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Biotinyl-high-density lipoproteins as a probe for the determination of high-density lipoprotein turnover in humans
311
Biochimica et Biophysics Acta, 1043 (1990) 311-317 Elsevier
BBALIP 53379
Biotinyl-~gh-density lipoproteins as a probe for the d~ter~nation of high-density lipoprotein turnover in humans Wolfgang H. Daerr, Wolfgang Pethke, Eberhard T.E. Windler and Heiner Greten Medizinische Kern-und Poliklinik, Uniuersiiiitskrankenhnw Eppendorf Hamburg (F.R.G.) (Received 7 November 1989)
Key words: Lipoprotein metabolism; HDL; Methylavidin,
[‘4C]-; Biotinyl-HDL,; (Human plasma)
A simple and reliable method has been developed for the determination of high-density lipoprotein (HDL) turnover in humans. In this method, complex formation of [‘4C]methylavidin with biotinyl-HDL, and subsequent precipitation of excess [ “C]methyiavidin with biotinyl-silica-gel is utilized for the detection of as little as 0.1 pg of biotinyl-HDL, (by protein) /ml of serum with high precision and repr~ucibili~, at 7.9 f 0.9% (n = 7) of the peptidyl lysine m~ified. In serial dilutions and quadruplicate determinations the intra- and interassay variations were less than 4.7% and 5.2% respectively (n = 5).. Recovery of biotinyl-HDL, averaged 92 f 5.7% throughout the working range of the assay (n = 4). Variations in the levels of HDL or very-low-density lipoprotein (VLDL) did not interfere with the measurement of biotinyf-ALL in serum. Also, results were not affected by storage of serum samples at -80°C for up to 4 weeks. After reinjection of autologous biotinyl-HDL, (0.12 mg protein/kg body wt.) into five normolipidemic male volunteers, typical decay curves were obtained. The mean half-life of biotinyl-HDL, was 5.1 f 0.5 days, no different from that reported for radiolabeled HDL or radiolabeled HDL a~li~proteins, Routine immunobinding anafysis did not reveal formation of antibodies with specificity towards HDL 4 weeks after the reinjection studies. From this it appears that biotinyl-HDL, is a suitable probe and a safe and reliable alternative for the determination of HDL turnover in humans when application of radiolabeled HDL is not desirable.
Introduction Among the procedures that are currently used in the study of the kinetic behavior of lipoproteins, radiotracer methods are the most common. Thus, radioiodination [1,2] has proved to be a with [ ‘2sI]iodomon~hlo~de powerful tool for the investigation of the metabolism of lipoproteins in plasma under a wide variety of differing experimental conditions [3-91. In a similar manner, modification of the protein moiety of lipoproteins with Bolton-Hunter reagent [lo], by acylation with ‘H-labeled acetic anbydride [ll] or by reductive methylation of a limited number of lysine residues [12,13] has been successfully employed for the study of lipoprotein turnover in humans, as well as other species. However, although these methods all are highly reproducible, sensitive and precise, when applied to the study of the metabolism of lipoproteins in humans, they imply the need for in-
Correspondence: W.H. Universit~tskra~enhaus burg 20, F.R.G. US-2760/90/$03.50
Daerr, Medizinische Kern- und Poiiklinik, Eppendorf, Martinistrasse 52, 2000 Ham-
travenous injection of radiolabeled compounds and hence cannot be used in larger population studies without reservation. On the other hand, reasonably simple and reliable alternatives which obviate the need for application of radioactivity to humans are not readily available. Indeed, retinol has, for instance, been used to delineate differences in the metabolic pathways of the triacylglycerol-rich lipoproteins of intestinal and hepatic origin [14-161. However, newly’ absorbed retinol is specifically transported in association with chylo~crons and their remnants, and therefore is not suited to the study of the metabolism of lipoproteins in general. Recently, it has been possible to demonstrate lipoprotein-receptor interactions with biotinylated lowdensity lipoproteins in vitro [17,18]. The present study was initiated in order to determine whether limited biotinylation of lipoproteins could also prove suitable for the study of lipoprotein metabolism in vivo. Here we report on the results obtained with biotin-modified high-density lipoproteins in humans. This work was presented in part at the Annual Scientific Meeting of the European Society for Clinical Investigation, Athens, April 1989.
0 1990 Elsevier Science PubIishers B.V. (Biomedical Division)
312 Materials and Methods Blood was obtained from healthy volunteers fasted previously for 12-14 h and, after clotting at room temperature, was separated from blood cells by lowspeed centrifugation. High-density lipoproteins, (HDL,) were prepared by rate zonal ultracentrifugation in a Kontron TZT 48.65 rotor at 400~ rpm and 14” C for 20 h with a discontinuous NaBr-gradient between the densities 1.0 and 1.4 g/ml according to the method described by Patsch [19]. Isolated peak fractions of HDL, were concentrated by ultracentrifugation (Amicon-Diaflow 10 PM-30, Lexington, U.S.A.), dialysed against several changes of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.5 mM KH,PO,, 0.9 mM CaCl,, 0.5 mM M&l,) (pH 7.4) and sterilized by passage through a Millipore filter (0.22 pm). For modification of lysine residues of HDL, with biotinyl-~-hydroxysuccinimide ester (BHSE, E-Y Laboratories, San Mateo, U.S.A.), concentrated HDL, preparations were diluted to 1.0 mg of protein per ml with phosphate-buffered saline (pH 7.4) and BHSE in dimethyl sulfoxide (DMSO; Merck, Darmstadt, F.R.G.) was added to give a final concentration of 0.05 mg/ml [ZO]. After 2 h at room temperature biotinylated HDL, was separated from excess reagents by exhaustive dialysis and sterilized by millipore filtration (0.22 pm). 3Hlabeled biotinyl-HDL, was typically prepared as follows. 2.0 mg of HDL, (by protein) was incubated with 0.1 mg of (6-[8,9-3H]BHSE (Amersham, Bucks., U.K.; specific activity 15.47 TBq/mmol) in 2.0 ml of phosphate-buffered saline (pH 7.4) for 2 h at room temperature. The reaction mixture was then passed over a Sephadex G 25-M column (PD-10; Pharmacia, Uppsala, Sweden). samples that contained the radiolabeled HDL, combined, sterilized as described above and the specific activity was determined (Tri-Carb A 300 C, Packard, U.S.A.). All lipoprotein preparations were stored at 4°C and used within 12 h of preparation.
