Apolipoprotein measurements in clinical biochemistry and their utility vis-a-vis conventional assays

Clinica Chimica Acta, 178 (1988) 1-34 Elsevier

CCA 04301

Critical Review

Apolipoprotein measurements in clinical biochemistry and their utility vis-a-vis conventional assays Paul S. Bachorik Lipid Research-Atherosclerosis

and Peter 0. Kwiterovich,

Unit, Departments of Pediatrics and Medicine, School of Medicine, Baltimore, MD (USA)

Jr.

The Johns Hopkins Unroersity

(Received 10 March 1988; revision received 13 June 1988; accepted after revision 15 June 1988) Key words: Apolipoprotein; Coronary disease; Coronary risk; Lipoprotein; Low density lipoprotein: High density lipoprotein; Apolipoprotein measurement: Apolipoprotein standardization

General

The major plasma lipids, cholesterol, cholesteryl esters, triglycerides and phospholipids are carried in plasma by the plasma lipoproteins, which normally constitute about 8 or 10% of the total circulating protein. Current procedures used to diagnose and treat the dyslipoproteinemias, and estimate the risk for coronary artery disease, include measurements of plasma lipids and lipoproteins. The lipoproteins are spherical complexes that contain lipid and protein (Fig. 1). Cholesteryl esters and triglycerides are in the core of the particle, and phosphatidylcholine, sphingomyelin, cholesterol and protein are oriented near the surface. The measurement of the total circulating mass of lipoprotein requires

Llpoproteln

portlcle

Fig. 1. FC, free cholesterol; CE, cholesteryl ester; PL, phospholipid; TG, triglycerides.

Correspondence to: Dr. P.S. Bachorik, Johns Hopkins University, Childrens Med./Surg. Baltimore, MD 21205, USA.

Center, 604,

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fairly sophisticated and time-consu~g methods, which are not suited to clinical purposes, however, and lipoprotein-cholesterol concentrations are usually used as an index of plasma lipoprotein concentration. The assumption that underlies the use of lipoprotein-cholesterol measurements for this purpose is that the composition of the lipoproteins is relatively invariant. This is not always true, however, and during the past few years attention has turned to the measurement of the protein components of the lipoproteins also. These components, when freed of lipoprotein lipids, are collectively termed the apolipoproteins. One of the important questions concerning apolipoprotein analysis is whether, and to what extent apolipoprotein measurements are better indicators of plasma lipoprotein levels and coronary risk than conventional lipoprotein cholesterol measurements. In this paper we discuss the clinical utility of plasma apolipoprotein measurements, as well as some of the laboratory methods used to measure lipoproteins and apolipoproteins. From a clinical standpoint, apolipoprotein measurements are made for two general purposes: (1) to aid in the diagnosis of several primary disorders of lipoprotein metabolism; and (2) to estimate coronary risk; each of these will be considered in turn. It might be mentioned at this point, however, that the use of apolipoprotein measurements for diagnostic purposes presentty remains within the purview of the specialized lipoprotein research center. In general medical practice, such measurements are presently most likely to be made as an aid to the assessment of coronary risk. For this purpose, much recent work has focused on apolipoproteins (APO) AI and B. Apo AI is the major apolipoprotein in high density lipoproteins (HDL), and Apo B is the major apolipoprotein in very low density lipoproteins (VLDL), low density lipoproteins (LDL), and chylomicrons. Plasma lipoproteins: structure, function and metabolism

Four major lipoprotein classes are found in plasma: chylomicrons, VLDL, LDL, and two major subfractions HDL, called I-IDL, and HDL,. The plasma lipoproteins have densities in the range of approximately 0.90 to 1.21 g/ml, compared with densities of above 1.30 g/ml for the other plasma proteins, and can be readily separated from plasma proteins by ultracentrifugation at d 1.21 g/ml. The lipoproteins can also be separated from each other based on differences in size, in their physical chemical properties such as density, flotation rate, and electrophoretic mobility. Plasma contains several other ~poproteins that are present in relatively low concentrations, including intermediate density lipoprotein (IDL) and Lp(a). Table I summarizes some of the properties of the plasma lipoproteins. Chylomicrons

Chylomicrons are synthesized in the intestine in response to a fat-containing meal, and serve to transport dietary fat. They are large particles that contain by weight about 90% triglycerides, 2% apolipoprotein (Table I) [1,21. Although chylomicrons are usually thought of in terms of triglyceride transport, they also transport all of the dietary cholesterol that is absorbed by the gut_ The large amount

3 TABLE

I

Some properties of human plasma lipoproteins Lipoprotein class

Density k/ml)

Flotation rate

Electrophoretic mobility ’

Chemical composition

Apolipoproteins

(I& by wt) FC+ CE

TG

PL

Protein

4

90

5

1

55

19

8

B-48, B-100, AIV, E, AI, AIL c’s B-100, C’s E, AI, AI1 B-100, C’s, E

Chylomicrons

< 1.006

S,>lOO

origin

VLDL

<1.006

S, 20-100

prebeta

20

$12-20 s,o-12

betaprebeta beta

Intermediate between VLDL and LDL 55 5 20 20

F,,,3.5-9

alpha

24

8

25

43

AI, AII, C’s, E

F,,,,O-3.5

alpha

21

2

23

55

AI, AIL C’s

prebeta

46

4

21

29

B-100, Lp(a)peptide

IDL 2DL HDL, HDL, Lp(a)

1.0061.19 1.0191.063 1.0631.12 1.121.21 1.051.12

B-100

a Agarose gel or paper electrophoresis.

of dietary fast that must be handled, on the order of up to 100 g/day, results in the synthesis of relatively large amounts of intestinal apolipoproteins [2]. The major apolipoproteins in chylomicrons include Apo B, Apo A-IV, Apo E, Apo A-I, Apo A-II and Apo C (Table I). It is thought that the intestine actually synthesizes two variants of Apo B, called Apo B-48 and Apo B-100, Apo A-I, Apo A-II, Apo A-IV and small quantities of Apo C-II [2,3]; Apo E and the bulk of the C apolipoproteins appear to exchange onto the chylomicrons from HDL as the chylomicron particles are metabolized. The chylomicrons are secreted into the lymphatic system and then enter the blood, where most of the triglyceride is hydrolyzed by lipoprotein lipase (LPL), an enzyme which is bound to the external surface of endothelial cells. During this process the particle loses about 96% of its mass, mostly as triglyceride, and the A and C apolipoproteins. A smaller, more cholesterol-rich chylomicron remnant particle results in which Apo E and Apo B are the major apolipoproteins. The chylomicron remnant is rapidly cleared from the circulation by the liver [4]. At least several apolipoproteins play key roles in the chylomicron pathway. Apo C-II is a required cofactor for lipoprotein lipase [5-71, and the other C apolipoproteins apparently inhibit hepatic uptake of chylomicrons themselves, thereby facilitating the adequate removal of triglycerides before the particle is cleared. Apo E apparently plays a role in targeting the chylomicron remnant receptor (Apo E receptor) which mediates the rapid hepatic clearance of the remnant particles [4,8]. The intestine also secretes ‘intestinal VLDL’, or ‘small chylomicrons’ in the fasting state, or after non-fat meals. In rats these particles have

4

been estimated to contribute about 10 or 20% to the circulating VLDL and triglyceride levels [9-111; however they are probably metabolized as chylomicrons

141. VLDL

VLDL are synthesized in the liver, secreted to the circulation, and serve to transport endogenously synthesized triglycerides. VLDL contains about 50% triglyceride, and the major apolipoproteins associated with the particle are Apo B-100, the C apolipoproteins, and Apo E. VLDL is initially catabolized in a manner similar to the chylomicrons. VLDL-triglycerides are hydrolyzed at the endothelial cell surface by lipoprotein lipase producing smaller, more cholesterol-enriched VLDL ‘remnant’ particles which are further metabolized to IDL. VLDL remnants can be cleared by the liver, and IDL is ultimately converted to LDL (Fig. 2) [12,13]. The details of the conversion of VLDL to LDL are not entirely understood, but they involve the progressive hydrolysis of VLDL-triglycerides, enrichment of cholesteryl

TABLE

II

Distribution of methods used in IUIS-CDC study

international collaborative apolipoprotein standardization

Apo AI (%) 198.3 ’ (55 laboratories) b RIA 6 ELISA RID 21 EIA 11 INAc 19 Total 51

APO B (W) 11 37 19 33

6 4 26 13 24 13

8 6 36 18 33

1986 d (85 laboratories) b RIA 10 ELISA 4 RID 25 EIA 23 INA’ 29 Total 91

11 4 2-l 25 32

8 6 28 24 28 94

9 6 30 26 30

1987 ’ (143 laboratories) RIA 15 ELISA 4 RID 29 EIA 24 INA = 65 Total 137

11 3 21 18 47

15 I 31 25 65 143

11 5 22 17 45

’ b ’ d ’

Ref. [113]. Some laboratories used more than one method. Includes, rate, endpoint, and turbidimetric methods. Ref. [115]. Ref. (1161.

