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A novel assay for the quantitative determination of lipoprotein-X (LP-X) in serum
Clinica Chimica Acta, 121 (1983) 383-390 Elsevier Biomedical Press
383
CCA 2400
A novel assay for the quantitative determination lipoprotein-X (LP-X) in serum Ebo S. Bos a,*, Antoon A. van der Doelen ‘, Ad E.H.C. Den&en Goverde a, Harry N. Magnani a and Gerhard M. Kostner
of
a, Bas C. b
a Organon Scientific Development Group, P. 0. Box 20, 5340 BH Oss (The Netherlands) and b Institute of Medical Biochemistry, (Received
University of Grar, Grar (Austria)
July 23rd; revision October
Sth, 1982)
Summary A new, fast and simple quantitative LP-X assay is described. The method selectively removes apoB-containing lipoproteins by precipitation with specific antibodies. To the supernatant, a precipitation system for LP-X, viz. SDS and MgCl, in final concentrations of 0.5 g/l and 0.2 mol/l respectively, is added and the turbidity measured at 360 nm. The assay is linear from 0.2 g/l-5.0 g/l and shows a high precision (inter-assay CV of less than 3%). The test correlates favourably with other quantitative LP-X assays, but has the particular advantage that it can be automated and that the time required for one series is only 2 h.
Introduction It is well established that the increase of the free cholesterol and phospholipid content in serum of patients suffering from obstructive jaundice coincides with the appearance of an abnormal low density lipoprotein [ 1,2]. This lipoprotein was initially designated ‘obstructive lipoprotein’ by Russ et al [3] and Switzer [4] and subsequently ‘Lipoprotein-X’ by Seidel et al [5]. LP-X contains 60% phospholipids, 30% free cholesterol and 6-10% protein [6,7], whereas the presence of esterified cholesterol is questionable [7]. The protein moiety of LP-X consists of 40% albumin (inside the vesicle) and 60% apoC-I (surface antigen) [S]. In contrast with other lipoproteins, LP-X migrates towards the cathode in agar electrophoresis [9]. Making use of its unique electrophoretic behaviour, Seidel developed a semi-quantitative determination of LP-X [9] which has been applied in the diagnosis of cholestasis in a number of laboratories [ 10,111.
* Author responsible for correspondence and reprints: Ebo S. Bos Ph.D., ment Group, P.O. Box 20, 5340 BH Oss, The Netherlands.
0009-8981/83/0000-0000/$03.00
0 1983 Elsevier Biomedical
Press
Organon
Scientific
Develop-
384
It has been suggested by several authors that the LP-X concentration could differentiate intra- from extrahepatic liver disease. Several methods for quantitative determination of LP-X have been developed. Most of them are time-consuming and laborious [7,12] or lack specificity [13]. In this paper a simple method for quantitating LP-X is described that can be performed within 90 min. Materials and methods Materials Fresh human blood for the isolation of LP-B was obtained from healthy volunteers after fasting for 12 h. Fresh human sera with a bilirubin concentration over 17 pmol/l were collected from the local hospital. They were stored at 4°C until assay which was carried out within 1 week of phlebotomy. Under these conditions LP-X levels remain stable for 2-3 weeks. Dextran sulphate 500 was purchased from Pharmacia (Uppsala, Sweden), sodium dodecyl sulphate (SDS) from Serva (Heidelberg, FRG), agar (Noble) from Difco (Detroit, MI, USA), and agarose (type HSB) from Litex (Glostrup, Denmark). Latex (type 21gC) was bought from Dow (Indianapolis, IN, USA) in a 300-g/1 suspension. All other chemicals used were of analytical grade from Merck (Darmstadt, FRG), Serva (Heidelberg, FRG) or Baker (Deventer, The Netherlands). Methods Isolation of LP-B. LP-B was isolated from freshly prepared human serum with two successive ultracentrifugation steps at a density of 1.025 g/cm3 and 1.06 g/cm3, respectively [14]. Minor contamination, with VLDL and HDL, of the main LDL fraction was removed by repeated gel filtration on Biogel A5m [15]. The purity of the eventual LP-B preparation was assessed by SDS-PAGE/(T = 3-26%, C = 3%) gels using the discontinuous buffer system of Laemmli [16] and by immunoelectrophoresis against monospecific anti-human apoC-I [ 171. Preparation and isolation of anti-human LP-B. Sheep were immunised with LP-B using the procedure of Schuurs et al [18], and were bled between 40 and 60 