In vivo metabolism of apolipoprotein S in humans. Comparison with apolipoprotein A-I metabolism

169

Clink Chimica Acta, 170 (1987) 169-180 Elsevier

CCA 03981

In vivo metabolism of apolipoprotein S in humans. Comparison with apolipoprotein A-I metabolism Claude L. Malmendier,

Jean-Fraqois

Lontie and Claude Delcroix

Research Foundation on Atherosclerosis, and Research Unit on A therosclerosrs, Faculiy OJMedicine, Uniuersith Libre de Bruxelles, Brussels (Belgam) (Received

26 March

Key words: Apohpoprotein

1987; revision received 1 July 1987; accepted

after revision

A-I; Apolipoprotein S; Metabolism; Normolipoproteinemia; Compartmental modeling

3 August

1987)

Plasma-Urine;

Summary A-I were 125I-labelled apolipoprotein (APO) S and ‘311-labelled apolipoprotein injected i.v. into healthy volunteers. Blood samples and daily urine collections were drawn periodically for 15 days. Ninety-eight percent of r3iI radioactivity and > 95% of 125I radioactivity were found in HDL after Superose gel chromatography of plasma. About 10% of each radioactivity was recovered in the d 1.250 infranate after one ultracentrifugation. Affinity chromatography on monoclonal anti-APO A-I Sepharose column separates two lipoprotein particles containing Apo S, one retained with Apo A-I (42.5%) and the other eluting without Apo A-I (57.5%). Kinetic parameters were calculated according to exponential curve fitting. Mean transit time was about 7.0 days for both Apo A-I and Apo S. FCR of Apo S was 50% higher than FCR of Apo A-I. Synthetic rate of Apo S was about 150 times smaller than for Apo A-I. As heterogeneity of HDL-S was suggested by both the results of affinity chromatography and the urinary data, a compartmental model was built which fitted adequately all data. Part of the model is common to HDL-A-I and HDL-S.

Introduction A new family of polypeptides, called apolipoprotein S, was found greatly increased in high density lipoproteins of neurological and postsurgery patients only fed by intravenous infusions of glucose [l]. This protein normally present in small

Correspondence to: Professor CL. Malmendier, Medicine, Universitt Libre de Bruxelles, Brussels,

0009-8981/87/$03.50

Research Belgium.

0 1987 Elsevier Science Publishers

Foundation

B.V. (Biomedical

on Atherosclerosis,

Division)

Faculty

of

170

quantities in plasma may represent in those patients as much as 40% of HDL proteins and replace both apoproteins (APO) A-I and A-II [2,3]. Apo S was shown able to bind lipids and form a stable complex. It possesses the same amphipathic helical segment of 26 residues as amyloid A protein [4]. Effectively apoprotein S is similar to Apo SAA as far as molecular weight, amino acid composition, partial amino acid sequence, immunological properties and association with HDL are concerned. Whereas the metabolism of Apoprotein A-I was studied by several authors [5-81 the metabolism of Apo S has not yet been investigated in human subjects. However a six-day kinetic study of i.v. injected ‘251-HDL Apo SAA was realized in monkeys

191. The present report describes kinetic of Apo A-I and Apo S, i.v. injected.

studies

designed

to compare

the catabolism

Material and methods Isolation of HDL High density ultracentrifugation

lipoproteins [8].

were

isolated

from

normal

plasma

by

sequential

Purification of apolipoprotein A-I Apo A-I was isolated from delipidated HDL and fractionated by column chromatography [8]. The purity was confirmed by polyacrylamide gel electrophoresis, amino acid composition and combined immunodiffusion/immunoelectrophoresis [8]. Isolation and purification of apoprotein S, HDL of abnormal composition from neurological patients infused with 50 g/l glucose [l] were isolated and delipidated as described in [2]. HDL apoproteins were fractionated on Sephacryl S-200 using 0.2 mol/l Tris-HCl, pH 8.2, containing 6 mol/l urea, 2 mmol/l sodium decylsulfate and 3 mmol/l sodium azide as elution buffer. The fraction V containing apoproteins C-II, C-III, D-2 and Apo S was dialyzed against 5 mmol/l ammonium bicarbonate, pH 8.0, concentrated in an Amicon Cell prior to lyophilization. Apo S, was isolated by chromatography of this fraction V on DEAE Sephacel as described previously [l] and the purity tested as for apoprotein A-I and by isoelectric focusing. The purity of Apo S, was 98%. Lubelling of apoproteins A-I and S, Lyophilized Apo A-I or Apo S, were dissolved in 1 mol/l glycine-NaOH buffer, pH 10.0, at a concentration of 3 mg/ml and radiolabelled respectively with 13tI and ‘25I by the iodine monochloride method as adapted by Shepherd et al [lo]. Labelling efficiency for both proteins was approximately 50%. Unbound iodine was removed by dialysis at 4°C for 48 h against 0.15 mol/l NaCl, 0.25 mmol/l EDTA, pH 7.4. Ninety-nine percent of the radioactivity remaining after dialysis was attached to the apoprotein.