5

VLDL

VLDL

Remnant

IDL

LDL

Per!pheral tissues

Fig. 2. VLDL metabolism.

esters, and loss of virtually all of the apolipoproteins except Apo B-100. It has been suggested that hepatic lipase may play a role in the conversion of IDL to LDL [14]. This enzyme is bound to the external surface of hepatic endothelial cells and has both lipase and phospholipase activity [14]. LDL

LDL carries most of the circulating cholesterol in man, and provides cholesterol to peripheral tissues for membrane biosynthesis and steroidogenesis. LDL-cholesterol is normally delivered to the tissues primarily via LDL receptor-mediated endocytosis [15]. The LDL receptor recognizes Apo B and after internalization of LDL, LDL-Apo B is degraded in the lysosomes. Half or more of the catabolism of LDL occurs in the liver, primarily in parenchymal cells [16-191; the rest occurs in peripheral tissues. HDL

The major sites of Apo A-I synthesis are the liver and the intestine [20]. HDL is believed to be secreted from the liver as a nascent, disc-shaped particle containing Apo A-I, cholesterol, phospholipid, and Apo E [21]. As discussed above, Apo A-I is also secreted from the intestine as part of the chylomicron. The nascent HDL particle rapidly accumulates cholesterol from ceil membranes and other lipoproteins and is converted to a mature spherical particle, in part through the action of lecithin : cholesterol acyltransferase (LCAT), which catalyzes cholesterol esterification in the circulation and results in the movement of the newly esterified cholesterol into the core of the HDL particle. As mentioned above, HDL participates in the catabolism of the triglyceride-rich lipoproteins, making Apo CII and Apo E available. It also seems to be involved in the process known as ‘reverse cholesterol transport’ [21]. In this process, HDL receives cholesterol from peripheral tissues, probably in the form of unesterified cholesterol from cell membranes, and transports it back to the liver for reutilization or disposal. This removal of tissue cholesterol may be facilitated in part by a high-affinity HDL receptor on the cell membrane [22,23]. Several mechanisms may be important in delivering this cholesterol back to the liver. Some of the esterified

6

cholesterol is transferred from HDL to the lower density lipoproteins, and subsequently taken up via the hepatic Apo E- and/or LDL receptors [24]. This transfer is probably catalyzed by specific transfer proteins that facilitate the removal of cholesteryl esters from the core of the HDL particle and move them to VLDL or LDL [24]. The direct uptake of cholesteryl esters from HDL without uptake of the entire HDL particle may also contribute to the ultimate hepatic removal of tissue cholesterol [25-271. At present it is not certain whether HDL receptor recognition of the HDL particle may involve one or more HDL apolipoproteins. Conventional

methods for measuring lipoprotein levels

Lipoprotein levels can be measured in terms of the circulating mass concentration of each lipoprotein, which includes the contribution of each of the lipid and apolipoproteiri components. Such measurements have usually been made with the analytical ultracentrifuge [28], but the methods are cumbersome, and not easily applied on a clinical level. It is also possible to calculate the mass concentration of the lipoproteins from individual assays of each of their components. This approach requires the quantitative isolation and pu~fication of each of the major lipoprotein classes before analysis, and again is much too tedious and time-consuming for clinical purposes. For this reason, plasma lipoprotein levels have usually been measured in terms of their cholesterol content. It might be mentioned that for the most part, the epidemiological data relating lipoprotein levels to cardiovascular risk are based on ~poprotein-chol~terol measurements. The relation of the plasma total cholesterol to lipoprotein cholesterol in the fasting state, may be summarized as follows: [total cholesterol] = [ VLDL chol] + [ LDL chol] + [ HDL chol] . Two methods are most commonly used to quantitate lipoprotein cholesterol. In the first, total plasma cholesterol and triglycerides are measured. In addition, HDLcholesterol is measured following removal of the other plasma lipoproteins by precipitation with a polyanion-divalent cation [29]. LDL-cholesterol concentration is then calculated from the empirical formula of FriedewaId et al. [30]: [LDL chol] = [total chol] - [HDL chol] - [TG/S], where the cholesterol and triglyceride concentrations are expressed in mg/dl. The factor [TG]/5 is an estimate of VLDL cholesterol concentration. (In a recent study, it was found that the factor [TG]/6.3 gives a somewhat better estimate of VLDLcholesterol in plasma [31].) This equation can be used to estimate the LDL cholesterol levels in most people, but the equation cannot be used in samples with triglycerides exceeding 400 mg/dl, in samples with a significant amount of chylomicrons, or in those with /3-VLDL (‘floating beta lipoproteins’, characteristic of type III hyperlipoproteinemia or dysbetalipoproteinemia), because the relative cholesterol and triglyceride compositions of these lipoproteins differ considerably

7

from VLDL. Specifically, chylomicrons have a triglyceride:cholesterol ratio of about 10 : 1. The application bf the formula would overestimate VLDL-cholesterol and thereby underestimate LDL cholesterol. /3-VLDL, on the other hand, is richer in cholesterol than normal VLDL, and use of the formula would give the impression of an increased LDL cholesterol concentration (i.e., a type II pattern) in a patient with type III hyperlipoproteinemia. The second procedure requires preparative ultracentrifugation and is used initially to establish the lipoprotein patterns in dyslipoproteinemic patients, in type III hyperlipoproteinemia, and in samples with high triglyceride concentrations and/or significant amounts of chylomicrons. In this procedure, cholesterol, triglyceride and HDL cholesterol are measured as above. A separate aliquot of plasma is ultracentrifuged at d 1.006 g/ml for 18 h, and the floating layer of VLDL is removed. The cholesterol concentration of the ultracentrifugal infranatant (d > 1.006 g/ml),

b

a

Origin

-

beta

-

pre-beta

-

C-J

Orlgln khylomlcrons If

beta

LDL

pre- beta

VLDL

alpha

H

HDL J

I

Lipoprotein

Origin

presed

-

electrophoresls

c

Fig. 3. Agarose gel ekctrophoresis illustrating: (a) migration of major lipoproteins; (b) patterns obtained in unfractionated plasma (left lane), d < 1.006 g/ml fraction (center lane), and d > 1.006 g/ml fraction (right lane) from a healthy subject; (c) patterns obtained in similar fractions from a patient with type III hyperlipoproteinemia.

8

which contains primarily LDL and HDL, is measured. The VLDL- and LDLcholesterol concentrations are calculated as follows. VLDL-chol = [total chol] - [d > 1.006 chol] LDL-chol

= [d > 1.006 chol] - [ HDL-chol]

The VLDL fraction will, of course, contain chylomicrons of /3-VLDL if present. When present in moderate amounts, chylomicrons would not influence the VLDLcholesterol measurement too much because of the relatively low cholesterol composition of chylomicrons. P-VLDL, however, is rich in cholesterol, and significantly increases the amount of cholesterol in the VLDL-containing fraction. fi-VLDL can be detected in the ultracentrifugal supematant (d < 1.006 g/ml) by agarose gel electrophoresis as a band with beta mobility, similar to that of LDL (Fig. 3). Furthermore, the presence of /3-VLDL influences the apparent ratio of VLDLcholesterol to plasma triglycerides. In the absence of /S-VLDL or chylornicrons, this ratio is generally 0.2 or less [30,31], but in the presence of j?-VLDL, the ratio exceeds 0.3. Apolipoprotein

measurement

Assay procedures have been devised for all of the known plasma apolipoproteins. Most of the methods are based on either electrophoretic or immunochemical techniques. Most of the apolipoproteins are assayed for research purposes, or as an aid to the diagnosis of several of the dyslipoproteinemias, some of which are relatively uncommon (see below). In such cases, the assays are performed in specialized research centers, and are not available in the routine clinical laboratory. The importance of quantitative Apo AI and Apo B measurements in evaluating cardiovascular risk is increasing, however (see below). Furthermore, initial multicenter efforts to standardize apolipoprotein assays are directed toward Apo AI and B, and for these reasons, our discussion of quantitative methods for apolipoprotein measurements focuses on these two apolipoproteins. Many of the factors that affect the measurement of Apo AI and Apo B, however, are relevant to the other apolipoproteins as well. Electrophoretic

methods

In general, electrophoresis, sometimes in combination with other methods, has been used for the qualitative identification of apolipoprotein variants as well as for estimating the relative proportion of apolipoproteins present in a mixture. A two-dimensional system of polyacrylamide gel electrophoresis and isoelectric focusing was used to establish the genetic basis for the three isoforms of Apo E (E2, E3, and E4) [32,33]. In this procedure, VLDL is first prepared by ultracentrifugation, after which it is lyophilized and redissolved in 9.5 mol/l urea containing a detergent, ampholines of the appropriate pH range, and /3-mercaptoethanol, and