days after the first injection. Prior to IgG isolation, dextran sulphate and MgCl, were added to the antiserum in a final concentration of 1 g/l and 0.1 mol/l, respectively, in order to remove ovine lipoproteins which might interfere with the eventual LP-X assay. IgG was isolated by precipitation with sodium sulphate (final concentration 1.07 mol/l) as described by Keckwick [ 191, and was finally dissolved in 0.1 mol/l NaCl and 0.2 mol/l Tris-HCl (pH 8.0). Each anti-(LP-B) preparation was assessed for its specificity by immunoelectrophoresis against several apolipoproteins. Purity of the IgG preparations was assessed by SDS-PAGE. Determination of anti-(LP-B) titre. 100 ~1 of normal human serum were added to 100 ~1 of a dilution series of anti-(LP-B) in saline (dilution factor 1.4). After 15 min of incubation at ambient temperature the immunoprecipitate was spun down at
385
15000 N/kg for 20 min. 50 ~1 of the supernatant were pipetted into each of two tubes, one for the test and one for the blank, and 500 pl of 0.2 mol/l MgCl, in saline were added to both tubes. To each blank, a further 50 ~1 of MgCl, solution were added. To the test solution 50 ~1 of a 5 g/l SDS solution were added whilst the reaction mixture was being vortexed. After 40-60 min the absorbance at 360 nm was read against the corresponding sample blank. The word titre used in this paper refers to the reciprocal of the maximum antiserum or immunoglobulin dilution at which, under the assay conditions, LDL and VLDL can be removed completely from NHS. The apoB-containing lipoproteins are considered to be removed completely if the difference between the reading of the test and blank value is less than 0.025 A,,,-units. 100 ~1 of an anti-(LP-B) solution of titre 2.7-5.4 was added to an LP-X assay. equal volume of the serum samples to be tested. After 15 min incubation at 25’C the immunoprecipitate was spun down at 15000 N/kg for 15 min. If the supernatant was opalescent or turbid, the immunoprecipitation reaction was repeated with the serum sample diluted with an equal volume of saline. From the clear supernate two aliquots of 50 ~1 were taken and treated as described for the determination of the anti-(LP-B) titre. If the absorbance of the final reaction mixture exceeded 1.50, reading was repeated after dilution with 0.2 mol/l MgCl,. Quantitation of LP-X. The quantitative determination of LP-X comprising an agar electrophoresis and a phosphorus determination on the cathodally migrating lipoprotein was carried out by the method of Magnani and Alaupovic [7]. In some experiments, LP-X was determined gravimetrically after isolation to purity by ultracentrifugation (G.M. Kostner, unpublished data). Results and discussion The principle of the assay design was as follows. LDL and VLDL both contain apoB as a major apoprotein and can be precipitated with antibodies raised against LP-B. HDL and LDL particles (like LP-X), that do not contain apoB, are left in solution. The LP-X can be selectively precipitated by one of the well-known combinations of (poly)anions and divalent cations. Prerequisites for this assay are that the antibodies are monospecific for apoB and do not cross-react with other apoproteins, especially apoC-I, the surface antigen of LP-X. LP-B isolated by ultracentrifugation and gel filtration showed a single band with an apparent molecular mass of around 275 000 (Fig. 1, lane B) and lacked the three apoC bands, which are clearly present in VLDL (Fig. 1, lane D). Hence this LP-B preparation was used for immunisation. Anti-(LP-B) sera prepared as described under ‘Methods’ usually had titres between 13 and 17. Immunoelectrophoresis and immunodiffusion demonstrated that the antibody was specific for the apoB protein, so that an interaction of the antibody with LP-X is highly unlikely. The IgG was shown to be free of ovine lipoproteins by SDS-PAGE (Fig. 1, lane E).
386
275 7
170
s X g
E 5 5 s r”
68 80
28
I,
17 8 A
8
G
D
E
Fig. 1. SDS-Polyacrylamide gel electrophoresis of LP-B and ovine anti-(LP-B)-IgG. Apparent molecular masses of the marker proteins are indicated in the margin. LDL and VLDL were analysed in different electrophoretic runs with the same serum preparation as a reference (lanes A, C). Lane A, C, 16 g/l normal human serum; lane B, 20 g/l human LP-B; lane D, 10 g/l human VLDL; lane E, 2.5 g/l ovine anti-(LP-B)-IgG.