34.4-71.6 GBq/mmol) at - 80 o C until use.
and stored
in multiple
aliquots
0.8 ml-aliquots of serum were added to 0.1 ml of a solution containing 6 pg of [‘4C]methylavidin and 0.2% (w/v) human serum albumin in phosphate-buffered saline (pH 7.4). The tubes (40 X 10 mm, Eppendorf, Hamburg, F.R.G.) were placed on a blood cell suspension rotator (Mixer A 257; Denley, U.K.) to provide end-over-end rotation at 4’ C for 1 h, after which 0.4 ml of a thoroughly mixed suspension of biotinyl-silica-gel (Si-60; Serva, Heidelberg, F.R.G.) in phosphate-buffered saline (pH 7.4) adjusted to 0.35 g of dry gel/ml, was added to each tube and the incubation was continued at 4°C for a further 30 min. The silica-gel was then sedimented at 9000 x g (12000 rpm) for 10 min (Beckman Microfuge B) and 0.2 ml of the clear supernatant was removed for determination of radioactivity. To estimate recovery [ 3H]biotinyl-HDL, at concentrations between 0.1 and 3.0 pg/mI was incubated with serum for 3 h at 37 * C and the soluble 3H-radioactivity was measured before and after precipitation of excess [‘4C]methylavidin as detailed above. Blank incubations without biotinyl-HDL, were routinely conducted and the values were subtracted from each experimental result. In our hands the 14C-radioactivity of the blanks did not exceed 6% of the initial radioactivity per tube if the biotinylated silica-gel was washed three to five times with phosphate-buffered saline (pH 7.4) prior to assay. Determination of NDL half& Plasma was obtained from five normolipe~c male volunteers and the HDL, isolated. 9-12 mg (0.12 mg/kg of body wt) of biotin-labeled, pyrogen-free and sterile HDL, (by protein) were reinjected within 48 h of preparation and blood samples withdrawn at 1, 3, 6, 12 h and at 12-h intervals thereafter. After clotting and centrifugation the serum was stored at - 80 a C and analyzed for biotinyl-HDL, within 2 weeks. During the reinjection studies, all subjects were on a free diet. Written consent was obtained.
A uidin
Avidin (Serva, Heidelberg, F.R.G.) was labeled with 14C ([ “C]formaldehyde, New England Nuclear DuPont, Boston, U.S.A.) by reductive methylation as described by Dottario [21]. Unbound radioactivity was removed by chromatography (Sephadex G-25 M, PD-10) and the fractions which contained the [‘4C]methylavidin rechromatographed on the same column. Under these conditions the [‘4C]methylavidin was electrophoresed as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels [23] and greater than 96% of the radioactivity comigrated with unlabeled avidin. The purified [‘4C]methylavidin was diluted with unlabeled avidin to provide a mean specific activity of 50 GBq/mmol (range
To analyze serum for the presence of antibodies directed against biotinyl-HDL,, biotinyl-HDL, was electrophoresed in 2-15% SDS polyacrylamide gradient slab gels and the apolipoproteins identified by an immunochemical blot transfer technique using polyclonal anti-HDL antibodies (Immuno, Vienna, Austria) and horseradish peroxidase-conjugated second antibody (goat-antirabbit IgG; Sigma, St. Louis, U.S.A.) as previously described [23]. Parallel incubations of nitrocellulose blots with dilutions of serum between 1 : 2 and 1: 100 (v/v) from each of the five subjects were analyzed with a peroxidase-conjugated anti-human IgG antibody
313 (goat-anti-human IgG; Sigma, St. Louis, U.S.A.). Blots of standard proteins (SDS-PAGE Molecular Weight Standard, high; Bio-Rad Laboratories, Richmond, U.S.A.) were stained with 0.1% (w/v) Amido Black B 1241. Total cholesterol and triacylglycerol Total cholesterol and triacylglycerol were measured enzymatically [25]. HDL-cholesterol was estimated after precipitation of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) with phosphotungstate and Mg2+ [26]. Protein was determined according to Lowry et al. [27]. c-Amino groups of lysine residues of HDL, were quantitated before and after exposure to BHSE according to the method of Habeeb [28]. Percentage biotinylation was calculated by difference. Biotinyl-HDL, residence time was calculated from standard log-linear blots using linear regression analysis as described by Jones [29]. Results Biotinyl-HDL, Incubation of a constant amount of HDL, with increasing amounts of BHSE produced a linear increase in the percentage biotinylation of the r-amino group of HDL, lysine residues. With several different HDL, preparations, the extent of lysine modification was 7.9 -t 0.9% (n = 7) at a final concentration of BHSE of 0.05 mg/ml. Higher degrees of biotinylation, with 24% and 50% of the amino groups modified, were obtained upon incubation of HDL, with BHSE at 0.15 mg/ml and 0.30 mg/ml, respectively. However, it has been shown previously that HDL, with less than 12% of its lysine residues modified is indistinguishable from native HDL, in terms of chemical composition, hydrated density, relative molecular weight, electrophoretic mobility in agarose-gel and structure [30]. Therefore, all further experiments were carried out using HDL, with the former low degree of biotinylation. Determination of biotinyl-HDL, in serum Biotinyl-HDL, was determined in serum by complex formation with [‘4C]methylavidin followed by precipitation of excess reactant with biotinylated silica-gel as outlined in Materials and Methods. Fig. 1 shows that the relation between complex formation and amount of biotinyl-HDL, was linear up to a concentration of approx. 3.0 pg/ml when increasing quantities of biotinylHDL, were added to serum and as little as 0.1 pg/ml could be reliably detected. The intraassay coefficient of variation ranged from 4.7% in the region below 0.5 pg/ml to 2.5% at higher concentrations. In seven experiments the interassay variation was 5.2%. Since lipoproteins can absorb to non-specific surfaces [31], the loss of biotinyl-HDL, during incubation with
0.1 0.5
1
2
3
[3H-lBIOTINVLHOL I,ugPROTEIN/mlSERUM1 Fig. 1. Determination of [3H]biotinyl-HDLs in serum. Indicated amounts of [3H]biotinyl-HDL, were added to normal serum and the 3H-activity measured (m). Aliquots from each sample were then incubated with a constant amount (6 pg) of [“‘C]methylavidin (specific activity 34.4 GBq/mmol) and the ‘H (0) and 14C activity (o), determined after precipitation of the surplus [14C]methylavidin with biotinyl-silica-gel as described in Materials and Methods. Each data point represents the mean f 1 SD. of triplicate determinations from two different experiments.