171

Preparation of radioactive solutions for in oiuo use Labelled apoproteins were sterilized by Millipore filtration (0.22 pm) and tested for pyrogen before reinjection [ll]. Total time from labelling to reinjection was < 48 h. Study protocol Thirty pCi of ‘251-Apo S (0.1 _+ 0.05 mg protein, i.e. < 5% of the normal pool) and 20 I_LC~of ‘311-labelled-Apo A-I (a mean of 0.2 f 0.1 mg of protein) dissolved in 0.15 mol/l NaCl were administered i.v. Twenty millilitres fasting blood samples were collected into EDTA (1 g/l) after 10 min, 30 min, 1, 2, 4, 8, 24 h and then every morning for up to 15 days after an overnight fast. Plasma was separated at 4 o C and 2 500 x g for 20 min in a refrigerated centrifuge. One millilitre was frozen at - 20 o C for Apo S and Apo A-I quantitation. The remaining plasma was kept at 4“C with 0.25 mmol/l EDTA and thimerosal. Twenty-four-hour urine was collected daily in plastic jars containing 1 ml of a preservative [12]. Subjects The four subjects, aged from 23-45 yr, participating in the turnover studies were normolipidemic (Table I). They had normal hepatic, renal, and thyroid functions, and were not on medication known to affect plasma lipids. Normal activity was permitted. They were maintained on an isocaloric diet (2500 Kcal) containing 20% protein, 35% fat and 45% carbohydrate, a P/S ratio of 0.3 to 0.4 and a cholesterol intake of 300 mg/day. All have been fasted for 12 h before the experiment. Potassium iodide (400 mg/day) was administered in two divided doses daily beginning 3 days prior to the study and extending throughout the study period. All subjects gave informed consent to the project. Analytical methods Lipid and lipoprotein Research Clinics [ 131.

analyses

were

prepared

Column chromatography Two millilitres graphed directly on Superose 6 Prep grade

by

the

method

of the

Lipid

portions of plasma were chromatocolumn FPLC system (Pharmacia) as

TABLE I Characteristics of the subjects Subject

Sex F M M M

1 2 3 4 Mean +

SD

Age

Body wt.

Height

(yr)

0%)

(cm)