When one volume of fresh normal human serum was mixed with an equal volume of anti-(LP-B) solution with a titre of at least 2.7, a bulky, colourless precipitate was formed which was easily spun down at low speed. When the clear supernate was subsequently treated with 1 g/l dextran sulphate, 0.1 mol/l MgCl, or 0.5 g/l SDS, 0.2 mol/l MgCl, no turbidity was observed. This indicates that VLDL- and LDL-containing LP-B were completely removed by the immunoprecipitation step. In previous experiments various combinations of precipitating agents, e.g. phosphotungstate, dextran sulphate, heparin, SDS and others in combination with CaCl,, MgCl,, CoCl, were tested. From all those combinations SDS/MgCl, proved to be optimal for the selective precipitation of residual non-LP-B-containing LDL (i.e. LP-X). A finely dispersed precipitate is formed, the absorbance of which reaches a constant level after 30 min and is stable for at least 90 min as shown in Fig. 2A. Because most sera from cholestatic patients show an elevated bilirubin concentration, and therefore an increased absorbance at 400 nm, turbidity was measured at 360 nm, where the bilirubin absorbance is at a minimum (Fig. 2B). Sera containing 200 pmol/l bilirubin give assay blanks of around 0.450 at 400 nm, which will definitely interfere with the precision of the assay at low LP-X concentrations, whereas the A,,,, -values never exceed 0.150. The commonly accepted poor stability of LP-X prevented the production of a standard preparation which could be used as an internal control/standard in the kit. However, it was observed in various calibration experiments, comprising gravimetric, ultracentrifugal and chemical anal-
387
1
;
,
,
0
,
,
20
40
Incubation
I
,
,;y, 60
120
time (min)
_.H’ I
500
450 Wavelength
400
350
(nm)
Fig. 2. Some characteristics of the turbidity assay. (A) Increase of turbidity with time after the addition of SDS. (LP-X)-positive sera were treated with anti(LP-B) as described under ‘Methods’. To 50 pl of clear supernate 500 81 of 0.2 mol/l MgCI, were added and subsequently, under vortexing, 50 gl of 5 g/l SDS. The increase of turbidity, measured as an increase of absorbance at 360 nm, was recorded immediately after the addition of the detergent and expressed as percentage of the final A,,,-value. The curve represents the mean of five experiments (CV Q 1%). (B) Spectra of bilirubin-containing sera. Bilirubin-containing sera were treated with anti-(LP-B) as described under ‘Methods’. To 50 gl of clear supemate 550 pl of 0.2 mol/l MgCl, were added and the spectrum between 340 nm and 550 nm was recorded. -, Serum 1 (56 pmol/l bilirubin); ---- - -, Serum 2 (24 pmol/l bilirubin); - .-. -, Serum 3 (8 pmol/l bilirubin).
ysis, that the turbidity caused by 1 g/l LP-X in our assay was constant in all tested. The absorbance at 360 nm for 1 g/l LP-X was 0.309 f 0.010 (n = 16) in calibrated by phospholipid determination and 0.310 + 0.004 in sera calibrated gravimetry after ultracentrifugation. This suggested the creation of a stable ondary ‘standard’, and a suspension of latex particles was found to be suitable. concentration of latex chosen gave an absorbance of 0.620 at 360 nm, equivalent
sera sera by secThe to
388
I
1
2
3
(LP-X)-concentration
g/l
LP-X (turbidimetric
4
5
(g/l)
assay)
Fig. 3. (A) Linearity of the LP-X assay. A pool of 3 LP-X positive sera from patients suffering from extrahepatic cholestasis was assessed for LP-X by gravimetry. A value of 15.4 g/l was found. From part of the serum LP-X was removed by ultracentrifugation at d. 1.075 g/cm3. With the bottom fraction the original LP-X serum was diluted to the given concentrations. Correlation between phospholipid determination and the turbidimetric assay. Sera of cholestatic patients which were found positive in the agar electrophoresis were analysed with the phosphohpid The correlation between both determination and turbidimetric assay as described under ‘Methods’. methods was determined plotting the results of the turbidimetric assay along the x-axis and those of the phosphohpid determination along the y-axis.