9

subjected to isoelectric focusing in the first dimension. The sample strip is then applied to an SDS-polyacrylamide gel for separation in the second dimension. Similar systems have also been used to separate genetic variants of Apo AI and E [34-371. Single dimension analytical isoelectric focusing in polyacrylamide gels has also been used to identify Apo E isoforms [38,39]. In this procedure, VLDL is first isolated and the lipids are extracted, after which the lipid-free protein (Apo VLDL) is dissolved in 8 mol/l urea, and applied to polyacrylamide gel and focused in the pH range 4-7. Under these conditions, a number of Apo E isoforms can be separated. Some of them reflect the genetic variation in the primary structure of Apo E [40-421 and others are due to post-translational modifications of Apo E such as variability in the number of sialic acid residues attached to the carbohydrate side chains [33] and the formation of dimers of Apo E or Apo E-APO AI1 through disulfide bridges [43,44]. For this reason, prior to isoelectric focusing, Apo E preparations are generally treated with dithiothreitol to break disulfide bonds, and with neuraminidase to remove sialic acid residues. The isoforms observed in such preparations then reflect primarily the amino acid changes in the polypeptide chains. It is evident that the kinds of electrophoretic analyses described above must be done under carefully controlled conditions. In addition, the proper interpretation of the patterns requires considerable experience. Apo B-48 and Apo B-100 SDS-polyacrylamide gel electrophoresis is used to separate Apo B-48 from Apo B-100 [3,45]. Triglyceride-rich lipoproteins are first isolated by ultracentrifugation. Preservatives such as gentamycin sulfate, chloramphenicol, EDTA, and sodium azide are used to prevent microbial, oxidative and proteolytic degradation of Apo B during sample preparation. The isolated lipoproteins are then extracted with an ethanol-diethyl ether mixture to remove lipids. The lipid-free precipitate is then extracted with 4.2 mol/l tetramethylurea to remove other apolipoproteins. Finally, the Apo B precipitate is dissolved in SDS buffer containing 2-mercaptoethanol, and subjected to electrophoresis under reducing conditions in 3% polyacrylamide gels, which separates Apo B-48 and Apo B-100. The ratio of the two forms is determined by scanning densitometry after the gels have been fixed and stained [3,45]. SDSpolyacrylamide gel electrophoresis in 3-30% gradient gels has also been used to separate Apo B-48 and Apo B-100 [46]. Apo C Polyacrylamide gel electrophoresis and isoelectric focusing has also been used to assess the presence of individual C apolipoproteins [7,47,48] of which Apo CII and Apo CIII are the major forms [49]. VLDL is first isolated by ultracentrifugation, then concentrated and subjected to isoelectric focusing in polyacrylamide gel either directly or after extraction to remove lipids. This procedure separates Apo CII (pZ - 4.9) as well as three isoforms of Apo CIII (Apo CIII,, pZ - 5.0; Apo CIII,, pZ - 4.8; Apo CIII,, pZ - 4.6) [48,49] that vary in the sialic acid content of their carbohydrate side chains.

Electrophoretic methods used in conjunction with scanning densitometry allow the determination of the relative proportions of the apolipoproteins as well as semiquantitative estimates of the amounts present. While they are useful for research and occasionally as a diagnostic acid in specialized lipoprotein laboratories, they have limited applicability for routine clinical use. Immunochemical

methods

Immunochemical methods have been used to measure the plasma concentrations of all the individual apolipoproteins. Because of their growing importance as predictors of coronary risk, the following discussion focuses on Apo AI and B. The factors that affect the measurement of these apolipoproteins, however, are relevant to the others also. Immunochemical

methods for measuring Apo AI and B

A number of different immunochemical methods have been devised for measuring Apo AI and B, including radioimmunoassay (RIA) [50-571, radial immunodiffusion (RID) [58-621, electroimmunoassay (EIA) [63-711, immunonepholometry (INA) [64,72-761, and enzyme-linked immunosorbant assay (ELISA) [77-791. At present there is no accepted reference method for apolipoprotein analysis, although RIA has been suggested as possibly the best current candidate method [80]. Not all of the methods in general use give the same results [80]. The immunochemical methods are influenced by a number of factors including the specificity of the antibodies for antigenic sites on the apolipoprotein and its various polymorphic forms [51,81], the accessibility of the apolipoprotein to the antibody, the presence of any particular apolipoprotein in more than one lipoprotein fraction, the time and temperature of the incubations, the nature of the standards used for the assays and other factors [51,82-851. The purpose here is to describe briefly the characteristics of these methods and consider various factors that can influence the interpretation of the measurements. General characteristics

of the methods

Radioimmunoassay RIA is a competitive binding assay in which the apolipoprotein in the sample to be measured competes with radiolabeled apolipoprotein for binding to a monospecific or monoclonal antibody. Conditions are established in which sufficient antibody is used to precipitate about half the radiolabeled apolipoprotein; the assay is most sensitive under these conditions. The amount of labeled apolipoprotein-antibody complex which forms in the absence of unlabeled apolipoprotein is arbitrarily set to 100%. Standard curves are constructed by including increasing amounts of the unlabeled apolipoprotein in the incubation system, which competes with the labeled apolipoprotein, and reduces the amount of labeled antigen bound by the antibody. The magnitude of the decrease depends on the concentration of unlabeled

11

apolipoprotein. Such standard curves are then used to quantitate the amount of apolipoprotein present in samples of unknown concentration. The amount of labeled apolipoprotein-antibody complex formed is measured following precipitation of the complex with a second antibody, one that reacts with the immunoglobulin in the antigen-antibody complex and with uncomplexed antibody, but not with uncomplexed apolipoprotein. The complex can also be measured using staphylococcus protein A as the precipitating agent [83]. RIA procedures are quite sensitive, and can measure Apo AI and Apo B in the OS- to lo-ng range. The results can be affected by the characteristics of both the antibodies and the apolipoproteins, and it is necessary to verify that under the conditions of the assay the samples and standard interact with the antibody in the same way, that is, that parallel displacement curves are obtained with both. The assays can be automated, but are fairly involved. They generally use radioiodinated ligands, which have relatively short half lives (i*‘I, t,,* = 60 days), and require a considerable amount of technical skill to operate properly. Furthermore, when used for apolipoproteins such as Apo AI and Apo B, which are present in relatively high concentrations in plasma, high sample dilutions must be used, which can contribute to the variability of the methods. It has been reported that only about 10% of plasma Apo AI is detected by RIA, unless steps are taken to maximize the exposure of the antigenic sites on Apo AI before the measurements are made [54,83]. Various denaturing techniques, such as heating [56], detergent-treatment [83], or treatment with tetramethylurea (TMU) and urea [86] have been used. Such treatment maximizes the detectability of Apo AI and gives a measure of total plasma Apo AI, without distinguishing its location in HDL and the post-HDL fraction of plasma. Apo AI, however, is at most a minor constituent of VLDL apolipoproteins [87], and only trace amount of Apo A-I have been detected in ultracentrifugally isolated apolipoproteins of d < 1.063 g/ml [86]. Very little non-lipoprotein associated Apo AI occurs in normal serum from fasting subjects, but up to 12% has been reported in hypertriglyceridemic patients. Non-associated Apo AI has also been found in the post-absorptive state [BB]. Apo AI can also appear in the d > 1.21 g/ml fraction artifactually following ultracentrifugation of plasma [89]. For Apo B, there appears to be less of a problem with masked antigenic sites [51,53,90], but some antisera can have different affinities for Apo B in different lipoprotein fractions [51,90], which can affect the measured values to an extent that depends on the relative amounts of the lipoproteins present. While Apo B is found to a greater or lesser extent in all of the lipoproteins except HDL, it is most highly concentrated in LDL, and plasma Apo B measurements in man generally reflect primarily the Apo B in this lipoprotein. RIA methods are performed in aqueous solution, and generally incorporate steps to maximize the exposure of the apolipoproteins, and minimize the variation in antibody affinity for different lipoproteins [91-931. When used in this way, RIA provides a measure of the total amount of Apo AI or Apo B in the sample. When it has been necessary to measure an apolipoprotein in a particular lipoprotein class, it is necessary first to isolate the lipoprotein fraction of interest.

