Pathology and Laboratory Medicine - part 8 potx

ABBREVIATIONS ACS, Acute coronary syndromes; AMI, acute myocardial infarction; CK, creatinekinase; CK-MB, MB isoenzyme of CK; cTnI, cTnT, cardiac troponins I and T; ECG,electrocardiogram

FABP as Marker of Myocardial Ischemia 325

costal pectoral muscles, and in whom the plasma myoglobin/FABP ratio increased from

8 to 60 during the first 24 h after AMI Finally, in situations where AMI patients show

a second increase of plasma concentrations of marker proteins, the ratio may be of help

to delineate whether this second increase was caused either by a recurrent infarction or

by the occurrence of additional skeletal muscle injury In the former case, the ratio willremain unchanged (38)

EARLY DIAGNOSIS OF AMI

The application of FABP especially for the early diagnosis of ACS is already cated from (1) its rapid release into plasma after myocardial injury, and (2) its relativelylow plasma reference concentration Several studies have now firmly established thatFABP is an excellent plasma marker for the early differentiation of patients with andthose without AMI, and that it even performs better than myoglobin A selection of thesestudies is discussed here

indi-Retrospective analyses of various marker proteins in plasma samples from patientswith AMI revealed that the diagnostic sensitivity for detection of AMI is better for FABPthan for myoglobin or CK-MB, especially in the early hours after the onset of symptoms

Fig 4 Mean plasma concentrations of myoglobin (MYO; l) and FABP ({) (left) and themyoglobin/FABP ratio (s) (right) in nine patients after AMI (and receiving thrombolytictherapy) (A), and in nine patients after aortic surgery (B) Data refer to means ± SEM (Adaptedfrom ref 38.)

326 Glatz et al.

For example, in a study including blood samples from 83 patients with confirmed AMI,taken immediately upon admission to the hospital (<6 h after chest pain onset), the diag-nostic sensitivity was significantly greater for FABP (78%, CI: 67–87%) than for myo-globin (53%, CI: 40–64%) or for CK-MB activity (57%, CI: 43–65%) (p < 0.05) (44)

In the last few years, larger studies have been done that allow for the proper ment of both the sensitivity and the specificity of FABP for AMI diagnosis In a (single-center) study with 165 patients admitted 3.5 h (median value) after the onset of chestpain, Ishii et al (43) found in admission blood samples diagnostic sensitivities and speci-ficities for FABP (>12 ng/mL) of 82% and 86%, respectively, and for myoglobin (>105ng/mL) of 73% and 76%, respectively (FABP vs myoglobin significantly different; p <0.05) A similar superior performance of FABP over myoglobin, in terms of both sensi-tivity and specificity of AMI diagnosis, was also observed in a prospective multicenterstudy consisting of four European hospitals and including 312 patients admitted 3.3 h(median value; range 1.5–8 h) after the onset of chest pain suggestive of AMI (EURO-CARDI Multicenter Trial) (49,50) For instance, specificities >90% were reached forFABP at 10 µg/L and for myoglobin at 90 ng/mL Using these upper reference concentra-tions in the subgroup of patients admitted within 3 h after onset of symptoms (n = 148),the diagnostic sensitivity of the first blood sample taken was 48% for FABP and 37%for myoglobin, whereas for patients admitted 3–6 h after AMI (n = 86), the sensitivitywas 83% for FABP and 74% for myoglobin (49,50) In addition, the areas under thereceiver operating characteristic (ROC) curves, constructed for the admission blood sam-ples from all patients, were 0.901 for FABP and 0.824 for myoglobin (significantly dif-ferent; p < 0.001) (Fig 5) This better performance of FABP over myoglobin for theearly diagnosis of AMI has also been reported in other smaller studies (27,51,52).More recently, Okamoto et al (53)confirmed the above findings by demonstrating,