that obtained with 2.0 g/l LP-X. The turbidity in the LP-X assay found with unknown samples was related to that of the latex suspension and hence the LP-X concentration could be easily derived. A linear relationship between LP-X con-
389 TABLE
I
INFLUENCE
OF OLEIC
ACID
ON THE LP-X DETERMINATION
Three LP-X positive sera and one LP-X negative serum were assessed for LP-X with the electrophoretic method [9] and our turbidimetric assay. To these sera, oleic acid was added to a final concentration of 0.8 mol/l and both assays were carried out again. Assay serum
1 2 3 4
- oleic acid turbidimetry WI) 5.1 4.6 2.2 0.1
+ oleic acid electrophoresis
turbidimetry
electrophoresis
+ + + _
(g/l) 5.6 4.5 2.2 0.1
_ _ _
centration and absorbance is observed in a concentration range of 0.2-5 g/l LP-X (Fig. 3A), when directly measured. The linear correlation can be extended to 11.0 g/l LP-X by diluting the turbid sample with 0.2 mol/l MgCl,. As is shown in Fig. 3B, a good corrrelation for the LP-X concentration in a series of samples (n = 16) was found between this new turbidimetric method and the reference phospholipid determination [7], the correlation coefficient being 0.99. When 12 sera which were LP-X negative in agar electrophoresis were analysed, an apparent LP-X concentration of 0.23-0.12 g/l was found with the turbidimetric assay, and 0.21 + 0.13 g/l with the phospholipid determination. Therefore, a value of 0.4 g/l was used as a cut-off level to differentiate positive and negative results and improve specificity. The intra-assay CV of our turbidity test was 1.5-3.0s (n = 10) and the inter-assay CV was 1.8-3.0%, determined with five sera in five consecutive analytical runs. It has been reported that LP-X loses its cathodal migration after incubation with post-heparin plasma, probably due to binding of free fatty acids liberated by heparin-induced lipases [20]. When oleic acid was added to (LP-X)-positive sera, LP-X could no longer be demonstrated by agar-electrophoresis although the characteristic LP-X particles were still visible under the electron microscope [21,22]. Table I shows that in three (LP-X)-positive sera LP-X could be normally determined with our assay at oleic acid concentrations which completely abolished cathodal migration. Thus, in post-prandial blood samples false negative results of the electrophoretic assay can be avoided. In summary, the assay described above is a fast, simple and reliable method for the quantitation of LP-X. It shows a good correlation with other quantitative tests and a high precision. It overcomes some of the disadvantages of the routinely applied electrophoretic method, e.g. its semi-quantitative character and false-negative results with post-heparin or post-prandial plasmas. Acknowledgements We are much indebted to Dr. A. Gribnau and his staff of the St. Anna Oss, for the collection and biochemical analysis of (LP-X)-positive sera.
hospital,
390
References 1 Kunkel HG, Slater RJ. Lipoprotein patterns of serum obtained by zone electrophoresis. J Clin Invest 1952; 3 1: 677-686. 2 Furman RH, Conrad LL. Ultracentrifugal characterizations of the lipoprotein spectrum in obstructive jaundice. Studies of serum lipid relationships in intra- and extrahepatic biliary obstruction, J Clin Invest 1957; 36: 713-722. 3 Russ EM, Raymont J, Barr DP. Lipoproteins in primary biliary cirrhosis. J Clin Invest 1956; 35: 133-144. 4 Switzer S. Plasma proteins in liver disease. I. Immunologically distinct low-density lipoproteins in patients with biliary obstruction. J Clin Invest 1967; 46: 1855-1866. 5 Seidel D, Alaupovic P, Furman RH. A lipoprotein characterizing obstructive jaundice. 1. Method for quantitative separation and identification of lipoproteins in jaundiced subjects. J Clin Invest 1969; 48: 1211-1223. 6 Seidel D, Alaupovic P, Furman RH, McConathy WJ. A lipoprotein characterizing obstructive jaundice. II. Isolation and partial characterization of the protein moieties of low density lipoproteins. J Clin Invest 1970; 49: 2396-2407. 7 Magnani HN, Alaupovic P. A method for the quantitative determination of the abnormal lipoprotein (LP-X) of obstructive jaundice. Clin Chim Acta 1972; 38: 405-411. 