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Enzyme-linked immunosorbant assay ELISA, like RIA, is a competitive binding procedure whose sensitivity is similar to that of RIA. The assays are generally performed in plastic tubes or plates containing sample wells. The tubes are coated with a solution of unlabeled apolipoprotein, which binds to the wall of the tubes. Standards or samples are then added along with unlabeled antibody. The antibody binds to the apolipoprotein in the standard or sample, as well as to the antigen bound to the wall of the tube. The greater the concentration of antigen in solution, the less antibody is bound to the antigen on the walls of the tubes. After the incubation, the incubation medium is removed, the tubes are washed extensively, and the amount of antibody bound to the apolipoprotein on the wall of the tubes is measured. This complex is measured using a second, labeled antibody that reacts with the immunoglobulin in the complex. The second antibody is labeled covalently with an enzyme such as peroxidase [78] or alkaline phosphatase [94]. The amount of labeled antibody bound is then quantitated by measuring the enzymatic activity. Non-competitive, two-site (‘sandwich’) assays have also been devised for apolipoprotein assay [95-981. In this procedure, unlabeled antibody is first coated onto the well walls of a plastic microtiter plate. The remaining unoccupied binding sites on the plastic are then blocked with a non-antibody protein such as bovine serum albumin or geletin [95,96]. The appropriate diluted sample or standard is then added to the wells. The antigen in the sample combines with the prebound unlabeled antibody, and the complex remains bound to the surface of the plastic. The amount of bound antigen depends on its concentration in the sample. The wells are then washed extensively to remove the remaining sample. The amount of antigen in the antigen-antibody complex is determined by adding antibody that has been complexed with peroxidase or alkaline phosphatase, as above. The enzyme-linked antibody binds to a second site on the antigen in the complex, and the amount of bound enzyme-linked antibody is determined by measuring the enzymatic activity. In addition to their sensitivity, ELISA assays have the advantage that the reagents are relatively stable and the assays can be reasonably inexpensive. Like RIA, ELISA assays can be automated, and because of their high sensitivity, ELISA assays can also require high sample dilutions when used to measure Apo AI and Apo B. Inasmuch as the ELISA assays depend on the same primary apolipoprotein-antibody interactions, they can be expected to be influenced similarly by such factors as the exposure of antigenic sites of Apo AI and the affinity constants for antibody-APO B interactions in different lipoprotein particles. Like RIA, ELISA assays theoretically provide a measure of the total Apo AI or Apo B concentration without distinguishing the lipoprotein location of the apolipoproteins. The lipoproteins must first be separated if the amount of apolipoprotein present in a particular lipoprotein class is to be measured. Radial immunodiffurion RID is based on the method devised by Mancini et al. [99]. The assays do not use radiolabeled apolipoproteins or antibodies. The antibody is uniformly incorporated into an agarose gel. The apolipoprotein, either in the standards or samples, is

13

applied to precut circular wells in the gel, and allowed to diffuse into the gel. The apolipoprotein is originally present in excess. As it diffuses into the gel, it eventually forms an immunoprecipitation ring, the area of which depends on the apolipoprotein concentration. At end point, the area of the precipitation ring is directly proportional to the apolipoprotein concentration. The apolipoprotein concentration of the sample is determined from standard curves that relate antigen concentration to ring area. RID assays are technically simpler to perform than RIA or ELISA assays. They are much less sensitive and do not require high dilutions of sample, but use relatively large quantities of antibody. RID assays are not easily automated. Because of the greater size homogeneity of Apo AI-containing particles and the virtual confinement of immunochemically detectable Apo AI to HDL and postHDL-containing fractions [86-891, Apo AI measurements with RID give values similar to those obtained with RIA or other methods [80]. The situation for Apo B is somewhat different, however. RID shows a reasonably high selectivity for Apo B in smaller Apo B-containing particles such as LDL, due to the limited movement of large Apo B-containing particles into the gel [84,85]. Thus, RID underestimates the Apo B in triglyceride-rich lipoproteins and gives lower Apo B measurements than RIA. This is illustrated from the findings of Lutalo-Bosa et al. [loo] who compared RIA and RID measurements of Apo B. In samples with triglyceride levels below 200 mg/dl these workers found that Apo B measurements in unfractionated plasma reflected primarily Apo B in the d> 1.019 g/ml fraction (LDL B). For both RIA and RID there was good agreement between Apo B values measured in either unfractionated plasma or the d > 1.019 g/ml fractions, and reasonably good agreement between plasma Apo B measurements with both methods. At triglyceride levels above 200 mg/dl, however, a greater proportion of plasma Apo B was in the lower density lipoproteins (IDL and VLDL). In those samples RID provided a more nearly accurate measure of Apo B in LDL, and RIA more accurately measured total plasma Apo B, as judged from comparative measurements of Apo B in plasma and the d> 1.019 fraction using both methods. Sniderman et al. [lOl-1031 took advantage of this property of RID to quantitate LDL-Apo B directly in unfractionated plasma. In this method, the samples are applied to 1.5% agarose gel containing anti-LDL, and allowed to diffuse for 18 h. Diffusion does not reach completion in this time, but under these conditions, VLDl does not migrate rapidly enough to affect the measurement of LDL-Apo B. We used a modification of this method in which diffusion was allowed to proceed for 72 h in a 2% agarose gel [104], under which conditions diffusion essentially reached equilibrium. We found that VLDL and IDL contributed an average of 13.8% to the Apo B measurements in plasma in a group of 46 fasting male and female spouses of patients undergoing coronary angiography at the Johns Hopkins Hospital. The mean (SD) cholesterol and triglyceride levels of these subjects were 207 [35] mg/dl and 109 [65] mg/dl, respectively [104]. Thus, even after diffusion reached endpoint, the measurements primarily reflected LDL-Apo B. Under conditions of very high triglyceride concentration, or when the concentration of smaller Apo B-containing particles such as IDL is increased, however, they would be expected to contribute

14

more to the Apo B values measured in unfractionated plasma, and the RID assay would be expected to lose some of its selectivity for LDL-Apo B. Aside from RID, there have as yet not been concerted efforts to exploit immunochemical or physicalchemical differences in lipoproteins to estimate apolipoprotein concentrations in one or another lipoprotein fraction directly in unfractionated plasma or serum. Electroimmunoassay EIA is based on the method originally described by Laurel1 [105]. As with RID, the antibody is uniformly impregnated into a gel composed of agarose [106,107], or a mixture of agarose and dextran sulfate [69,108], or agarose and hydroxymethyl cellulose [109]. The standards or samples are applied to circular wells at one end of the plate and then subjected to electrophoresis, which moves the lipoproteins into the antibody-containing gel. As it moves into the gel, the apolipoprotein forms a precipitin line which has a triangular or ‘rocket’ shape. The area of the rocket is proportional to the antigen concentration. Electrophoresis takes 3 or 4 h. Like RID, EIA is not as sensitive as RIA or ELISA. It requires relatively large quantities of antibody and is not readily automated. It is, however, relatively rapid and uses fairly low dilutions of sample. In contrast to RIA, Apo AI values measured by EIA were about the same whether or not the lipids were extracted from serum before the assays were performed [69]. As with the other methods, Apo AI measurements with EIA reflect primarily Apo AI in HDL and post-HDL plasma, and only one or two percent of the total plasma Apo AI can be detected in the lower density lipoproteins. For Apo B measurement, however, the EIA assay apparently does not detect Apo B equally in all lipoprotein fractions [llO]. This is probably due to differential rates of electrophoretic migration of different lipoproteins into the gel. Pretreatment of the samples with lipase reduces the larger Apo B-containing particles to a more uniform size similar to LDL, and this treatment has been used for Apo B measurements with EIA [110]. Immunonephelometry In INA the apolipoprotein and antibody are allowed to react to form an insoluble complex that remains in a stable suspension long enough to measure the turbidity of the reaction system. The assay is performed under conditions in which the amount of antibody is not limiting. Both the rate of formation of the complex, and the total amount of complex formed, are proportional to the apolipoprotein concentration, and both rate- and endpoint INA methods have been devised [ill]. The antigen concentration in the sample is calculated from standard curves relating turbidity to concentration. In many cases a polyanion, such as polyethylene glycol, is included in the reaction mixture to enhance aggregation [112]. Rate nephelometric methods have the advantage that they are very rapid; Apo AI or Apo B measuremeasurements ments can be made within several minutes [113], whereas endpoint require several hours of incubation. Apolipoprotein measurements by INA can be influenced by the turbidity of the triglyceride-rich lipoprotein themselves when they are present in sufficient con-

