of this kind. As each batch of labelled protein must be used within 4--6 weeks, the number of studies which can be
75
performed must be limited. Because of the duration of the experiments and the high degree of cooperation required from the participants, we have n o t involved the same subjects in studies with different batches. We have therefore presented the experimental data with calculated means and standard deviations only, without statistical comparisons between groups. The integrated analysis m e t h o d (Nosslin, 1973) is generally useful for protein turnover studies and has also been employed in some reports on C3 metabolism. In our hands, probably due to the difficulties encountered in the initial part of the studies, this method has not been found suitable. The presented procedure has standardized the method fairly well. It is nevertheless advisable to include normal controls for each batch of labelled C3 used in patient studies. REFERENCES Alper, C.A. and F. Rosen, 1967, J. Clin. Invest. 46, 2021. i~erson, S.A., R.S. Yalow, S.S. Schreiber and J. Post, 1953, J. Clin. Invest. 32,746. Berson, S.A. and R.S. Yalow, 1957, Fed. Proc. 16, 13S. Carpenter, C.B., S. Ruddy, I.H. Shehadeh, H.J. Miiller-Eberhard, J.P. Merrill and K. Frank Austen, 1969, J. Clin. Invest. 48, 1495. Charlesworth, J.A., D. Gwyn Williams, E. Sherington, P.J. Lachmann and D.K. Peters, 1974a, J. Clin. Invest. 53, 1578. Charlesworth, J.A., D. Gwyn Williams, E. Sherington and D.K. Peters, 1974b, Clin. Sci. Molec. Med. (Oxf.) 4 6 , 2 2 3 , Charlesworth, J.A., D.G. Williams, P. Naish, P.J. Lachmann and D.K. Peters, 1974c, Clin. Exp. Immunol. 16,445. Lachmann, P.J., M.J. Hobart and W.P. Aston, 1973, In: Handbook of Experimental Immunology, ed. D.M. Weir (Blackwell Scientific Publications, Oxford) p. 5.1. Mancini, G., A.O. Carbonara and J.F. Heremans, 1965, Immunochemistry 2, 235. Matthews, C.M.E., 1957, Phys. Med. Biol. 2, 36. McConahey, P.J. and F.J. Dixon, 1966, Int. Arch. Allergy 29,185. Nosslin, B., 1973, In: Protein Turnover, CIBA Foundation Symposium 9. (Elsevier, Amsterdam) p. 113. Petz, L.D., D.S. Fink, E.A. Letsky, H.J. Fudenberg and H.J. Miiller-Eberhard, 1968, J. Clin. Invest. 47, 2469. Potter, B.J., E. Elias and E. Anthony Jones, 1976, J. Lab. Clin. Med. 88,427. Ruddy, S., C.B. Carpenter, K.W. Chin, J.N. Knostman, N.A. Soter, O. GStze, H.J. MiillerEberhard and K.F. Austen, 1975, Medicine 54,165. Sliwinski, A.J. and N.J. Zvaifler, 1972, Clin. Exp. Immunol. 11, 21.