8 Alaupovic P, Seidel D, McConathy WJ, Furman RH. Identification of the protein moiety of an abnormal human plasma low density lipoprotein in obstructive jaundice. FEBS Lett 1969; 4: 113- 115. 9 Seidel D. A new immunochemical technique for a rapid semiquantitative determination of the abnormal lipoprotein (LP-X) characterizing cholestasis. Clin Chim Acta 1971; 31: 225-229. 10 Seidel D, Gretz H, Ruppert C. Significance of the LP-X test in differential diagnosis of jaundice. Clin Chem 1973; 19: 86-91. 11 Ross A, Murphy GM, Wilkinson PA, Mills Gl, Sherlock S. Occurrence of an abnormal lipoprotein in patients with liver disease. Gut 1970; 11: 1035- 1037. 12 Kostner GM, Petek W, Holasek A. Immunochemical measurements of lipoprotein-X. Clin Chem 1974; 20: 676-685. 13 Talafant E, Tovarek J. Turbidimetric determination of lipoprotein-X in serum. Clin Chim Acta 1979; 96: 261-264. 14 Kostner GM, Laggner P, Prexl HJ, Holasek A, Ingolic E, Geymeyer W. Investigation of the abnormal low-density lipoprotein occurring in patients with obstructive jaundice. Biochem J 1976; 157: 401-407. 15 Miiller K, Laggner P, Glatter 0, Kostner GM. The structure of human plasma low-density lipoprotein-B. An X-ray small angle scattering study. Eur J B&hem 1978; 82: 73-80. 16 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. 17 Kostner GM, Patsch JR, Sailer S, Braunsteiner H, Holasek A. Polypeptide distribution of the main lipoprotein density classes separated from human plasma by rate zonal ultracentrifugation. Eur J Biochem 1974; 45: 61 I-621. 18 Schuurs AHWM, de Jager E, Homan JDH. Studies on human chorionic gonadotrophin. III. Immunochemical characterisation. Acta Endocrinol (Copenhagen) 1968; 59: 120- 138. 19 Keckwick RA. The serum proteins in multiple myelomatosis. Biochem J 1940; 34: 1248-1257. 20 Sauar J, Ritland S, Home R, Horn R. The effect of lipoprotein lipase and hepatic lipase on the electrophoretic mobility of lipoprotein-X. Clin Chim Acta 1978; 88: 461-467. 21 Muckle TJ, Edwards JA, Auckland P. Influence of oleic acid on serum lipoprotein-X in vitro. Clin Chem 1977; 23: 2302-2305. 22 Ras MR, Masden S, Frison JC, Rubies-Prat J. Disappearing lipoprotein-X. Clin Chem 1978; 24: 840-841.
383
CCA 2400
A novel assay for the quantitative determination lipoprotein-X (LP-X) in serum Ebo S. Bos a,*, Antoon A. van der Doelen ‘, Ad E.H.C. Den&en Goverde a, Harry N. Magnani a and Gerhard M. Kostner
of
a, Bas C. b
a Organon Scientific Development Group, P. 0. Box 20, 5340 BH Oss (The Netherlands) and b Institute of Medical Biochemistry, (Received
University of Grar, Grar (Austria)
July 23rd; revision October
Sth, 1982)
Summary A new, fast and simple quantitative LP-X assay is described. The method selectively removes apoB-containing lipoproteins by precipitation with specific antibodies. To the supernatant, a precipitation system for LP-X, viz. SDS and MgCl, in final concentrations of 0.5 g/l and 0.2 mol/l respectively, is added and the turbidity measured at 360 nm. The assay is linear from 0.2 g/l-5.0 g/l and shows a high precision (inter-assay CV of less than 3%). The test correlates favourably with other quantitative LP-X assays, but has the particular advantage that it can be automated and that the time required for one series is only 2 h.
Introduction It is well established that the increase of the free cholesterol and phospholipid content in serum of patients suffering from obstructive jaundice coincides with the appearance of an abnormal low density lipoprotein [ 1,2]. This lipoprotein was initially designated ‘obstructive lipoprotein’ by Russ et al [3] and Switzer [4] and subsequently ‘Lipoprotein-X’ by Seidel et al [5]. LP-X contains 60% phospholipids, 30% free cholesterol and 6-10% protein [6,7], whereas the presence of esterified cholesterol is questionable [7]. The protein moiety of LP-X consists of 40% albumin (inside the vesicle) and 60% apoC-I (surface antigen) [S]. In contrast with other lipoproteins, LP-X migrates towards the cathode in agar electrophoresis [9]. Making use of its unique electrophoretic behaviour, Seidel developed a semi-quantitative determination of LP-X [9] which has been applied in the diagnosis of cholestasis in a number of laboratories [ 10,111.
* Author responsible for correspondence and reprints: Ebo S. Bos Ph.D., ment Group, P.O. Box 20, 5340 BH Oss, The Netherlands.