15

centration [112,114]. In one report, this interference increased the apparent concentration of serum apo AI by about 5% in samples with triglyceride levels below 175 mg/dl [114]. Lipoprotein turbidity could be reduced by treating the samples with a nonionic detergent (Tween 20) and could be eliminated by first precipitating the Apo B-containing lipoproteins with phosphotungstate and MgCl, [114]. Manipulations such as detergent treatment, heating, delipidation, and treatment with 8 mol/l urea or 6 mol/l guanidine hydrochloride increase immunodetectable Apo AI [75,76,112,113]. Treatment with 8 mol/l urea or guanidine hydrochloride were more effective than delipidation or heating [75]. Apo B measurements are affected by some of the same factors that influence Apo AI measurements, such as the ability of VLDL and LDL themselves to scatter light [73]. One approach has been to treat samples with a detergent or with lipase or a mixture of lipase and cholesteryl esterase [74,111,113] in order to convert the lipoproteins to a smaller, more homogeneous population of particles. The sensitivity of INA procedures is similar to that of EIA and RID methods. When used under optimal conditions, INA gives an estimate of total plasma Apo AI or Apo B concentrations. Apolipoprotein

standardization

As mentioned above, there is at present no available reference method for apolipoprotein analysis and no existing standardization program for these measurements. For the past several years there has been an ongoing collaborative initiative by the Standardization Committee, International Union of Immunological Societies, and the Centers for Disease Control to develop standardization procedures, serumbased reference materials, and quality control materials for the standardization of Apo AI and Apo B measurements [80,115,116]. In this program, the various sources of variation in apolipoprotein measurements are being investigated, including method-related differences, variation in antibody preparations, stability of reference materials and a number of other questions. Over 100 laboratories around the world currently participate in these efforts, and all of the methods discussed above are represented. Table II summarizes the distribution of methods used by these laboratories during different phases of the study [115,117,118]. In 1983, 55 laboratories in 15 countries analyzed Apo AI and Apo B in a lyophilized serum pool that was prepared as a proposed reference material. Each laboratory used at least one method, and several used more than one [115]. In 1986, data were reported from 85 laboratories [117] and in 1987, 143 laboratories participated [118]. Although this was not a survey of methods used in different laboratories, per se, the participants in this study can be assumed to be seriously interested in clinical apolipoprotein measurements, and the table therefore gives some estimate of the most widely used methods. It is clear that EIA, RID and INA were by far the most widely used methods. This is perhaps not too surprising, since these are technically simpler than RIA and ELISA. Based on these data, it might be anticipated that much of the population-based data that will be gathered in the foreseeable future will probably be based on these three methods.

16 TABLE

III

Apo AI and Apo B values measured in IUIS-CDC proposed reference materials Apo AI

Apo B

No. of Analyses

Pool mean

No. of analyses

Pool mean

(mg/dl) 1983 a Pool no. 1883 RIA 44 ELISA RID 176 EIA 96 1986 b Pool LB RIA ELISA RID EIA INA Pool LC RIA ELISA RID EIA INA

(mg/dI)

122 _ 117 122

30 24 112

70 70 59 74

60 24 145 138 174

252 217 259 215 247

48 36 168 144 168

235 165 158 186 229

60 24 145 138 174

303 266 322 262 305

48 36 168 144 168

299 204 192 230 289

121 123 129 125 119

48 36 167 144 168

70 63 54 58 71

Pool LD (same as Pool 1883, above) RIA 60 ELISA 24 RID 145 EIA 138 INA 168

44

a Ref. 11131. b Ref. [115]. TABLE

IV

Apo AI and Apo B values measured in IUIS-CDC

proposed reference materials a

Pool means Apo B

Apo AI Pool A

B

C

lb

Pool A

B

C

lb

RIA ELISA RID EIA INA

136 130 138 131

107 108 112 106

97 93 98 96

110 136 125 132

88 89 68 78

68 70 57 61

65 66 56 59

66 61 56 56

Kinetic Endpoint Turbidimetric

146 137 140

111 104 109

100 93 99

114 119 115

95 102 102

69 76 77

66 71 72

64 71 68

a Ref. (1161. b Same as Pool no. 1883 in Table III

17

Tables III and IV illustrate the mean values, by method, of the serum control pools that were analyzed in these studies. In general, all of the methods gave similar mean Apo AI values for all of the pools, although the ELISA and EIA values were lower in the pools with Apo AI levels above 250 mg/dl (Table III). For apolipoprotein B there was a greater divergence of values with different methods, particularly in the pools with abnormally high (150-300 mg/dl) Apo B levels. Interestingly, the ELISA values were considerably lower than the RIA and INA values in these pools, even though all three methods would be expected to measure total serum Apo B. There was better correspondence between the methods in the pools with an Apo B level in the normal range. As might be expected, the RID and EIA methods tended to give somewhat lower values than the other three methods, even in samples with low Apo B levels. This may have reflected the relative selectivity of these methods for smaller Apo B-containing particles due to the differential migration of the lipoproteins in the supporting media, as discussed above. One of the major findings of these studies was that the greatest source of variation for both Apo AI and Apo B was laboratory-to-laboratory variation. Method-to-method variability was negligible for Apo AI, but was up to 25% of the variability for Apo B [115,117,118]. Although these studies did not address the question of differential lipoprotein specificity of the methods, the greater variability of Apo B measurements, particularly at high Apo B levels, probably reflected the polydispersity of Apo B-containing lipoproteins. Twenty to 30% of the laboratoryto-laboratory variability for Apo AI and Apo B was due to differences in the kinds of calibrators and reagents used [118]. Interestingly, differences in antisera accounted for only 5% and 8% of the variability of Apo AI and Apo B measurements, respectively [116]. These studies are ongoing and should ultimately lead to the development of stable reference and quality control materials for apolipoprotein analysis. Equally importantly, they should provide a better understanding of the various apolipoprotein methods, their relative lipoprotein specificities and other factors that influence the accuracy and precision of the methods. Utility of apolipoprotein assays Assessment of coronary risk Heiss and Tyroler [119] reviewed 18 studies which were published between 1976 and 1982, and which examined the association between apolipoproteins and ischemic heart disease (IHD), as defined by coronary angiography, or survival of a myocardial infarction (MI). Most of these studies were concerned with apolipoproteins AI and B, and rather consistently indicated an inverse relationship between Apo AI level and cardiovascular risk, and a direct relationship between Apo B level and risk. These findings were not unexpected because similar relationships were established previously for HDL, which carries most plasma Apo AI, and LDL, which carries most plasma Apo B, using the respective lipoprotein cholesterol measurements as indices of lipoprotein concentration. Furthermore, in several of the studies in which the apolipoproteins and their corresponding lipoproteins (Apo AI and HDL-