24 23 45 26

49 75 75 64

160 186 176 179

66+12

175*111

TG

TC

HDL-C

LDL-C

Apo B 81 62 112 94

(mUdI) 89 70 119 90 92k20

183 138 214 213

60 37 36 57

105 87 154 138

187*35

47.5 f 13

121 f 30

87+21

described in [14]. The absorbance was monitored continuously at 280 nm. All subfractions were assayed in a Beckman gamma 5 500 counting system. The radioactivity profiles were compared to the protein profiles. Total plasma lipoproteins were isolated from 5 ml plasma by ultracentrifugation for 20 h at 12°C at a density of 1.250 g/ml. The supematant (in 2 ml) was chromatographed without prior dialysis on the same column and with the same conditions as above. Similarly, radioactivity and protein profiles were compared. Isolation of lipoproteins by immunoaffinity chromatography Specific monoclonal IgG was covalently bound to cyanogen-bromide activated Sepharose CL4B as described by Bukberg et al [15]. One millilitre of plasma was transferred to disposable plastic Econo-column (0.7 id. X 10 cm) (BioRad) containing 1 ml of the immunosorber: anti-APO A-I Sepharose (theoretical binding capacity of 1860 pg). The sample was eluted with the equilibrium buffer 0.1 mol/l borate, 0.1 mol/l NaCl (pH 8.0). After the unretained fraction was eluted, 3 mol/l sodium thiocyanate was applied to desorb the retained lipoproteins. The column was then washed successively with 2 x 2 ml borate buffer (pH 8.0) 2 X 2 ml sodium thiocyanate, 2 x 2 ml borate and finally 2 X 2 ml 3 mol/l sodium thiocyanate before being reequilibrated [16]. After each passage, the eluate was counted for ‘*‘I and 13’1 radioactivity. Apoprotein determination Apo A-I was quantitated by sandwich ELISA technique [17]. Pure Apo A-I was used as primary standards for calibration. Secondary standards were made of delipidated plasma. Dilution was 1 : 800000. The CVs were 5.2% (intraassay) and 8.9% (interassay). Apo S was quantitated using a sandwich ELISA [18]. Pure Apo SAA was used as primary standard. The antisera were prepared by injecting New Zealand white rabbits with two chemically synthesized short fragments of Apo SAA corresponding to residues 58-69 and 95-104 of Apo SAA covalently linked to tetanus toxoid by use of glutaraldehyde [19]. The produced antibodies recognized both peptides and Apo SAA. Dilution was 1 : 100. The coefficients of variation were 4.5% (interassay) and 7.0% (interassay). Mathematical analysis Considering the hypothesis that all particles constitute an homogeneous pool, the mean residence time (T) for all material entering and leaving the system was determined from the area under the plasma decay curve by a multiexponential computer curve fitting technique [20,21] T = EAi/af/ZA,/a, where Ai and a, are the coefficients and exponents of a sum of exponential terms fitted to the response data. FCR was calculated according to the method described by Matthews [22] as X.A/C, /a,. The production rate was calculated from the following formula (assuming steady-state conditions): Apo A-I(or

Apo S) cone X plasma

vol x FCR/wt.

173

Plasma volume was calculated as 4.1% of the subject’s body weight which values in good agreement with those obtained from the isotope dilution occurred over the first 10 min after injection.

gave that

Model building The SAAM 27 program for multicompartmental analysis [21] was used to analyse the data. A multicompartmental model was built to account for the tracer and tracee (non-labelled) kinetic data. All plasma data (Apo A-I and Apo S) were fitted simultaneously to the model. Urinary excretions of labelled iodide released as a were included. The urine data serve as an result of lipoprotein degradation, additional independent constraint on the model by requiring that all the label be accounted for. The urinary subsystem was built by connecting the iodide subsystem of Berman [21] on all the plasma outputs. The daily excretion rate was obtained by multiplying the iodide activity by the fraction excreted daily and by the plasma volume. Results Distribution of 13’1 and ‘251 radioactivity Most i3iI-Ape A-I radioactivity (> 98%) was found in the HDL peak when the supernatant of the ultracentrifugation at d 1.250 was chromatographed on Superose 6 column (Fig. 1). The amount of radioactivity recovered in the d 1.250 infranate was 11.0 f 2.0%. When the lipoproteins were directly separated from total plasma without prior separation by ultracentrifugation on the same column, all the radioactivity was found in a peak corresponding in location to HDL, keeping in mind that the profile of the lipoproteins is masked by the presence of other plasma proteins (Fig. 1). Most of ‘251-Apo S radioactivity (about 95%) was found in the HDL peak after column chromatography of the d 1.250 supernatant. The amount found in the infranate of d 1.250 was 8.9 + 1.1%. When the plasma was directly chromatographed, 93% was recovered in the zone corresponding to HDL. The remaining radioactivity was found in the peak corresponding to albumin or similar size proteins. The affinity chromatography on monoclonal anti-A-I Sepharose CL-4B column allowed to separate two lipoprotein particles containing Apo S, one retained by the column with Apo A-I, the other eluting directly without Apo A-I. The respective retained and unretained fractions were 42.5 and 57.5%. Kinetic analysis and parameters HDL-Apo A-I plasma curves and HDL-Apo S curves are shown in Fig. 2. Initial decay of radioiodinated apolipoprotein S was faster than that observed for Apo A-I but after the 2nd day, the curves of both apoproteins are parallel. Twenty-four hours after the injection of Apo S, < 5% of the radioactivity of the fast component remained in the plasma compartment.

174 IO

08

06

-1

50000.

30000.

10000 : 0

IO 20 30 40 TUBE NUMBER

50

Fig. 1. Column elution profile of plasma and lipoproteins. a. Lipoproteins were isolated by ultracentrifugation at density 1.25 g/ml from 5 ml plasma. A final volume of 2 ml was injected onto a column of Superose 6 Prep Grade Pharmacia (56 x 1.6 cm) and eluted at 0.75 ml/min with 0.15 mot/l sodium chloride, 0.01% Na,EDTA and 0.02% sodium azide, pH 7.2. b. 2 ml of plasma were applied directly to ‘3’I-labelled Apo A-I radioactivity (X the column under the same conditions. x13 ‘251-labelled Apo-S radioactivity (0 0).