0009-8981/83/0000-0000/$03.00
0 1983 Elsevier Biomedical
Press
Organon
Scientific
Develop-
384
It has been suggested by several authors that the LP-X concentration could differentiate intra- from extrahepatic liver disease. Several methods for quantitative determination of LP-X have been developed. Most of them are time-consuming and laborious [7,12] or lack specificity [13]. In this paper a simple method for quantitating LP-X is described that can be performed within 90 min. Materials and methods Materials Fresh human blood for the isolation of LP-B was obtained from healthy volunteers after fasting for 12 h. Fresh human sera with a bilirubin concentration over 17 pmol/l were collected from the local hospital. They were stored at 4°C until assay which was carried out within 1 week of phlebotomy. Under these conditions LP-X levels remain stable for 2-3 weeks. Dextran sulphate 500 was purchased from Pharmacia (Uppsala, Sweden), sodium dodecyl sulphate (SDS) from Serva (Heidelberg, FRG), agar (Noble) from Difco (Detroit, MI, USA), and agarose (type HSB) from Litex (Glostrup, Denmark). Latex (type 21gC) was bought from Dow (Indianapolis, IN, USA) in a 300-g/1 suspension. All other chemicals used were of analytical grade from Merck (Darmstadt, FRG), Serva (Heidelberg, FRG) or Baker (Deventer, The Netherlands). Methods Isolation of LP-B. LP-B was isolated from freshly prepared human serum with two successive ultracentrifugation steps at a density of 1.025 g/cm3 and 1.06 g/cm3, respectively [14]. Minor contamination, with VLDL and HDL, of the main LDL fraction was removed by repeated gel filtration on Biogel A5m [15]. The purity of the eventual LP-B preparation was assessed by SDS-PAGE/(T = 3-26%, C = 3%) gels using the discontinuous buffer system of Laemmli [16] and by immunoelectrophoresis against monospecific anti-human apoC-I [ 171. Preparation and isolation of anti-human LP-B. Sheep were immunised with LP-B using the procedure of Schuurs et al [18], and were bled between 40 and 60 days after the first injection. Prior to IgG isolation, dextran sulphate and MgCl, were added to the antiserum in a final concentration of 1 g/l and 0.1 mol/l, respectively, in order to remove ovine lipoproteins which might interfere with the eventual LP-X assay. IgG was isolated by precipitation with sodium sulphate (final concentration 1.07 mol/l) as described by Keckwick [ 191, and was finally dissolved in 0.1 mol/l NaCl and 0.2 mol/l Tris-HCl (pH 8.0). Each anti-(LP-B) preparation was assessed for its specificity by immunoelectrophoresis against several apolipoproteins. Purity of the IgG preparations was assessed by SDS-PAGE. Determination of anti-(LP-B) titre. 100 ~1 of normal human serum were added to 100 ~1 of a dilution series of anti-(LP-B) in saline (dilution factor 1.4). After 15 min of incubation at ambient temperature the immunoprecipitate was spun down at
385
15000 N/kg for 20 min. 50 ~1 of the supernatant were pipetted into each of two tubes, one for the test and one for the blank, and 500 pl of 0.2 mol/l MgCl, in saline were added to both tubes. To each blank, a further 50 ~1 of MgCl, solution were added. To the test solution 50 ~1 of a 5 g/l SDS solution were added whilst the reaction mixture was being vortexed. After 40-60 min the absorbance at 360 nm was read against the corresponding sample blank. The word titre used in this paper refers to the reciprocal of the maximum antiserum or immunoglobulin dilution at which, under the assay conditions, LDL and VLDL can be removed completely from NHS. The apoB-containing lipoproteins are considered to be removed completely if the difference between the reading of the test and blank value is less than 0.025 A,,,-units. 100 ~1 of an anti-(LP-B) solution of titre 2.7-5.4 was added to an LP-X assay. equal volume of the serum samples to be tested. After 15 min incubation at 25’C the immunoprecipitate was spun down at 15000 N/kg for 15 min. If the supernatant was opalescent or turbid, the immunoprecipitation reaction was repeated with the serum sample diluted with an equal volume of saline. From the clear supernate two aliquots of 50 ~1 were taken and treated as described for the determination of the anti-(LP-B) titre. If the absorbance of the final reaction mixture exceeded 1.50, reading was repeated after dilution with 0.2 mol/l MgCl,. Quantitation of LP-X. The quantitative determination of LP-X comprising an agar electrophoresis and a phosphorus determination on the cathodally migrating lipoprotein was carried out by the method of Magnani and Alaupovic [7]. In some experiments, LP-X was determined gravimetrically after isolation to purity by ultracentrifugation (G.M. Kostner, unpublished data). Results and discussion The principle of the assay design was as follows. LDL and VLDL both contain apoB as a major apoprotein and can be precipitated with antibodies raised against LP-B. HDL and LDL particles (like LP-X), that do not contain apoB, are left in solution. The LP-X can be selectively precipitated by one of the well-known combinations of (poly)anions and divalent cations. Prerequisites for this assay are that the antibodies are monospecific for apoB and do not cross-react with other apoproteins, especially apoC-I, the surface antigen of LP-X. LP-B isolated by ultracentrifugation and gel filtration showed a single band with an apparent molecular mass of around 275 000 (Fig. 1, lane B) and lacked the three apoC bands, which are clearly present in VLDL (Fig. 1, lane D). Hence this LP-B preparation was used for immunisation. Anti-(LP-B) sera prepared as described under ‘Methods’ usually had titres between 13 and 17. Immunoelectrophoresis and immunodiffusion demonstrated that the antibody was specific for the apoB protein, so that an interaction of the antibody with LP-X is highly unlikely. The IgG was shown to be free of ovine lipoproteins by SDS-PAGE (Fig. 1, lane E).