18

cholesterol; Apo B and LDL- or VLDL-cholesterol) were both measured, there was an indication that the apolipoproteins might be somewhat stronger risk predictors than the lipoprotein cholesterol measurements [ 1191. In the past several years, a number of additional studies have appeared in which the relative strength of the association between apolipoprotein and lipoprotein cholesterol measurements and CAD has been investigated. In one study, DeBaker et al. [72] compared 70 MI survivors and 70 controls. They found that both Apo AI and Apo B were predictors of disease. HDL-cholesterol seemed to be a marginally better discriminator than Apo AI. The relative discriminating power of LDLcholesterol and Apo B were not reported. Kostner [120] found that MI survivors were classified more accurately from Apo AI and Apo B measurements than from LDL- or HDL-cholesterol levels. Hamsten et al. [65] found that a group of 116 MI survivors had higher VLDL, LDL and Apo B levels and lower HDL, HDL,, HDL,, Apo AI and Apo AI1 levels than matched controls. They observed proportionate group mean increases in Apo B and LDL whereas the mean percent decrease in HDL-cholesterol in the CAD patients was about twice that of Apo AI. This appeared to be due to the stronger association of low HDL-cholesterol with CAD in hypertriglyceridemic patients. Others [63] found that Apo AI and Apo B better distinguished relatives of CAD patients from controls than did HDL-cholesterol, total cholesterol or triglycerides. In univariate analyses between CAD patients and controls, Apo B was the best discriminator followed by HDL-cholesterol. Apo B was the best discriminator in male patients, while Apo AI was better in women. Schmidt et al. [SS] found that LDL-cholesterol and LDL-Apo B about equally separated male CAD patients from controls, whereas HDL-cholesterol was a better indicator than Apo AI. Although similar trends were observed in women, they were not statistically significant in that study. Conversely, Maciejko et al. [121] reported that Apo AI was a better marker for angiographically documented CAD than was HDL-cholesterol. Kukita et al. [66] found that male CAD patients had lower Apo AI and higher Apo B levels than matched controls. This relationship was independent of serum triglyceride level. HDL-cholesterol also discriminated CAD from controls in patients with normal triglyceride levels but not in patients with elevated triglycerides. Sniderman et al. [101,122] and others [123-1251 reported that the plasma level of Apo B in LDL (LDL-B) better discriminated patients with angiographically documented CAD than LDL cholesterol levels. Sedlis et al. [50], comparing the ability of lipoprotein lipids and apolipoproteins to predict the severity of coronary artery disease found that although the apolipoproteins were better predictors than the corresponding lipoproteins, neither were strong predictors of the severity of disease. Lehtonen and co-workers [64] found that HDL-cholesterol and Apo AI were about equally sensitive in correctly classifying about 70% of the coronary artery bypass patients with three vessel disease and controls that they studied. Similarly, LDL-cholesterol and Apo B were equally sensitive in classifying accurately about 60% of the patients and the controls. Kempen et al. [126] examined the relation between the extent of coronary artery stenosis and left ventricular ejection fraction, and various parameters including

19

LDL- and HDL-cholesterol and Apo AI and AII. They found weak correlations between LDL-cholesterol or HDL-cholesterol, but not between APO AI or Apo AIL and the extent of stenosis. However, they reported strong correlations (r = 0.5, p < 0.001) between Apo AI or Apo AI1 levels and left ventricular function, and lesser but still significant correlations between HDL cholesterol (r = 0.40, p < 0.01) and ventricular function. The reason for the relationship between ventricular function and the apolipoprotein levels was not clear. Van Stiphout et al. [59] found that of the lipoproteins and apolipoproteins, the Apo B/APO AI ratio in the sons (but not in the daughters) of patients with coronary atherosclerosis best discriminated those whose fathers had 3 vessel disease (> 70% stenosis) from those without significant disease (< 20% stenosis), while the HDL-cholesterol/LDL-cholesterol ratio was the best discriminators in the fathers themselves. A similar association appears to exist in the general population also. Freedman et al. [127] found that children whose fathers reported that they had suffered a myocardial infarction had significantly lower levels of Apo AI and lower ratios of LDL cholesterol to Apo B, as well as significantly higher ratios of Apo B to Apo AI than children whose fathers who had not had a heart attack. In children whose mothers had reported having an MI, the LDL cholesterol to Apo B ratio was unaltered, but they had higher Apo B to Apo AI ratios. Interestingly, serum lipoprotein cholesterol measurements were not related to the MI history of either the fathers or the mothers. Although the results of these studies are not all entirely consistent, they nonetheless generally indicate (1) that Apo AI and Apo B are strong risk predictors, (2) that at least in some cases the apolipoprotein levels, particularly Apo B, seem to be more strongly associated with cardiovascular risk than LDL- or HDL-cholesterol levels, and (3) that the apolipoproteins are better indicators of the presence or absence of disease than of the degree of stenosis. Apo AI and Apo B primarily reflect the plasma concentrations of LDL and HDL, respectively. The fact that they differ somewhat from LDL-cholesterol and HDL-cholesterol in the strength of their relation to risk, however, reflects the varying cholesterol-apolipoprotein composition of these lipoproteins in vivo. This was not entirely unexpected, because various workers have found less than a perfect correlation between the levels of these apolipoproteins and the two lipoprotein cholesterol levels. For example, most workers who have measured the correlation between HDL cholesterol and Apo AI have reported correlation coefficients of approximately 0.6 to 0.7 [60,86,121,126,128,129]. In our laboratory, we have observed that plasma Apo AI:HDL-cholesterol ratios vary from about 2 : 1 to 4 : 1. This is similar to the findings of Friedman et al. [130]. Apo AI and cholesterol are components of both HDL, and HDL,, and each of these fractions can be separated into several subfractions that vary in their chemical and apolipoprotein composition [131,132]. This undoubtedly contributes to the wide variation in the relative amounts of Apo AI and HDL-cholesterol present in plasma. The basis for the apparent compositional variation in LDL is better understood. Several years ago, Sniderman et al. [loll found that the majority of the patients they

20

studied with angiographically documented CAD had elevated levels of LDL-B and normal levels of LDL cholesterol. They coined the term ‘hyperapobeta-lipoproteinemia’, or ‘hyperapoB’, to distinguish this condition from hyperbetalipoproteinemia (Type II hypercholesterolemia), in which both the cholesterol and Apo B

TABLE

V

Apolipoprotein Method

AI and B levels in normal n

men Apo AI

Age (yr)

Apo B

Ref.

100 (35)

_

[541

109 (24)

_ _

Mean (SD) (mg/dl) RIA

41

37 a

19

(13-58) _

65 d 17 115

RID

EIA

133 (32) 130 (20)

27 b _

116 (30) _

20-29

58

30-39

_

78 (19)

56

40-49

_

88 (21)

47

50-59

_

85 (18)

2

60-65

_

50

20-29

117 (18)

90 _

77

30-39

117 (19)

_

77

40-49

120 (10)

_

22

50-59

125 (22)

_

4

126 (20)

172

50-65 _

95

40-49

35

53 h

137 (23) _

90d

22 b

_

72 (17)

38 d

10 b

_

72 (19) _

120 (20)

123 (21)

31 d

_ _ 83 (11)

146d

_

138 (12)

98 (19)

40

52 ’ _

121 (30) 143 (24)

132 (28) _

10 (3.4) c

141 (22)

84 (21)

77

51(11)

c

130 (18)

84 (20)

80

35 (10) c

132 (20)

83 (13)

116

40 (4) = 50 (11) c _

126 (19)

105 (20)

129 (20)

86 (18) _

19

140 31 d 115 70 3032 12

_

125 (21)

18-65

136 (23)

29b

139 (15) _

’ Mean (range). b Mean. ’ Mean (SD). Males and females combined

127 (28) _

50 (9) =

35 d

d

81 (19)

26

2277 d

INA

_

or sex not indicated.

112 (21) 113 (23) _ _ 159 (59)

~1291 v31 1751 1641 [901 [901 [901 I901 PI [@‘I [601 [hOI PI [601 Pfd I1501 P221 v221 (1221 [761 PO91 11511 [691 [1271 F31 Lb31 1651 WI [761 Lb41 ~721 [114, 1281 [751 (731

21

components of LDL are elevated [122]. HyperapoB has also been described in a large percentage of MI patients, patients with familial combined hyperlipidemia and in phytosterolemia [122]. The latter is a condition in which plant sterols, which are not normally absorbed in man, accumulate in the blood and tissues. Several subclasses of LDL have been identified which differ somewhat in their densities. Most normolipidemic subjects have a predominance of one of the less dense subfractions, while in a few one of the more dense fractions predominate [122]. In hyperapoB, the increased LDL-B level results from the presence of an increased level of LDL particles that are smaller, denser, cholesteryl ester depleted, and relatively enriched in Apo B [104]. Since the LDL particle is depleted in cholesteryl esters, the patient is usually normocholesterolemic or has only a borderline-high cholesterol level. Because of the altered composition of LDL, the elevated LDL levels in hyperapoB can be detected more accurately by measuring LDL-B than by measuring LDL cholesterol. In contrast, in type II hypercholesterolemia, the increased LDL levels are revealed by measuring either LDL-B or LDL cholesterol. For this reason it is perhaps not surprising that LDL-B is a better risk predictor than LDL cholesterol; the latter does not accurately reflect the increased LDL levels in hyperapoB patients. A predominance of a more dense LDL subfrac-

TABLE VI Apo I and B levels in healthy women Method

RIA

RID

EIA INA

’ Mean (range). b Mean. ’ Mean (SD).

n

Age (yr) 34

21 35 66 29 38 25 2 44 14 27 12 2 188 104 19 74 1387 14

29 a (16-58) 33 b 20-29 30-39 40-49 50-59 60-65 20-29 30-39 40-49 50-59 60-65 40-49 36 (10) = 18-65 30 b

Apo AI

Apo B mean (SD)

Ref.