The parameters obtained after mathematical analysis of the HDL-Apo A-I and Apo S curves according to Matthews [22] are shown in Table II. FCR and mean transit time through the whole system of HDL Apo A-I are similar to those found in the literature [8,23,24]. The synthesis rate is also in the normal range. FCR of HDL-Apo S (0.403 + 0.067 pools/day) are significantly different from those observed for Apo A-I (0.250 + 0.013) but the mean transit times are almost identical (respectively 7.02 rt_0.31 and 7.10 f 0.38 days for Apo S and Apo A-I) (Table II). The synthesis rates of Apo S in these healthy subjects are about 200 times smaller

175

DAYS

0.1 0

2

6

6 DAYS

IO

12

14

16

Fig. 2. Radioactivity decay curves normalized to the lo-min point of ‘3’I-HDL-Apo A-I (0) and ‘X I-HDL-Apo S (0) in plasma. Mean ( f SD) decay curves of 4 healthy subjects were plotted as a fraction of initial radioactivity obtained ten minutes after injection.

than for Apo A-I, due to their differences of concentrations in the plasma. As we are in steady-state conditions, the very small pool of Apo S did not show significant modifications for the duration of the experiment. The mean U/P ratio (0.361 + 0.037) is close but different from the mean FCR calculated from the plasma curve (0.403 f 0.067). If the FCR calculated from the area under the plasma radioactivity curve and the U/P ratio do not agree, the catabolism does not occur from the plasma compartment alone or there is a polydisperse collection of particles. In that case, the FCR give a composite picture of an ‘average’ FCR. In fact, the acute biphasic shape of the U/P ratio curve (Fig. 2) suggests a kinetic heterogeneity in HDL-Apo A-I and Apo S. Model In comparison with the values obtained from the classical mathematical analysis of the curves according to Matthews, a better insight in the lipoprotein metabolism may be obtained from compartmental modeling (Fig. 3). The decaying curve of HDL-Apo S was 3-exponential and that of HDL-Apo A-I was 2-exponential but the terminal slopes of both HDL-Apo A-I and Apo S were parallel. As the exponents of the two slowest exponentials were close, we fit this part of the curves by two common exponentials. A best fit of the observed data was obtained when these

176 TABLE

II

Kinetic parameters curve fitting Subject

of HDL-Apo

A-I and S in healthy

HDL cone

subjects

derived

a

from multiexponential

FCR

T1/2 (days)

T (days)

computer

Production rate

(mg/dI)

(% pool/ day)

A-I S

138 0.72

0.247 0.351

5.66 5.66

7.10 7.06

14.1 0.104

A-I S

116 0.46

0.236 0.446

6.33 6.33

7.62 7.52

11.3 0.085

A-I S

125 0.51

0.247 0.325

5.47 5.47

6.72 6.74

12.8 0.068

A-I S

134 0.66

0.268 0.489

5.80 5.80

6.99 6.78

14.8 0.133

Means f SD A-I

128*10

0.25OkO.013

5.81 kO.37

7.10 + 0.38

13.2+ 1.5

0.403 f 0.067

5.81*0.37

7.02 * 0.31

S a Calculated

0.59*0.12 from the terminal

(mg/kg

slope of plasma

4 13

per day)

0.098 k 0.027

HDL decay curve

4 0

HDL

lodure ----

--Urine

Fig. 3. Multicompartmental model describing the HDL-Apo A-I (dotted lines) and Apo S (solid lines) metabolism in human subjects. Tracer inputs: * ‘3’I-Apo A-I (in 2 and 4), * * ‘251-Apo S (in 1 and 2). Triangles 13 and 8 are respective summers for plasma. 75% of Apo A-I (from Triangle 13) followed a common pathway with 40% (from Triangle 8) of Apo S. The remaining 25% and 60% of apoproteins A-l and S, respectively, have an independent fate. Compartments 1, 2, and 4 represent plasma pools, compartments 3 and 15 extravascular pools, and compartments 5, 6, and ‘I=.iodide pool. Triangles 14 and 9 are respective summers for daily urine data. This model accounts for the shape of U/P ratio curves. All catabolism is assumed to occur from plasma pools. Kinetic parameters derived from the model: mean f SD. The exchange rate constants (La,, and L,,,) and the catabolic rate constants (L,,, L 6.4 and L,,,) are in U/day. L,,, = 0.50+0.28; L,,, = 0.24*0.01; L,,, = 0.194kO.015; L,, =1.18* 0.57; L,., = 4.2 f 1.4.