386
275 7
170
s X g
E 5 5 s r”
68 80
28
I,
17 8 A
8
G
D
E
Fig. 1. SDS-Polyacrylamide gel electrophoresis of LP-B and ovine anti-(LP-B)-IgG. Apparent molecular masses of the marker proteins are indicated in the margin. LDL and VLDL were analysed in different electrophoretic runs with the same serum preparation as a reference (lanes A, C). Lane A, C, 16 g/l normal human serum; lane B, 20 g/l human LP-B; lane D, 10 g/l human VLDL; lane E, 2.5 g/l ovine anti-(LP-B)-IgG.
When one volume of fresh normal human serum was mixed with an equal volume of anti-(LP-B) solution with a titre of at least 2.7, a bulky, colourless precipitate was formed which was easily spun down at low speed. When the clear supernate was subsequently treated with 1 g/l dextran sulphate, 0.1 mol/l MgCl, or 0.5 g/l SDS, 0.2 mol/l MgCl, no turbidity was observed. This indicates that VLDL- and LDL-containing LP-B were completely removed by the immunoprecipitation step. In previous experiments various combinations of precipitating agents, e.g. phosphotungstate, dextran sulphate, heparin, SDS and others in combination with CaCl,, MgCl,, CoCl, were tested. From all those combinations SDS/MgCl, proved to be optimal for the selective precipitation of residual non-LP-B-containing LDL (i.e. LP-X). A finely dispersed precipitate is formed, the absorbance of which reaches a constant level after 30 min and is stable for at least 90 min as shown in Fig. 2A. Because most sera from cholestatic patients show an elevated bilirubin concentration, and therefore an increased absorbance at 400 nm, turbidity was measured at 360 nm, where the bilirubin absorbance is at a minimum (Fig. 2B). Sera containing 200 pmol/l bilirubin give assay blanks of around 0.450 at 400 nm, which will definitely interfere with the precision of the assay at low LP-X concentrations, whereas the A,,,, -values never exceed 0.150. The commonly accepted poor stability of LP-X prevented the production of a standard preparation which could be used as an internal control/standard in the kit. However, it was observed in various calibration experiments, comprising gravimetric, ultracentrifugal and chemical anal-
387
1
;
,
,
0
,
,
20
40
Incubation
I
,
,;y, 60
120
time (min)
_.H’ I
500
450 Wavelength
400
350
(nm)
Fig. 2. Some characteristics of the turbidity assay. (A) Increase of turbidity with time after the addition of SDS. (LP-X)-positive sera were treated with anti(LP-B) as described under ‘Methods’. To 50 pl of clear supernate 500 81 of 0.2 mol/l MgCI, were added and subsequently, under vortexing, 50 gl of 5 g/l SDS. The increase of turbidity, measured as an increase of absorbance at 360 nm, was recorded immediately after the addition of the detergent and expressed as percentage of the final A,,,-value. The curve represents the mean of five experiments (CV Q 1%). (B) Spectra of bilirubin-containing sera. Bilirubin-containing sera were treated with anti-(LP-B) as described under ‘Methods’. To 50 gl of clear supemate 550 pl of 0.2 mol/l MgCl, were added and the spectrum between 340 nm and 550 nm was recorded. -, Serum 1 (56 pmol/l bilirubin); ---- - -, Serum 2 (24 pmol/l bilirubin); - .-. -, Serum 3 (8 pmol/l bilirubin).