104 (34)

1541

122 (27) 149 (30)

11291 1751 WI WI WI WI WI WI 1601 1601 WI 1601 WI [1501 (691 W31 [114,128]

74 71 82 86 121 125 126 132 139 146 135 137 146 139 147 147

(18) (18) (21) (16)

(20) (22) (20) (40) (25) (23) (78) (17) (25) (24)

_ 82 (12)

1751

22 TABLE

VII

Primary hyperlipoproteinemias:

assessment of apolipoproteins

of diagnostic utility

Disorder

Lipoprotein pattern

Use of apolipoprotein measurement

Familial hypercholesterolemia

IIa (occasional IIB) Normal; IIa (occasional IIb)

LDL B markedly elevated with a high ratio of LDL cholesterol to LDL B ( > 1.6) LDL B elevated with a low ( < 1.2) or normal (1.2 to 1.6) ratio of LDL cholesterol to LDL B Total plasma Apo B elevated with a low ratio of LDL cholesterol to LDL B LDL B elevated with a normal LDL cholesterol level; low ratio of LDL cholesterol to LDL B ( -z 1.2) Apo E phenotype E,, E, Apo C-II levels barely detectable

Sitosterolemia

Familial combined hyperlipidemia

IIa, IIb, IV

Hyperapobetalipoproteinemia

Normal: IV

Dysbetalipoproteinemia Apo C-II deficiency

I, V

III

tion is also associated with familial combined hyperlipidemia [122]. In cases such as these, knowledge of both the LDL-B and LDL cholesterol level gives more information than either measurement alone. Tables V and VI illustrate the Apo AI and Apo B levels that have been reported in healthy subjects in various studies. Diagnosis of primary lipoprotein disorders Primary hyperlipoproteinemias The primary hyperlipoproteinemias in which an assessment of poproteins can be useful diagnostically are summarized in Table VII.

the apoli-

Familial hypercholesterolemia Familial hypercholesterolemia (FH) is a Mendelian dominant disorder characterized by significant elevations of plasma total and LDL cholesterol; the plasma triglyceride level is usually normal (type IIa), but can be elevated (type IIb) (Table V). FH is due to a deficiency in the LDL (B,E) receptor [133]. In patients with heterozygous FH, the diagnosis in adults is often suspected because of the presence of xanthomas in the extensor tendons of the hands, Achilles tendons, elbows and tibia1 tuberosity. In patients without tendon xanthomas, the diagnosis may be suspected if the ratio of LDL cholesterol to LDL B is high, that is, greater than 1.6. The larger, cholesteryl ester-enriched LDL are secondary to the catabolic defect in FH which results in an increased residence time of LDL in plasma [134]. A high ratio of LDL cholesterol to LDL B is not always present in patients with FH, however. Assays of LDL receptor activity in cultured fibroblasts or lymphocytes can be useful, particularly in detecting individuals who are FH homozygotes, but there can be considerable overlap in LDL receptor activity in cells from FH heterozygotes and normals [133].

23

Sitosterolemia Sitosterolemia is a metabolic disorder characterized by the abnormal presence of elevated levels of plant sterols in blood. Sitosterolemia is due to the double dose of a mutant allele, and in the recessive state the disorder is usually expressed as large, tendon and tuberous xanthomas in the first decade of life [135]. The metabolic defects in sitosterolemia include abnormal absorption of plant sterols from the diet, decreased removal of sitosterol from the blood, and decreased conversion of sitosterol into bile acids [136]. Sitosterolemia was initially described by Bhachattyra and Connor [137] as a disorder of lipid and lipoprotein metabolism in which the lipids are normal, but it is now generally appreciated that sitosterolemia is often accompanied by elevations in LDL cholesterol and LDL B protein [135]. While sitosterolemia is a rare trait (about 18 cases in the world’s literature) [136], patients with sitosterolemia have been mistakingly diagnosed as FH heterozygotes [138]. In the original two patients described by Bhachattyra and Conner, in whom the lipoprotein patterns tend to be normal, and in six patients from a large Amish kindred, in which the lipoprotein patterns tend to be Type IIa, or occasionally IIb, an elevation of LDL B protein was found with a low (< 1.2) or normal (1.2-1.6) ratio of LDL cholesterol to LDL B protein [135]. Thus, the presence of an elevated LDL B protein, particularly in a normolipidemic or mildly hypercholesterolemic patient with xanthomas, suggests the presence of sitosterolemia. The definitive diagnosis of sitosterolemia is made by demonstrating the presence of plant sterols in plasma. Familial combined hyperlipidemia Familial combined hyperlipidemia (FCH) was initially described in 1973 by Goldstein and co-workers [139] in survivors of myocardial infarction and their families. Affected relatives had either significant hypercholesterolemia (type IIa lipoprotein pattern), significant hypertriglyceridemia (type IV lipoprotein pattern), or both hypercholesterolemia and hypertriglyceridemia (type IIb lipoprotein pattern). FCH apparently results from an abnormality in a single gene (Mendelian dominant trait). In contrast to FH, tendon xanthomas are not present in FCH. FCH results from an overproduction of VLDL in liver [140,141]. More recently, a low ratio of LDL cholesterol to LDL B has been found in patients with FCH [142]. Increased LDL levels in FCH result from the overproduction of VLDL and the subsequent enhanced conversion of VLDL to LDL [140,141]. The LDL receptor in FCH is normal [142]. Thus, FCH is an overproduction defect rather than a defect in the catabolism of LDL. Many patients with significant elevations of LDL but who do not have tendon xanthomas have FCH rather than FH. The definitive diagnosis of FCH in a given patient can be difficult. If first degree relatives are available for study, the presence of FCH can be confirmed by documenting the various lipoprotein patterns that characterize this disorder in affected first-degree relatives. Hyperapobetalipoproteinemia (hyperapoB) HyperapoB was initially described by Sniderman et al. [loll in patients undergoing elective coronary arteriography (see also above). About half of the patients with

24

angiographically documented coronary atherosclerosis had an elevated LDL B level (> 120 mg/dl) but a normal or near normal level of LDL cholesterol (majority between 130 mg/dl and 160 mg/dl). A low ratio of LDL cholesterol to LDL B is present in patients with hyperapoB. Initially described as a disorder of lipoprotein metabolism in which the lipids were normal, hyperap was subsequently found to be present in both normolipidemic and hypertriglyceridemic survivors of myocardial infarction (1031. While the normolipidemic lipoprotein pattern found in hyperapoB was not originally described in FCH, a possible relation between FCH and hyperapoB was suggested by the finding of hyperapoB in h~ert~glyceride~c survivors of myocardial infarction and in a significant proportion of patients with type IV lipoprotein patterns (normal LDL cholesterol) studied in a lipid clinic ]103]. HyperapoB, like FCH, is due to the overproduction of VLDL in liver with a subsequent overproduction of LDL B in plasma [134]. The measurement of Apo B in patients with coronary artery disease who have normal lipoprotein patterns is particularly useful, and the finding of an elevated LDL B in such patients indicates that the patient has a primary lipoprotein disorder which needs to be followed and treated. Patients with hyperapoB also have a defect in the clearance of postprandial lipoproteins following a fat load [143]. This defect is accompanied by a decrease in HDL,. HDL cholesterol level and apolipoprotein AI levels are often low in patients with hyperapoB [ 1221.

Dysbetalipoproteinemia (type III hyperlipoproteinemia) is a lipoprotein disorder which often presents as marked increases in both plasma total cholesterol and triglyceride [144]. The hyperlipidemia results from the presence of increased amounts of VLDL remnants that contain both cholesterol and triglyceride. VLDL from patients with dysbe~poproteine~a migrate as beta- rather than as prebeta lipoproteins on agarose electrophoresis, and thus has been called ‘floating beta lipoproteins’ or &VLDL. As discussed above, lipoproteins isolated in the VLDL density range ordinarily have a ratio of cholesterol to triglyceride of about 0.2, while the VLDL in dysbetalipoproteinemia have a cholesterol to triglyceride ratio that is usually > 0.3. Many patients with dysbet~ipoproteine~a have distinctive orange deposits in the creases of the palms of the hands (planar palmar xanthomas), and tuberous xanthomas over the elbows, knees, and buttocks. Tendon xanthomas can also appear in patients with dysbetalipoproteinemia. Dysbetalipoproteinemia seems to result from the confluence of two factors: (1) the presence of Apo E,/E, phenotype for Apo E; and (2) presumably another ‘gene’ associated with ove~r~uction of VLDL 11451. Thus, in patients suspected of having dysbet~poproteine~a, the demonstration of the E,/E, phenotype is a useful confirmatory test. Dysbetalipoproteinemia results from a slow clearance of the VLDL remnants because apolipoprotein E, has a decreased affinity for the LDL (B,E) receptor [145]. In addition, the clearance of chylomicron remnants is also delayed in patients with dysbetalipoproteinemia because the chylomicron remnants that contain only the E, isoform of Apo E are cleared more slowly than normal through the hepatic chylomicron remnant (Apo E) receptor. While relatively

25

uncommon (prevalence about 1 in 1500 poproteinemia is important because it is premature CAD and peripheral vascular table with diet and fibric acid derivatives

to 1 in 2000), the diagnosis of dysbetaliassociated with a marked predilection to diseases [144,145] but is eminently treaor nicotinic acid.