biotinylated silica-gel was also estimated. Comparison of the values of [ 3Hjbiotinyl-HDL3 determined in serum with those after complex formation with avidin and addition of silica-gel showed that recovery of biotinylHDL, averaged 92 + 5.7% (n = 4) throughout the working range of the assay (Fig. 1).
Effects of freezing and variations in lipoprotein composition on the determination of biotinyl-HDL, To ensure that the values of biotinyl-HDL, were not affected by the conditions of storage, biotinyl-HDL, was initially measured in fresh serum. Nearly identical curves were obtained for the same dilutions of biotinylHDL, in serum stored frozen at - 80 o C for up to 4 weeks prior to being assayed (Fig. 2). Similarly, curves made from dilutions of biotinyl-HDL, in sera which contained varying amounts of VLDL triacylglycerol (between 60 mg/ml and 220 mg/dl) but a constant amount of HDL cholesterol (42 mg/dl) or vice versa (between 24 mg/dl and 68 mg/dl HDL-cholesterol, 120 mg/dl VLDL triacylglycerol) were indistinguishable (data not shown). These results indicate that neither freezing of serum and storage at - 80 o C nor variations in the levels of HDL or VLDL interfere with the measurement of biotinyl-HDL, in serum.
314
;
Exponential
;
(slope
5. c
‘;
1
I
= b, 1 =062days
.i i_
WD HDL-CHOL=
i
35mg
dl-’
c
;
a.1
L
BIOTINVL-HDL [pg PROlElN/mlSERUM]
50
Fig. 2. Effect of freezing and storage on the determination of biotinylHDL, in serum. Biotinyl-HDL, was added to normal serum and determined by complex formation with [‘4C]methylavidin (specific activity 71.6 GBq/mmol) before (0) and after (0) freezing and storage at - 80 o C for 4 weeks as outlined in Materials and Methods. The results of one of three experiments are shown. Each data point is the mean k 1 S.D. of quadruplicate determinations.
L
J
I
100 150 200 TIME (hours1
250
Fig. 3. Typical biotinyl-HDL, plasma decay cmve with exponentials and y-axis intercepts in a normolipidemic male. Each data point is the mean * 1 S.D. of quadruplicate determinations.
travascular and extravascular compartments and the terminal linear component (r, - 0.95 to - 0.99) reflecting the metabolic clearance of biotinyl-HDL, from the body [32]. From the curves it can be seen that equilibration was complete by day 2 of the studies. At this time, 74 f 5% of the biotin label was found in the intravascular compartment. The mean half-life of biotinyl-HDL, was 5.1 rt 0.5 days. There was no correlation between the level of HDL-cholesterol and the half-life of biotinyl-HDL,.
Metabolism of biotinyl-HDL, in normal adult males After reinjection of autologous biotinyl-HDL, into normolipemic male volunteers typical decay curves were obtained. Figs. 3 and 4 show that the time-course of biotinyl-HDL, clearance from the plasma in each of the five subjects could be resolved into two exponential functions, the first and faster component relating to the between the inrate of equilibration of biotinyl-HDL,
1.0
1.0
0.5
WP
HDL Chol
’
t112
58mgidl
L8days
01
1.0
0.5
HDL Chol 70mgldl
150
TIME
Fig. 4. Plasma decay curves of biotinyl-HDL,
200
(hours)
in normolipemic
..-~,I
0.1
--4
100
250
50
,1
.~_~~
100 TIME
150
200
250
Ihoursl
males. Each data point is the mean + 1 S.D. of quadruplicate
determinations.
315
S
P
12345
Fig. 5. Immunobinding assay for IgG antibodies with specificity towards biotinyl-HDL,. Biotinyl-HDL, (50 pg of protein/lane) was electrophoresed in 2-15% polyacrylamide gradient slab gels containing 0.1% SDS. The proteins were transferred to nitrocellulose membranes and incubated with a 1: 100 dilution of anti human HDL rabbit serum (lane P) or with 1: 2 dilutions of human sera obtained 4 weeks after the reinjection studies (lanes l-5). Following the first incubation, the blots were then incubated with peroxidase-conjugated goat-anti-rabbit IgG (lane P) or goat-anti-human IgG (lanes l-5) and developed with diaminobenzidine. The molecular weight markers were fi-galactosidase, M, 116000; phosphorylase b, M, 97400; bovine serum albumin, MC 66000; ovalbumin, M, 45000 and carbonic anhydrase, M, 29000 (lane S).