assess-in a sassess-ingle-center study consistassess-ing of 189 patients admitted to hospital withassess-in 12 h afterthe onset of symptoms, that the area under the ROC curve of FABP was 0.921, whichwas significantly (p < 0.05) greater than that of myoglobin (0.843) and CK-MB activ-ity (0.654) In addition, a multicenter study consisting of three North American hospi-tals and including 460 consecutive patients, reported by Ghani et al (28), also revealed abetter diagnostic performance of FABP over myoglobin during the first 4 h after admis-sion, the areas under the ROC curves being 0.80 for FABP and 0.73 for myoglobin.Strikingly, the area under the ROC curve of CK-MB mass was 0.79, and that of cTnIwas 0.91 (28), which caused the authors to conclude that in their study neither FABP normyoglobin show the sensitivity and specificity necessary to detect AMI significantlyearlier than do the existing markers This conclusion seemingly contradicts the well-documented poor diagnostic performance of CK-MB mass and cTnT or cTnI in the veryearly hours after infarction (cf Fig 6) (13,39) The discrepancy is explained by the factthat the hospital delay time, which was not given, has to be added to the admission time.When assuming a hospital delay of 3–4 h, the study results would apply to the period up

to 7 or 8 h after the onset of symptoms, whereas FABP and myoglobin are useful cially in the preceding hours

espe-In some of these above-mentioned studies, investigators evaluated whether the nostic performance of FABP as early plasma marker of myocardial injury could fur-ther improve when the criterion of a plasma myoglobin/FABP ratio <10 (or <14), that

diag-FABP as Marker of Myocardial Ischemia 327

Fig 5 ROC curves for detection of AMI in 238 patients with chest pain suggestive of AMI,and admitted to hospital within 6 hours from the onset of symptoms, comparing the concentra-tions of FABP (l) and myoglobin (n), and the myoglobin/FABP ratio (U) in the admissionblood sample ROC curves were constructed by plotting the sensitivity (% true positives) forthe confirmed AMI group (135 patients) against 100 - specificity (% false positives) for thenon-AMI group (103 patients) The areas under the ROC curves are 0.874 for FABP, 0.780 formyoglobin, and 0.870 for the myoglobin/FABP ratio (FABP vs myoglobin, and FABP vs theratio significantly different; p < 0.001) (Data obtained from the EUROCARDI MulticenterTrial [49,50].)

Fig 6 ROC curves for detection of AMI in patients having either AMI (n = 15) or unstableangina pectoris (UAP; n = 10), comparing selected markers of muscle necrosis (FABP, myo-globin [Mb], and TnT) and markers of activated blood coagulation (fibrin monomers [FM] andTpP) Median hospital delay was 2.8 h (range 0.8–6 h) ROC curves were obtained from double-logarithmic plots Lack of discrimination by TnT is apparent from its coincidence with the line ofidentity Arrows indicate optimal cutoff values For a combined test, that is when either FABP >

6 ng/mL or TpP >7 mg/L as diagnostic for AMI, the sensitivity was 87% and the specificity 80%.(Adapted from Hermens et al [67].)

328 Glatz et al.

is, the exclusion of skeletal muscle as source of FABP, is taken as an additional eter (28,43,50,52) In each of these study populations, there were a few cases in whichboth myoglobin and FABP were elevated in the admission plasma sample, but in whichthe myoglobin/FABP ratio was >10 (or >14) Without this latter result, these patientswould be falsely diagnosed as having had myocardial injury However, because the prev-alence of skeletal muscle injury in these study populations was very low (<1% of cases),this additional parameter did not significantly alter the ROC curve for FABP (Fig 5).Therefore, the routine measurement of the myoglobin/FABP ratio in samples from patientssuspected for MI does not seem justified because it does not add value to the measure-ment of FABP alone In addition, the myoglobin/FABP ratio cannot provide absolutecardiac specificity (3)

param-At first sight it may be surprising that FABP appears as an earlier marker for AMIdetection than does myoglobin, even though the two proteins show similar plasmarelease curves However, these findings can be explained when realizing that the myo-cardial content of FABP (0.57 mg/g wet wt) is four- to fivefold lower than that of myo-globin (2.7 mg/g wet wt), yet the plasma reference concentration of FABP (1.8 ng/mL)

is 19-fold lower than that of myoglobin (34 ng/mL) (Table 1) This means that afterinjury the tissue to plasma gradient is almost fivefold steeper for FABP than for myo-globin, making plasma FABP rise above its upper reference concentration at an earlierpoint after AMI onset than does plasma myoglobin, thereby permitting an earlier diag-nosis of AMI