Apo C-II deficiency Apo C-II deficiency is a rare metabolic disorder of lipoprotein metabolism which usually presents in adulthood as profound hypertriglyceridemia [7,146]. The lipoprotein pattern is type I (increased chylomicrons alone) or type V (increased chylomicrons and VLDL). Patients may have a history of pancreatitis, or present with markedly creamy plasma. The levels of Apo C-II, a required co-factor for lipoprotein lipase, are barely detectable using immunochemical assays [7,146]. Thus, an assessment of the plasma levels of Apo C-II in patients with either type I or type V lipoprotein patterns may provide useful etiologic information, and help to distinguish those with Apo C-II deficiency from the classic patients with lipoprotein lipase deficiency (type I) or patients with a more common form of type V hyperlipoproteinemia, in which Apo C-II is not deficient and lipoprotein lipase activity is either normal or low, but not absent. Primary hypolipoproteinemias In Table VIII are summarized the primary hypolipoproteinemias assessment of apolipoproteins can be of diagnostic utility.

in which the

Familial hypoalphalipoproteinemia Familial hypoalphalipoproteinemia was described [147,148] in families with premature CAD. Familial hypoalphalipoproteinemia is characterized by very low levels of HDL cholesterol and Apo AI. The lipoprotein pattern is hypoalpha (low HDL cholesterol, normal LDL cholesterol and LDL B) and results from the

TABLE VIII Primary hypolipoproteinemias: assessment of apolipoproteins of diagnostic utility Disorder

Lipoprotein pattern

Use of apolipoprotein measurement

Familial hypoalphalipoproteinemia Apo AI variants

hypoalpha; type IV hypoalpha; normal

Abetalipoproteinemia

abeta

Hypobetalipoproteinemia

hypobeta

Apo AI low; confirms low HDL-C level. LDL B level normal. Low Apo AI levels with low or normal HDL cholesterol levels; abnormal electrophoretic migration of Apo AI mutants. Document absence of immunochemically detectable Apo B-100; assess presence or absence of Apo B-48. Document presence of small amounts of Apo B-100

26

apparent influence of a Mendelian dominant trait. Triglyceride levels may be low, normal, or mildly elevated. The measurement of Apo AI levels are useful to confirm that a low HDL level is present. The etiology of familial hypoalphalipoproteinemia has not been clearly established and defects of decreased production of Apo AI or enhanced removal of HDL from plasma are under investigation in a number of laboratories. Since low HDL cholesterol levels are often secondary to hypertriglyceridemia, the diagnosis of familial hypoalpha can be difficult to make. In patients who have normal levels of cholesterol, triglyceride and LDL B protein but who have low levels of HDl cholesterol and apoA1, the diagnosis can be more clearly established. Thus, the measurement of LDL B protein is also useful in assessing patients with low HDL cholesterol levels to assess the presence of either hyperapoB or FCH (see above). Hypoalphalipoproteinemia - Apo AI variants A number of variants of Apo AI have been described, specifically Apo AIGiesanr Apo AIMit,_,, Apo AfMlarburg, and Apo AIBaltimore 11471. Tw~dimensional polyacrylamide gel electrophoresiscan be useful in detecting these variants in patients with low levels of HDL cholesterol and Apo AI. Molecular biological studies of the apolipoprotein AI gene have also been useful as a diagnostic tool to identify such patients. In affected members from these families, the HDL cholesterol is not always low and can in fact be normal. The apolipoprotein AI levels, however, are usually low. In some families, Apo AI variants have been associated with CAD, while in others, this seems not to be the case. At present, the usefulness of detecting Apo AI variants in the general population has not been established. Abetalipoproteinemia Ab~ta~poproteine~a is a primary hypolipoproteine~a characterized by the absence of i~un~he~cally detectable Apo B-100 in plasma [87]. The abeta lipoprotein pattern (no detectable VLDL or LDL present in plasma) results from the expression of a double dose of a mutant allele. Clinical findings include fat malabsorption, steatorrhea, retinitis pigmentosa, cerebellar ataxia and other neurological problems. Abetalipoproteinemia is most often due to the complete absence of both Apo B-100 and Apo B-48 and hence these patients cannot mobilize dietary fat from the intestine producing deposition of fat droplets in the intestinal cells. Furthermore, VLDL cannot be mobilized from the liver, and VLDL and LDL are therefore absent from the plasma. Because of the inability of these patients to absorb and transport fat-soluble vitamins, the clinical findings are believed to be secondary to deficiencies in fat-soluble vitamins. Immunochemical assay is critical to document the absence of Apo B-100 in plasma. After a fatty meal, the presence or absence of Apo B-48 can be determined by polyacrylamide gel electrophoresis (see above), since some variants of abetalipoproteinemia have been described in which Apo B-48 is present and Apo B-100 absent [149]. Hypobetalipoproteinemia H~obeta~poproteine~a is a Mendelian dominant condition which ordinarily presents as low levels of LDL cholesterol in blood 1871. The immun~he~c~ assay

21

useful to demonstrate that Apo B-100 is present, which definitively rules out abetalipoproteinemia. Some patients have a double dose of the mutant allele, and thus are homozygous for a dominant trait. Their clinical presentation is often similar to that of patients with classic abetalipoproteinemia. In such patients, both of the parents will have hypobeta, while the parents of patients with abetalipoproteinemia will usually have normal lipoprotein patterns. is

Summary Qualitative and quantitative measurements of apolipoproteins such as Apo CII and Apo E, as well as Apo AI and Apo B can aid in the diagnosis of specific lipoprotein metabolic defects. When used in this way, their usefulness will probably remain more or less confined to specialized research centers which have the expertise to perform such analyses and interpret the measurements. In addition, however, Apo AI and Apo B also promise to be useful for the estimation of cardiovascular risk both in individuals and in populations, and both of these apolipoprotein measurements will probably supplement rather than replace conventional lipoprotein measurements. As with the lipoproteins, the usefulness of Apo AI and Apo B measurements will depend on the accuracy and precision with which they can be measured. The current standardization efforts should lead to the development of reference methods for the measurement of total plasma levels of Apo AI and Apo B, the availability of reference materials for these apolipoproteins, and eventually, to the establishment of methods that can specifically measure Apo AI and Apo B in particular lipoprotein subfractions. Acknowledgements This work was supported in part by NIH grants 5ROl HL31450 and 2ROl HL31497. The authors are indebted to Ms. Carol McGeeney for preparing the manuscript. References Jackson RL, Morrisett JD, Gotto Jr AM. Lipoproteins and lipid transport: structural and functional concepts. In: RXkind BM, Levy RI, eds. Hyperlipidemia: diagnosis and therapy. New York: Grune and Stratton, 1977;1-16. Bisgaier CL, Glickman RM. Intestinal synthesis, secretion, and transport of lipoproteins. Ann Rev Physiol 1983;45:625-636. Kane JP, Hardman DA, Paulus HE. Heterogeneity of apolipoprotein B: Isolation of a new species from human chylomicrons. Proc Nat1 Acad Sci (USA) 1980;77:2465-2469. Green PHR, Glickman RM. Intestinal lipoprotein metabolism. J Lipid Res 1981;22:1153-1173. LaRosa JC, Levy RI, Herbert P, Lux SE, Fredrickson DS. A specific apoprotein activator for lipoprotein lipase. Biochem Biophys Res Commun 1970:41:57-62. Brown WV, Baginsky ML. Inhibition of lipoprotein lipase by an apoprotein of human very low density lipoprotein. Biochem Biophys Res Commun 1972;46:375-382. Breckenridge WC, Little LA, Steiner G, Chow A, Poapst M. Hypertriglyeridemia associated with deficiency of apolipoprotein C-II. N Engl J Med 1978;298:1265-1273.

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