Immunobinding
analysis
Because of the importance of recognizing the occurrence of antibodies directed against biotinyl-HDL, serum from each of the five subjects was analyzed with an immunoblot test for detection of IgG antibodies with specificity towards biotinyl-HDL, 4 weeks after the reinjection studies. Fig. 5 shows that polyspecific antiserum directed to human HDL reacted with three different bands, of 28, 42 and 90 kDa, on the protein blots of biotinyl-HDL,, corresponding to apolipoproteins A-I, A-IV and A-I multimer, respectively (lanes S and P). In contrast, none of the sera from the five subjects was reactive with biotinyl-HDL, apolipoproteins at dilutions between 1 : 2 and 1 : 100 (lanes l-5). From this it appears that antibodies specific to HDL are not formed in humans upon single exposure to autologous biotinyl-HDL,. Discussion In the present investigation, we set out to develop a novel tracer method for the determination of the kinetic
behavior of HDL that could be readily used in clinical, metabolic and epidemiologic studies but would avoid the intravenous application of radioactivity to humans. Taking advantage of biotins ability to bind to avidin with high affinity and specificity [33], the strategy eventually adopted was to isolate HDL, from serum, react the c-amino group of HDL, lysine residues with biotinyl-N-hydroxysuccinimide ester, reinject the modified autologous HDL, and then identify the biotin label in serum samples by complex formation with [r4C]methylavidin. This approach appeared feasible for several reasons. First, the major apolipoprotein constituents of HDL,, apolipoproteins A-I and A-II, which comprise greater than 90% of the total HDl, protein, are readily exchangeable among the different HDL subfractions [34-351. Therefore, it is reasonable to assume that the decay of the biotin label after injection of biotinyl-HDL, would adequately describe the decay of the total HDL in plasma. Second, controlled attachment of biotinylN-hydroxysuccinimide ester to lysine residues can be achieved with great ease with no apparent effect on the structure of HDL, [30]. Third, detection of the biotin label in serum does not rely primarily on the extent of biotinylation of HDL,. Instead, by using highly labeled avidin it is possible to keep the number of biotin-derivatized lysine residues small with no loss of sensitivity of detection. Finally, and most importantly, modification of the r-amino group of lysine residues does not abolish the ability of HDL, to bind to specific cell surface receptors [36,37] and, hence, limited biotinylation of HDL, lysine residues would not be expected to interfere with the receptor-mediated clearance of HDL, from the plasma. However, the successful implementation of the biotin-avidin system as a tool for the determination of the plasma residence time of HDL obviously required a method that would allow the [‘4C]methylavidin-biotinyl-HDL, complexes to be reliably separated from excess [r4C]methylavidin. Ideally, this method should be simple and rapid and permit simultaneous processing of a large number of samples. In these respects, the method described in the present paper appears to be particularly advantageous. Using biotinyl-silica-gel to precipitate free [i4C]methylavidin, separation of the surplus [14C]methylavidin from biotinyl-HDL,-bound [14C] methylavidin in serum was achieved with high precision and reproducibility with a minimum of time and labor. Co-precipitation of biotinyl-HDL, was negligible and there was no indication for binding of [‘4C]methylavidin to serum constituents unrelated to its biotinbinding properties [38]. However, even though these results were obtained with sera of widely differing lipoprotein compositions, unspecific precipitation of biotinyl-HDL, in sera with grossly abnormal lipoprotein concentrations cannot be entirely ruled out. For this reason, the specificity of the precipitation procedure
316 need be confirmed when applying the biotin-a~din system to the study of the turnover of HDL under these pathologic conditions. When injected intravenoudy, the kinetic behavior of autologous biotinyl-HDL, was in general agreement with the published data for radiolabeled HDL [39-411 or radiolabeled HDL apolipoproteins [42-443. As observed by others, we also found biphasic HDL decay. After an initial rapid disappearance phase (t,,,2, 0.62 days) the decay of the biotin label was first-order over the time intervals studied and with 5.1 + 0.5 days in our group of healthy male subjects the mean half-life for irreversible degradation of biotinyl-HDL~ was not different from that reported for radiolabeled HDL. Likewise, our estimate of the size of the equilibrating extravascular pool, 26% of that of the intravascular pool, compared favorably with that of similar experiments using radiolabeled HDL. These results clearly show that biotin modification of a limited number of HDL, lysine residues does not affect the rate of removal of HDL from the plasma. They are consistent with the notion of a lysine-independent cellular recognition and uptake system for the bulk of human HDL [36,373. An issue of critical importance for the use of biotinylated HDL, in humans concerns the potential immunogenicity of chemically derivatized lipoproteins. That modifications as subtle as, for instance, methylation of the e-amino group of lysine residues of homologous LDL can render these lipoproteins immunogenic has recently been demonstrated [45]. Therefore, the possiblity that addition of biotin would also lead to the appearance of specific antibodies directed against HDL, had to be seriously considered. However, analysis of the sera from our subjects 4 weeks after the reinjection studies failed to reveal the presence of antibodies with specificity towards biotinyl-HDL~, suggesting that autologous biotinyl-HDL, does not provoke an immune response in humans. This lack of immunogenicity of biotinyl-HDL, may be related to the low degree of modification achievd in the present study. Alternatively, it may be due to differences in the presentation of biotin on the surface of HDL, compared with LDL. On the other hand, single exposure to a weak antigenic stimulus may not elicit an antibody response sufficiently strong as to permit it to be detected even by a method as sensitive as immunoblotting. Therefore, the formation of antibodies against biotinyl-HDL~ cannot be unambiguously excluded and hence, repeated injections of biotinyl-HDL, into humans must be avoided. In summary, taking this latter restriction into account, our results show that biotinyl-HDL, is a reliable and safe probe for the study of HDL turnover in humans. It is anticipated that it will prove to be expedient whenever application of radiolabeled HDL is undesirable.