177

exponentials were incorporated in a two-compartmental subsystem common to Apo A-I and Apo S. 75 _t 8% of Apo A-I particles decay by this slow route but only 40 f 3% of Apo S particles (pathway 2 to 5 of Fig. 3). The fast decay route accounts for 25 _+ 8% of Apo A-I (pathway 4 to 6) and is about 20 times slower than the first exponential of Apo S which accounts for 60 * 3% of the Apo S decay (pathway 1 to 7). The simultaneous fit of the Apo A-I and Apo S plasma radioactivity curves accounts for the daily excretion of radioactivity in urine, and thus for the cumulative urine activity and for the urine/plasma activity ratio. The parameters calculated using the model are given in the legend of Fig. 3. The mean residence times of the different particles through the system were respectively 7.65 days for the particle common to Apo A-I and Apo S (subsystem 2-3) 0.5 days for the particle specific to apo A-I (compartment 4) and 0.24 days for the particle specific to Apo S (compartment 1). Obviously these residence times are completely independent and differ from the T values given in Table II, the latter assuming an homogeneous behaviour of all particles. The FCR is the reciprocal of the T. Discussion Distribution of radioactivity in lipoprotein fractions The fact that the decay curve within the d 1.25 g/ml infranate parallels the plasma curve and the HDL curve suggested that the Apo A-I found in this fraction was dissociated during ultracentrifugation. This was confirmed by the observation that all Apo A-I plasma radioactivity was found in HDL zone after Superose column chromatography of total plasma without previous ultracentrifugation. The recovery of all radioactivity in HDL implies that the injected free Apo A-I is quickly and completely associated with this lipoprotein and followed the fate of HDL. Almost all Apo S (about 95%) was associated with HDL after chromatography and the effect of ultracentrifugation in releasing Apo S was similar to that found for Apo A-I. Kinetics At the beginning the Apo S decays much more rapidly than Apo A-I but the influence of the fast component is evidently short-lived as the mean residence times through the system calculated according to Rescigno [20] are the same for both apoproteins. Thus the presence of this fast component lasting about 24 h increases the initial slope but has not much effect on the analysis of the whole curve. The mean residence times for the HDL-Apo A-I and Apo S are identical (about 7 days) indicating a slow ‘global’ catabolism. The slow catabolism of HDL-Apo S was already suspected after i.v. [‘4C]leucine administration to a neurological patient with a high plasma level of Apo S. The disappearance of Apo S was parallel to that of Apo A-I and Apo A-II for the first 24 h [3]. The quick appearance of Apo S after gastro-intestinal surgery contrasted with the delay of disappearance of high plasma levels of Apo S even after resuming a completely normal diet [3]. The possibility of a persistent induction of Apo S

178

synthesis long after the surgical procedure seems less likely than a slow catabolism. We compared our results to those of Parks and Rude1 [9] involving the injection of labelled HDL-Apo SAA in monkeys, notwithstanding the difference in the duration of experiment. The decay curves followed over 6 days by these authors were biexponential with t,,, values of 0.39 days for the initial phase and 2.5 days for the second phase [9]. Using the same procedure to analyze our data for the same period of time we obtained, respectively, t,,, of 0.15 days and 3.25 days. Restricting oneself to these two components, HDL-Apo S seems to be metabolized more rapidly than HDL-Apo A-I. Extending the experiments to 15 days, it appears that the second exponential and a third slower exponential are common to HDL-Apo S and HDL-Apo A-I. The existence of these common exponentials suggests that lipoproteins containing Apo A-I and Apo S are catabolized as a whole particle. The kinetic differences can be explained by two populations of HDL-S particles with different fates. Simultaneous fit of plasma and urine data constraints us to discard the two-pool or three-pool model of Matthews. This is because the plasma compartment would actually contain two separate pools of HDL-Apo S instead of one. Therefore these pools could have different turnover rates. The same conclusion applies to HDL-Apo A-I as was already postulated by Zech et al [25]. Model Kinetics of heterogeneous particles requires a more complex model than the Matthews model. The model shown in Fig. 3 has several important implications. First it is consistent with the hypothesis that there is a part of the metabolic pathway common to HDL-Apo A-I and Apo S indicating that 40% of Apo S particles in HDL is removed at the same rate as 70% of Apo A-I particles. Second, a population of HDL-S (not linked to Apo A-I) is removed directly from the plasma and degraded about 20 times faster than the other one. In the development of the model the two moieties were necessary to account for the rate of appearance of iodine label in the urine as depicted in particular by the shape of the U/P ratio curve. The validity of the proposed model was confirmed by the results of affinity chromatography, separating 2 populations of HDL-S, one associated to Apo A-I, the other not containing Apo A-I. The relative proportions of the 2 types of particles (42.5 and 57.5%, respectively) correspond to those derived from the model (40 and 60%). In a study of induction of Apo SAA in mice by endotoxin, Hoffman and Benditt [26] found by immunoprecipitation an Apo SAA binding to a specific subpopulation of lipoprotein particles containing Apo A-I. They confirmed in mouse hepatocyte culture [27] that the formation of particles containing both Apo SAA and Apo A-I occurs subsequently to Apo SAA secretion. Conclusions This paper presenting the first kinetic study performed with Apo S in healthy subjects show the existence of different Apo S-containing HDL particles with different turnover rates. The particles containing also Apo A-I (proposed as LP-A-I : S) have a much slower turnover rate than the particles not linked to Apo