It is now firmly documented that the subgroup of patients with unstable angina toris who show a significantly increased plasma concentration of cTnT (>0.2 ng/mL)have a prognosis as serious as do patients with definite AMI (54,55) This observationmost likely relates to the presence of minor myocardial cell necrosis In those patients

pec-in whom unstable angpec-ina pectoris is pec-in fact acute mpec-inor MI, the advantage of FABP forearly assessment of injury may be used Recently, Katrukha et al (56) measured FABPand cTnI in serial plasma samples from 31 patients with unstable angina and showed that

in the admission sample cTnI was elevated (cutoff value 0.2 ng/mL) in 13% and FABP(cutoff value 6 ng/mL) in 54% of patients, whereas at 6 h after admission cTnI waselevated in 58% and FABP in 52% of patients Importantly, all patients who had an ele-vated FABP concentration at 6 h showed an elevated cTnI value at 12 h after admission(56) These preliminary data suggest that FABP may identify (acute) minor MI withsimilar sensitivity as cTnI, but at an earlier point after admission of the patient.EARLY ESTIMATION OF MYOCARDIAL INFARCT SIZE

Myocardial infarct size is commonly estimated from the serial measurement of cardiacproteins in plasma and calculation of the cumulative release over time (plasma curvearea), taking into account the elimination rate of the protein from plasma (57) Thisapproach requires that the proteins are completely released from the heart after AMIand recovered quantitatively in plasma Complete recovery is well documented for CK,LDH, and myoglobin (but does not apply for the structural proteins cTnT and cTnI [39]),and could also be shown for FABP (37,58) Figure 2 (lower panel) presents the cumu-lative release patterns of these four proteins, expressed in gram-equivalents (g-eq) ofhealthy myocardium per liter of plasma (i.e., infarct size) The release of FABP and myo-

FABP as Marker of Myocardial Ischemia 329

globin is completed much earlier than that of either CK or LDH, but despite this kineticdifference for each of the proteins, the released total quantities yield comparable esti-mates of the mean extent of myocardial injury when evaluated at 72 h after the onset ofAMI (Fig 2)

This method to estimate infarct size has proven its value when applied to the tion of early thrombolytic therapy in patients with AMI (59) With the (classically used)enzymatic markers, the method has the drawback that the data on infarct size in the indi-vidual patient become available relatively late (72 h), that is, too late to have an influence

evalua-on acute care (60) For the more rapidly released markers FABP and myoglobin, infarctsize estimation for individual patients is hampered by the fact that these proteins arecleared by the kidneys, and the patients often suffer from renal insufficiency, whichwould lead to overestimation of infarct size De Groot et al (61) recently suggested theuse of individually estimated clearance rates for FABP and myoglobin to measure myo-cardial infarct size within 24 h These individual clearance rates are calculated using glo-merular filtration rates (estimated from plasma creatinine concentrations and correctedfor age and gender) and plasma volume (corrected for age and gender) This impliesthat a reliable estimate of myocardial infarct size becomes available when the patient

is still in the acute care department, if frequent blood samples are taken and analyzedrapidly

FABP AS REPERFUSION MARKER

The application of FABP as a plasma marker for the early detection of successful onary reperfusion in patients with AMI has been investigated by three groups (62–64).Ishii et al (62) studied 45 patients treated with intracoronary thrombolysis or direct per-cutaneous transluminal coronary angioplasty (PTCA), in whom coronary angiographywas performed every 5 min to identify the onset of reperfusion Both plasma FABPand myoglobin were found to rise sharply after the onset of reperfusion, and the rela-tive first-hour increase rates of both markers showed a predictive accuracy of >93%.Subsequently, in a study consisting of 58 patients, de Lemos et al (63) also demon-strated that following successful reperfusion plasma FABP and myoglobin rise sharply,whereas in patients with failed reperfusion these markers rise at a much slower rate Inthis study the patency of the infarct-related artery was determined from a single-pointangiogram, and could be predicted from either plasma FABP or myoglobin with a sens-itivity of approx 60% and a specificity of approx 80% This minor performance of themarkers in this study when compared with that of Ishii et al (62) may be explained bythe strict inclusion criteria in the latter