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Biochimica et Biophysics Acta, 1043 (1990) 311-317 Elsevier
BBALIP 53379
Biotinyl-~gh-density lipoproteins as a probe for the d~ter~nation of high-density lipoprotein turnover in humans Wolfgang H. Daerr, Wolfgang Pethke, Eberhard T.E. Windler and Heiner Greten Medizinische Kern-und Poliklinik, Uniuersiiiitskrankenhnw Eppendorf Hamburg (F.R.G.) (Received 7 November 1989)
Key words: Lipoprotein metabolism; HDL; Methylavidin,
[‘4C]-; Biotinyl-HDL,; (Human plasma)
A simple and reliable method has been developed for the determination of high-density lipoprotein (HDL) turnover in humans. In this method, complex formation of [‘4C]methylavidin with biotinyl-HDL, and subsequent precipitation of excess [ “C]methyiavidin with biotinyl-silica-gel is utilized for the detection of as little as 0.1 pg of biotinyl-HDL, (by protein) /ml of serum with high precision and repr~ucibili~, at 7.9 f 0.9% (n = 7) of the peptidyl lysine m~ified. In serial dilutions and quadruplicate determinations the intra- and interassay variations were less than 4.7% and 5.2% respectively (n = 5).. Recovery of biotinyl-HDL, averaged 92 f 5.7% throughout the working range of the assay (n = 4). Variations in the levels of HDL or very-low-density lipoprotein (VLDL) did not interfere with the measurement of biotinyf-ALL in serum. Also, results were not affected by storage of serum samples at -80°C for up to 4 weeks. After reinjection of autologous biotinyl-HDL, (0.12 mg protein/kg body wt.) into five normolipidemic male volunteers, typical decay curves were obtained. The mean half-life of biotinyl-HDL, was 5.1 f 0.5 days, no different from that reported for radiolabeled HDL or radiolabeled HDL a~li~proteins, Routine immunobinding anafysis did not reveal formation of antibodies with specificity towards HDL 4 weeks after the reinjection studies. From this it appears that biotinyl-HDL, is a suitable probe and a safe and reliable alternative for the determination of HDL turnover in humans when application of radiolabeled HDL is not desirable.
Introduction Among the procedures that are currently used in the study of the kinetic behavior of lipoproteins, radiotracer methods are the most common. Thus, radioiodination [1,2] has proved to be a with [ ‘2sI]iodomon~hlo~de powerful tool for the investigation of the metabolism of lipoproteins in plasma under a wide variety of differing experimental conditions [3-91. In a similar manner, modification of the protein moiety of lipoproteins with Bolton-Hunter reagent [lo], by acylation with ‘H-labeled acetic anbydride [ll] or by reductive methylation of a limited number of lysine residues [12,13] has been successfully employed for the study of lipoprotein turnover in humans, as well as other species. However, although these methods all are highly reproducible, sensitive and precise, when applied to the study of the metabolism of lipoproteins in humans, they imply the need for in-
Correspondence: W.H. Universit~tskra~enhaus burg 20, F.R.G. US-2760/90/$03.50
Daerr, Medizinische Kern- und Poiiklinik, Eppendorf, Martinistrasse 52, 2000 Ham-
travenous injection of radiolabeled compounds and hence cannot be used in larger population studies without reservation. On the other hand, reasonably simple and reliable alternatives which obviate the need for application of radioactivity to humans are not readily available. Indeed, retinol has, for instance, been used to delineate differences in the metabolic pathways of the triacylglycerol-rich lipoproteins of intestinal and hepatic origin [14-161. However, newly’ absorbed retinol is specifically transported in association with chylo~crons and their remnants, and therefore is not suited to the study of the metabolism of lipoproteins in general. Recently, it has been possible to demonstrate lipoprotein-receptor interactions with biotinylated lowdensity lipoproteins in vitro [17,18]. The present study was initiated in order to determine whether limited biotinylation of lipoproteins could also prove suitable for the study of lipoprotein metabolism in vivo. Here we report on the results obtained with biotin-modified high-density lipoproteins in humans. This work was presented in part at the Annual Scientific Meeting of the European Society for Clinical Investigation, Athens, April 1989.
0 1990 Elsevier Science PubIishers B.V. (Biomedical Division)
312 Materials and Methods Blood was obtained from healthy volunteers fasted previously for 12-14 h and, after clotting at room temperature, was separated from blood cells by lowspeed centrifugation. High-density lipoproteins, (HDL,) were prepared by rate zonal ultracentrifugation in a Kontron TZT 48.65 rotor at 400~ rpm and 14” C for 20 h with a discontinuous NaBr-gradient between the densities 1.0 and 1.4 g/ml according to the method described by Patsch [19]. Isolated peak fractions of HDL, were concentrated by ultracentrifugation (Amicon-Diaflow 10 PM-30, Lexington, U.S.A.), dialysed against several changes of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.5 mM KH,PO,, 0.9 mM CaCl,, 0.5 mM M&l,) (pH 7.4) and sterilized by passage through a Millipore filter (0.22 pm). For modification of lysine residues of HDL, with biotinyl-~-hydroxysuccinimide ester (BHSE, E-Y Laboratories, San Mateo, U.S.A.), concentrated HDL, preparations were diluted to 1.0 mg of protein per ml with phosphate-buffered saline (pH 7.4) and BHSE in dimethyl sulfoxide (DMSO; Merck, Darmstadt, F.R.G.) was added to give a final concentration of 0.05 mg/ml [ZO]. After 2 h at room temperature biotinylated HDL, was separated from excess reagents by exhaustive dialysis and sterilized by millipore filtration (0.22 pm). 3Hlabeled biotinyl-HDL, was typically prepared as follows. 2.0 mg of HDL, (by protein) was incubated with 0.1 mg of (6-[8,9-3H]BHSE (Amersham, Bucks., U.K.; specific activity 15.47 TBq/mmol) in 2.0 ml of phosphate-buffered saline (pH 7.4) for 2 h at room temperature. The reaction mixture was then passed over a Sephadex G 25-M column (PD-10; Pharmacia, Uppsala, Sweden). samples that contained the radiolabeled HDL, combined, sterilized as described above and the specific activity was determined (Tri-Carb A 300 C, Packard, U.S.A.). All lipoprotein preparations were stored at 4°C and used within 12 h of preparation.