179

A-I (or LP-S). The heterogeneity justifies the building of a compartmental model fitting at best simultaneously the plasma and urine data. As in some pathological conditions the plasma concentration of Apo S may increase as much as 80 times the normal value, future studies will be focused especially on the distinct metabolic changes of each particle entity. These studies might bring practical informations about the regulation of Apo S-containing particles. Acknowledgements We wish to thank Professor J-C. Fruchart for apolipoprotein plasma. This work was supported by the Research Foundation Belgium.

S determination in on Atherosclerosis,

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180 16 McConathy WJ, Koren E, Wieland H, Campos EM, Lee DM, Kloer HU, Alaupovic P. Evaluation of immunoaffinity chromatography for isolating human lipoproteins containing apolipoprotein B. J Chromatogr 1985;342:47-66. 17 Dubois DY, Cantraine F, Malmendier CL. Comparison of different sandwich enzyme immunoassays for the quantitation of human apolipoproteins A-I and A-II. J Immunol Methods 1987;96:115-120. 18 Fruchart J-C, Fievet C, Puchois P. Apolipoproteins. In: Bergmeyer, ed. Methods of enzymatic analysis, Vol. VIII. Weinheim: Verlag Chemie, 1985;126-138. 19 Gesquiere JC, Delpierre CC, Tartar AL. Synthetic peptides as alternative antigens in the production of antibodies against human apolipoproteins. Clin Chem 1985:31:784-785. 20 Rescigno A, Gurpide E. Estimation of average times of residence, recycle and interconversion of blood-borne compounds using tracer methods. J Clin Endocrinol Metab 1973;36:263-276. 21 Berman M, Weiss MF. SAAM Manual. U.S. DHEW Publ. NIH No 75-180, 1978 22 Matthews CME. The theory of tracer experiments with ‘3’1-labelled plasma proteins. Phys Med Biol 1957;2:36-53. 23 Shepherd J, Packard CJ, Patsch JR, Gotto Jr AM, Taunton OD. Metabolism of apolipoproteins A-I and A-II and its influence on the high density lipoprotein subfraction distribution in males and females. Eur J Clin Invest 1978;8:115-120 24 Schaefer EJ, Zech LA, Jenkins LL, Bronzert TJ, Rubalcaba EA, Lindgren FT. Aamodt RL, Brewer Jr HB. Human apo~poprotein A-I and A-II metabolism. J Lipid Res 1982;23:850-862. 25 Zech LA, Schaefer ET, Bronzert TJ, Aamodt RL, Brewer Jr HB. Metabolism of human apolipoproteins A-I and A-II: compartmental models. J Lipid Res 1983;24:60-71. 26 Hoffman JS, Benditt EP. Changes in high density lipoprotein content following endotoxin administration in the mouse. Formation of serum amyloid protein-rich subfractions. J Biol Chem 1982;257:10510-10517. 27 Hoffman JS, Benditt EP. Secretion of serum amyloid protein and assembly of serum amyloid protein-rich high density lipoprotein in primary mouse hepatocyte culture. J Biol Chem 1982;257:10518-10522.