cor-In a multicenter study consisting of 115 patients with confirmed AMI and receivingthrombolytic agents, and who underwent coronary angiography within 120 min of thestart of thrombolysis, de Groot et al (64) also observed that FABP and myoglobinperform equally well as markers to discriminate between reperfused and nonreperfusedpatients Similar to the study of de Lemos et al (63), these investigators found relativelylow sensitivities and specificities (approx 70%), which, however, could be improved(to approx 80%) by normalization to infarct size (64) These data indicate the equalsuitabilities of FABP and myoglobin as noninvasive reperfusion markers, especially inretrospective studies in which infarct size is known

330 Glatz et al.

NEW APPROACHES TO INCREASE

FURTHER THE DIAGNOSIC PERFORMANCE OF FABP

A limitation of the use of markers of cell necrosis for assessment of tissue injury isthe time lag between the onset of necrosis and the appearance of the marker proteins inplasma This explains why up to 2–3 h after the onset of AMI, the performance of suchmarkers generally is insufficient for clinical decision making Therefore, approacheshave been presented to increase further the diagnostic performance of the plasma mark-ers in these early hours after AMI

To circumvent the problem of the upper reference concentration that is defined forpopulations and used for individual cases, it has been suggested to collect two (or more)serial blood samples during the first hours after admission and express the difference inmarker concentration or activity in these samples This approach has been applied espe-cially to identify low-risk patients who would show no ECG abnormalities as well as twonegative results for protein markers (hence, no significant change with time), and for whomearly discharge would be a safe option (65) In a second EUROCARDI Multicenter Trial,

we studied whether in patients admitted for suspected AMI without ECG changes, AMIcan be ruled out by assay of FABP, myoglobin, or CK-MB mass in two serial blood sam-ples, collected on admission and 1–3 h thereafter, respectively For comparison, cTnTwas measured in a third sample taken 12–36 h after admission Preliminary results fromthis study revealed that two negative marker concentrations within 3 h from admissionruled out AMI with very high negative predictive values (>90%) with the highest valuefound for FABP (negative predictive value 98%), being similar to that of cTnT elevation(³0.1 ng/mL) in the sample taken 12–36 h after admission (B Haastrup et al., unpublisheddata, 1999) A similar conclusion was also reached in a subsequent single-center studyconsisting of 130 patients admitted for suspected AMI with no significant ST-segmentelevation (66) These data indicate the excellent utility of FABP for early triage and riskstratification of patients with chest pain

Another approach to increase further the diagnostic performance of FABP in the earlyhours after onset of chest pain is its use in combination with markers of activated bloodcoagulation (67) Because intracoronary formation of blood clots on ruptured arterio-sclerotic plaques is considered the main cause of AMI, detection of activated blood coag-ulation potentially allows for the early diagnosis of AMI (68) Various (small-size) studieshave indicated that in the very early hours (0–3 h) after AMI onset, coagulation markersshow a higher sensitivity and specificity for AMI detection than necrosis markers (69,70) In addition, a tendency toward higher marker concentrations was observed for shorterhospital delays, a finding related to the fact that the acute thrombotic event precedescoronary occlusion and muscle necrosis In a pilot study consisting of 25 patients witheither AMI or unstable angina pectoris, we showed that combining a marker of musclecell necrosis (FABP) and a marker of activated blood coagulation (thrombus precursorprotein [TpP]) yielded a markedly higher sensitivity and specificity for AMI detectionthan either of the markers alone (Fig 6) (67) Moreover, the performance of such a com-bined test is expected to be relatively insensitive to hospital delay because TpP will per-form better in patients who are admitted earlier, whereas FABP will perform better inpatients who are admitted later (38,70) At present, we are investigating other markers

of activated blood coagulation, a.o tissue factor and soluble fibrin, which, in

combina-FABP as Marker of Myocardial Ischemia 331

tion with FABP, could bridge the diagnostic time gap of the first few hours after onset ofsymptoms in patients with ACS (71) A general problem in this field of research is thatthe tight physiological control of blood coagulation, required to prevent thrombolysis,

is affected by a large number of feedback mechanisms and inhibitors that may easilyobscure the relationship between the extent of prothrombolytic activation and the con-centrations of activated products in plasma