34.4-71.6 GBq/mmol) at - 80 o C until use.
and stored
in multiple
aliquots
0.8 ml-aliquots of serum were added to 0.1 ml of a solution containing 6 pg of [‘4C]methylavidin and 0.2% (w/v) human serum albumin in phosphate-buffered saline (pH 7.4). The tubes (40 X 10 mm, Eppendorf, Hamburg, F.R.G.) were placed on a blood cell suspension rotator (Mixer A 257; Denley, U.K.) to provide end-over-end rotation at 4’ C for 1 h, after which 0.4 ml of a thoroughly mixed suspension of biotinyl-silica-gel (Si-60; Serva, Heidelberg, F.R.G.) in phosphate-buffered saline (pH 7.4) adjusted to 0.35 g of dry gel/ml, was added to each tube and the incubation was continued at 4°C for a further 30 min. The silica-gel was then sedimented at 9000 x g (12000 rpm) for 10 min (Beckman Microfuge B) and 0.2 ml of the clear supernatant was removed for determination of radioactivity. To estimate recovery [ 3H]biotinyl-HDL, at concentrations between 0.1 and 3.0 pg/mI was incubated with serum for 3 h at 37 * C and the soluble 3H-radioactivity was measured before and after precipitation of excess [‘4C]methylavidin as detailed above. Blank incubations without biotinyl-HDL, were routinely conducted and the values were subtracted from each experimental result. In our hands the 14C-radioactivity of the blanks did not exceed 6% of the initial radioactivity per tube if the biotinylated silica-gel was washed three to five times with phosphate-buffered saline (pH 7.4) prior to assay. Determination of NDL half& Plasma was obtained from five normolipe~c male volunteers and the HDL, isolated. 9-12 mg (0.12 mg/kg of body wt) of biotin-labeled, pyrogen-free and sterile HDL, (by protein) were reinjected within 48 h of preparation and blood samples withdrawn at 1, 3, 6, 12 h and at 12-h intervals thereafter. After clotting and centrifugation the serum was stored at - 80 a C and analyzed for biotinyl-HDL, within 2 weeks. During the reinjection studies, all subjects were on a free diet. Written consent was obtained.
A uidin
Avidin (Serva, Heidelberg, F.R.G.) was labeled with 14C ([ “C]formaldehyde, New England Nuclear DuPont, Boston, U.S.A.) by reductive methylation as described by Dottario [21]. Unbound radioactivity was removed by chromatography (Sephadex G-25 M, PD-10) and the fractions which contained the [‘4C]methylavidin rechromatographed on the same column. Under these conditions the [‘4C]methylavidin was electrophoresed as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels [23] and greater than 96% of the radioactivity comigrated with unlabeled avidin. The purified [‘4C]methylavidin was diluted with unlabeled avidin to provide a mean specific activity of 50 GBq/mmol (range
To analyze serum for the presence of antibodies directed against biotinyl-HDL,, biotinyl-HDL, was electrophoresed in 2-15% SDS polyacrylamide gradient slab gels and the apolipoproteins identified by an immunochemical blot transfer technique using polyclonal anti-HDL antibodies (Immuno, Vienna, Austria) and horseradish peroxidase-conjugated second antibody (goat-antirabbit IgG; Sigma, St. Louis, U.S.A.) as previously described [23]. Parallel incubations of nitrocellulose blots with dilutions of serum between 1 : 2 and 1: 100 (v/v) from each of the five subjects were analyzed with a peroxidase-conjugated anti-human IgG antibody
313 (goat-anti-human IgG; Sigma, St. Louis, U.S.A.). Blots of standard proteins (SDS-PAGE Molecular Weight Standard, high; Bio-Rad Laboratories, Richmond, U.S.A.) were stained with 0.1% (w/v) Amido Black B 1241. Total cholesterol and triacylglycerol Total cholesterol and triacylglycerol were measured enzymatically [25]. HDL-cholesterol was estimated after precipitation of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) with phosphotungstate and Mg2+ [26]. Protein was determined according to Lowry et al. [27]. c-Amino groups of lysine residues of HDL, were quantitated before and after exposure to BHSE according to the method of Habeeb [28]. Percentage biotinylation was calculated by difference. Biotinyl-HDL, residence time was calculated from standard log-linear blots using linear regression analysis as described by Jones [29]. Results Biotinyl-HDL, Incubation of a constant amount of HDL, with increasing amounts of BHSE produced a linear increase in the percentage biotinylation of the r-amino group of HDL, lysine residues. With several different HDL, preparations, the extent of lysine modification was 7.9 -t 0.9% (n = 7) at a final concentration of BHSE of 0.05 mg/ml. Higher degrees of biotinylation, with 24% and 50% of the amino groups modified, were obtained upon incubation of HDL, with BHSE at 0.15 mg/ml and 0.30 mg/ml, respectively. However, it has been shown previously that HDL, with less than 12% of its lysine residues modified is indistinguishable from native HDL, in terms of chemical composition, hydrated density, relative molecular weight, electrophoretic mobility in agarose-gel and structure [30]. Therefore, all further experiments were carried out using HDL, with the former low degree of biotinylation. Determination of biotinyl-HDL, in serum Biotinyl-HDL, was determined in serum by complex formation with [‘4C]methylavidin followed by precipitation of excess reactant with biotinylated silica-gel as outlined in Materials and Methods. Fig. 1 shows that the relation between complex formation and amount of biotinyl-HDL, was linear up to a concentration of approx. 3.0 pg/ml when increasing quantities of biotinylHDL, were added to serum and as little as 0.1 pg/ml could be reliably detected. The intraassay coefficient of variation ranged from 4.7% in the region below 0.5 pg/ml to 2.5% at higher concentrations. In seven experiments the interassay variation was 5.2%. Since lipoproteins can absorb to non-specific surfaces [31], the loss of biotinyl-HDL, during incubation with
0.1 0.5
1
2
3
[3H-lBIOTINVLHOL I,ugPROTEIN/mlSERUM1 Fig. 1. Determination of [3H]biotinyl-HDLs in serum. Indicated amounts of [3H]biotinyl-HDL, were added to normal serum and the 3H-activity measured (m). Aliquots from each sample were then incubated with a constant amount (6 pg) of [“‘C]methylavidin (specific activity 34.4 GBq/mmol) and the ‘H (0) and 14C activity (o), determined after precipitation of the surplus [14C]methylavidin with biotinyl-silica-gel as described in Materials and Methods. Each data point represents the mean f 1 SD. of triplicate determinations from two different experiments.