ysis, that the turbidity caused by 1 g/l LP-X in our assay was constant in all tested. The absorbance at 360 nm for 1 g/l LP-X was 0.309 f 0.010 (n = 16) in calibrated by phospholipid determination and 0.310 + 0.004 in sera calibrated gravimetry after ultracentrifugation. This suggested the creation of a stable ondary ‘standard’, and a suspension of latex particles was found to be suitable. concentration of latex chosen gave an absorbance of 0.620 at 360 nm, equivalent
sera sera by secThe to
388
I
1
2
3
(LP-X)-concentration
g/l
LP-X (turbidimetric
4
5
(g/l)
assay)
Fig. 3. (A) Linearity of the LP-X assay. A pool of 3 LP-X positive sera from patients suffering from extrahepatic cholestasis was assessed for LP-X by gravimetry. A value of 15.4 g/l was found. From part of the serum LP-X was removed by ultracentrifugation at d. 1.075 g/cm3. With the bottom fraction the original LP-X serum was diluted to the given concentrations. Correlation between phospholipid determination and the turbidimetric assay. Sera of cholestatic patients which were found positive in the agar electrophoresis were analysed with the phosphohpid The correlation between both determination and turbidimetric assay as described under ‘Methods’. methods was determined plotting the results of the turbidimetric assay along the x-axis and those of the phosphohpid determination along the y-axis.
that obtained with 2.0 g/l LP-X. The turbidity in the LP-X assay found with unknown samples was related to that of the latex suspension and hence the LP-X concentration could be easily derived. A linear relationship between LP-X con-
389 TABLE
I
INFLUENCE
OF OLEIC
ACID
ON THE LP-X DETERMINATION
Three LP-X positive sera and one LP-X negative serum were assessed for LP-X with the electrophoretic method [9] and our turbidimetric assay. To these sera, oleic acid was added to a final concentration of 0.8 mol/l and both assays were carried out again. Assay serum
1 2 3 4
- oleic acid turbidimetry WI) 5.1 4.6 2.2 0.1
+ oleic acid electrophoresis
turbidimetry
electrophoresis
+ + + _
(g/l) 5.6 4.5 2.2 0.1
_ _ _
centration and absorbance is observed in a concentration range of 0.2-5 g/l LP-X (Fig. 3A), when directly measured. The linear correlation can be extended to 11.0 g/l LP-X by diluting the turbid sample with 0.2 mol/l MgCl,. As is shown in Fig. 3B, a good corrrelation for the LP-X concentration in a series of samples (n = 16) was found between this new turbidimetric method and the reference phospholipid determination [7], the correlation coefficient being 0.99. When 12 sera which were LP-X negative in agar electrophoresis were analysed, an apparent LP-X concentration of 0.23-0.12 g/l was found with the turbidimetric assay, and 0.21 + 0.13 g/l with the phospholipid determination. Therefore, a value of 0.4 g/l was used as a cut-off level to differentiate positive and negative results and improve specificity. The intra-assay CV of our turbidity test was 1.5-3.0s (n = 10) and the inter-assay CV was 1.8-3.0%, determined with five sera in five consecutive analytical runs. It has been reported that LP-X loses its cathodal migration after incubation with post-heparin plasma, probably due to binding of free fatty acids liberated by heparin-induced lipases [20]. When oleic acid was added to (LP-X)-positive sera, LP-X could no longer be demonstrated by agar-electrophoresis although the characteristic LP-X particles were still visible under the electron microscope [21,22]. Table I shows that in three (LP-X)-positive sera LP-X could be normally determined with our assay at oleic acid concentrations which completely abolished cathodal migration. Thus, in post-prandial blood samples false negative results of the electrophoretic assay can be avoided. In summary, the assay described above is a fast, simple and reliable method for the quantitation of LP-X. It shows a good correlation with other quantitative tests and a high precision. It overcomes some of the disadvantages of the routinely applied electrophoretic method, e.g. its semi-quantitative character and false-negative results with post-heparin or post-prandial plasmas. Acknowledgements We are much indebted to Dr. A. Gribnau and his staff of the St. Anna Oss, for the collection and biochemical analysis of (LP-X)-positive sera.
hospital,
390
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