biotinylated silica-gel was also estimated. Comparison of the values of [ 3Hjbiotinyl-HDL3 determined in serum with those after complex formation with avidin and addition of silica-gel showed that recovery of biotinylHDL, averaged 92 + 5.7% (n = 4) throughout the working range of the assay (Fig. 1).
Effects of freezing and variations in lipoprotein composition on the determination of biotinyl-HDL, To ensure that the values of biotinyl-HDL, were not affected by the conditions of storage, biotinyl-HDL, was initially measured in fresh serum. Nearly identical curves were obtained for the same dilutions of biotinylHDL, in serum stored frozen at - 80 o C for up to 4 weeks prior to being assayed (Fig. 2). Similarly, curves made from dilutions of biotinyl-HDL, in sera which contained varying amounts of VLDL triacylglycerol (between 60 mg/ml and 220 mg/dl) but a constant amount of HDL cholesterol (42 mg/dl) or vice versa (between 24 mg/dl and 68 mg/dl HDL-cholesterol, 120 mg/dl VLDL triacylglycerol) were indistinguishable (data not shown). These results indicate that neither freezing of serum and storage at - 80 o C nor variations in the levels of HDL or VLDL interfere with the measurement of biotinyl-HDL, in serum.
314
;
Exponential
;
(slope
5. c
‘;
1
I
= b, 1 =062days
.i i_
WD HDL-CHOL=
i
35mg
dl-’
c
;
a.1
L
BIOTINVL-HDL [pg PROlElN/mlSERUM]
50
Fig. 2. Effect of freezing and storage on the determination of biotinylHDL, in serum. Biotinyl-HDL, was added to normal serum and determined by complex formation with [‘4C]methylavidin (specific activity 71.6 GBq/mmol) before (0) and after (0) freezing and storage at - 80 o C for 4 weeks as outlined in Materials and Methods. The results of one of three experiments are shown. Each data point is the mean k 1 S.D. of quadruplicate determinations.
L
J
I
100 150 200 TIME (hours1
250
Fig. 3. Typical biotinyl-HDL, plasma decay cmve with exponentials and y-axis intercepts in a normolipidemic male. Each data point is the mean * 1 S.D. of quadruplicate determinations.
travascular and extravascular compartments and the terminal linear component (r, - 0.95 to - 0.99) reflecting the metabolic clearance of biotinyl-HDL, from the body [32]. From the curves it can be seen that equilibration was complete by day 2 of the studies. At this time, 74 f 5% of the biotin label was found in the intravascular compartment. The mean half-life of biotinyl-HDL, was 5.1 rt 0.5 days. There was no correlation between the level of HDL-cholesterol and the half-life of biotinyl-HDL,.
Metabolism of biotinyl-HDL, in normal adult males After reinjection of autologous biotinyl-HDL, into normolipemic male volunteers typical decay curves were obtained. Figs. 3 and 4 show that the time-course of biotinyl-HDL, clearance from the plasma in each of the five subjects could be resolved into two exponential functions, the first and faster component relating to the between the inrate of equilibration of biotinyl-HDL,
1.0
1.0
0.5
WP
HDL Chol
’
t112
58mgidl
L8days
01
1.0
0.5
HDL Chol 70mgldl
150
TIME
Fig. 4. Plasma decay curves of biotinyl-HDL,
200
(hours)
in normolipemic
..-~,I
0.1
--4
100
250
50
,1
.~_~~
100 TIME
150
200
250
Ihoursl
males. Each data point is the mean + 1 S.D. of quadruplicate
determinations.
315
S
P
12345
Fig. 5. Immunobinding assay for IgG antibodies with specificity towards biotinyl-HDL,. Biotinyl-HDL, (50 pg of protein/lane) was electrophoresed in 2-15% polyacrylamide gradient slab gels containing 0.1% SDS. The proteins were transferred to nitrocellulose membranes and incubated with a 1: 100 dilution of anti human HDL rabbit serum (lane P) or with 1: 2 dilutions of human sera obtained 4 weeks after the reinjection studies (lanes l-5). Following the first incubation, the blots were then incubated with peroxidase-conjugated goat-anti-rabbit IgG (lane P) or goat-anti-human IgG (lanes l-5) and developed with diaminobenzidine. The molecular weight markers were fi-galactosidase, M, 116000; phosphorylase b, M, 97400; bovine serum albumin, MC 66000; ovalbumin, M, 45000 and carbonic anhydrase, M, 29000 (lane S).
Immunobinding
analysis
Because of the importance of recognizing the occurrence of antibodies directed against biotinyl-HDL, serum from each of the five subjects was analyzed with an immunoblot test for detection of IgG antibodies with specificity towards biotinyl-HDL, 4 weeks after the reinjection studies. Fig. 5 shows that polyspecific antiserum directed to human HDL reacted with three different bands, of 28, 42 and 90 kDa, on the protein blots of biotinyl-HDL,, corresponding to apolipoproteins A-I, A-IV and A-I multimer, respectively (lanes S and P). In contrast, none of the sera from the five subjects was reactive with biotinyl-HDL, apolipoproteins at dilutions between 1 : 2 and 1 : 100 (lanes l-5). From this it appears that antibodies specific to HDL are not formed in humans upon single exposure to autologous biotinyl-HDL,. Discussion In the present investigation, we set out to develop a novel tracer method for the determination of the kinetic
behavior of HDL that could be readily used in clinical, metabolic and epidemiologic studies but would avoid the intravenous application of radioactivity to humans. Taking advantage of biotins ability to bind to avidin with high affinity and specificity [33], the strategy eventually adopted was to isolate HDL, from serum, react the c-amino group of HDL, lysine residues with biotinyl-N-hydroxysuccinimide ester, reinject the modified autologous HDL, and then identify the biotin label in serum samples by complex formation with [r4C]methylavidin. This approach appeared feasible for several reasons. First, the major apolipoprotein constituents of HDL,, apolipoproteins A-I and A-II, which comprise greater than 90% of the total HDl, protein, are readily exchangeable among the different HDL subfractions [34-351. Therefore, it is reasonable to assume that the decay of the biotin label after injection of biotinyl-HDL, would adequately describe the decay of the total HDL in plasma. Second, controlled attachment of biotinylN-hydroxysuccinimide ester to lysine residues can be achieved with great ease with no apparent effect on the structure of HDL, [30]. Third, detection of the biotin label in serum does not rely primarily on the extent of biotinylation of HDL,. Instead, by using highly labeled avidin it is possible to keep the number of biotin-derivatized lysine residues small with no loss of sensitivity of detection. Finally, and most importantly, modification of the r-amino group of lysine residues does not abolish the ability of HDL, to bind to specific cell surface receptors [36,37] and, hence, limited biotinylation of HDL, lysine residues would not be expected to interfere with the receptor-mediated clearance of HDL, from the plasma. However, the successful implementation of the biotin-avidin system as a tool for the determination of the plasma residence time of HDL obviously required a method that would allow the [‘4C]methylavidin-biotinyl-HDL, complexes to be reliably separated from excess [r4C]methylavidin. Ideally, this method should be simple and rapid and permit simultaneous processing of a large number of samples. In these respects, the method described in the present paper appears to be particularly advantageous. Using biotinyl-silica-gel to precipitate free [i4C]methylavidin, separation of the surplus [14C]methylavidin from biotinyl-HDL,-bound [14C] methylavidin in serum was achieved with high precision and reproducibility with a minimum of time and labor. Co-precipitation of biotinyl-HDL, was negligible and there was no indication for binding of [‘4C]methylavidin to serum constituents unrelated to its biotinbinding properties [38]. However, even though these results were obtained with sera of widely differing lipoprotein compositions, unspecific precipitation of biotinyl-HDL, in sera with grossly abnormal lipoprotein concentrations cannot be entirely ruled out. For this reason, the specificity of the precipitation procedure
316 need be confirmed when applying the biotin-a~din system to the study of the turnover of HDL under these pathologic conditions. When injected intravenoudy, the kinetic behavior of autologous biotinyl-HDL, was in general agreement with the published data for radiolabeled HDL [39-411 or radiolabeled HDL apolipoproteins [42-443. As observed by others, we also found biphasic HDL decay. After an initial rapid disappearance phase (t,,,2, 0.62 days) the decay of the biotin label was first-order over the time intervals studied and with 5.1 + 0.5 days in our group of healthy male subjects the mean half-life for irreversible degradation of biotinyl-HDL~ was not different from that reported for radiolabeled HDL. Likewise, our estimate of the size of the equilibrating extravascular pool, 26% of that of the intravascular pool, compared favorably with that of similar experiments using radiolabeled HDL. These results clearly show that biotin modification of a limited number of HDL, lysine residues does not affect the rate of removal of HDL from the plasma. They are consistent with the notion of a lysine-independent cellular recognition and uptake system for the bulk of human HDL [36,373. An issue of critical importance for the use of biotinylated HDL, in humans concerns the potential immunogenicity of chemically derivatized lipoproteins. That modifications as subtle as, for instance, methylation of the e-amino group of lysine residues of homologous LDL can render these lipoproteins immunogenic has recently been demonstrated [45]. Therefore, the possiblity that addition of biotin would also lead to the appearance of specific antibodies directed against HDL, had to be seriously considered. However, analysis of the sera from our subjects 4 weeks after the reinjection studies failed to reveal the presence of antibodies with specificity towards biotinyl-HDL~, suggesting that autologous biotinyl-HDL, does not provoke an immune response in humans. This lack of immunogenicity of biotinyl-HDL, may be related to the low degree of modification achievd in the present study. Alternatively, it may be due to differences in the presentation of biotin on the surface of HDL, compared with LDL. On the other hand, single exposure to a weak antigenic stimulus may not elicit an antibody response sufficiently strong as to permit it to be detected even by a method as sensitive as immunoblotting. Therefore, the formation of antibodies against biotinyl-HDL~ cannot be unambiguously excluded and hence, repeated injections of biotinyl-HDL, into humans must be avoided. In summary, taking this latter restriction into account, our results show that biotinyl-HDL, is a reliable and safe probe for the study of HDL turnover in humans. It is anticipated that it will prove to be expedient whenever application of radiolabeled HDL is undesirable.
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