OTHER APPLICATIONS OF THE PLASMA MARKER FABP

FABP was also found to be useful for the early detection of postoperative myocardialtissue loss in patients undergoing coronary bypass surgery (3,72–74) In these patients,myocardial injury may be caused by global ischemia/reperfusion and, in addition, bypostoperative MI In our study,we found that in such patients, plasma CK, myoglobin,and FABP are already significantly elevated 0.5 h after reperfusion In the patients whodeveloped postoperative MI, a second increase was observed for each plasma markerprotein, but a significant increase was recorded earlier for FABP (4 h after reperfusion)than for CK or myoglobin (8 h after reperfusion) (72) These data suggest that FABPwould allow for an earlier exclusion of postoperative MI, thus permitting the earlier trans-fer of these patients from the intensive care unit to the ward Recently, both Hayashida

et al (73) and Petzold et al (74)also reported that FABP is an early and sensitive markerfor the diagnosis of myocardial injury in patients undergoing cardiac surgery

Antibodies directed against FABP have been shown to be useful for the chemical detection of very recent MIs (75–77) Partial depletion of FABP was observed

immunohisto-in cardiomyocytes with a post-immunohisto-infarction immunohisto-interval of <4 h (75), immunohisto-indicatimmunohisto-ing that FABP nostaining can confirm the clinical diagnosis or suspicion of early MI in routine autopsypathology

immu-Finally, besides the application of FABP in early diagnosis of myocardial injury inpatients, the marker is now also applied for evaluating MI after coronary artery ligationand for estimating infarct size in experimental animals such as mice and rats (78–81).CONCLUSION

The early diagnosis of ACS is important because it may improve patient treatmentand reduce complications Biochemical markers of myocardial cell damage continue

to be important tools for differentiating patients with AMI from those without AMI,because specific ST-segment changes in the admission ECG remain absent in a greatnumber of patients with AMI (1,3) FABP is a novel biochemical marker that showsrelease characteristics from injured myocardium and elimination rates from plasma thatare similar to those of myoglobin, which at present is regarded as the preferred earlyplasma marker of cardiac injury (82–85) Experimental studies indicate that this resem-blance relates to the similar molecular masses of FABP (14.5 kDa) and myoglobin (17.6kDa) Several clinical studies with patients suspected of having AMI reveal a superiorperformance of FABP over myoglobin (as well as other marker proteins) for the earlydetection of AMI This finding most likely relates to marked differences in tissue con-tents of FABP and myoglobin in cardiac and skeletal muscles that result in a relativelylow upper reference concentration in plasma for FABP compared with that for myoglo-bin These differences in tissue contents are also reflected in the plasma concentrations

332 Glatz et al.

of these proteins after either cardiac or skeletal muscle injury, in such a manner that theratio of the plasma concentrations of myoglobin and FABP can be applied to discrimi-nate myocardial from skeletal muscle injury

Limitations of the use of FABP as a diagnostic plasma marker in the clinical settinginclude (1) the relatively small diagnostic window, which extends to only 24–30 h afterthe onset of chest pain, and (2) its elimination from plasma mainly by renal clearance,possibly causing falsely high values in case of kidney malfunction These drawbackscan, however, be overcome by the simultaneous measurement in plasma of a late markersuch as cTnT or cTnI and assay of plasma creatinine to identify patients with renal insuf-ficiency and to calculate a corrected FABP concentration It is important to note thatthese same limitations also apply to myoglobin, which is now recommended by both theNational Academy of Clinical Biochemistry (NACB) Committee on Standards of Labor-atory Practice (82) and the International Federation of Clinical Chemistry (IFCC) Com-mittee on Standardization of Markers of Cardiac Damage (83) as preferred early marker

of MI, to be used in combination with cTnT or cTnI In spite of the recognition that, todate, relatively few centers have investigated the performance of FABP for early diagno-sis of AMI, the uniformly observed superiority of FABP over myoglobin indicates thatthe optimal set of biochemical markers of muscle necrosis for assessment of ACS may

be FABP together with cTnT or cTnI (86)

ACKNOWLEDGMENTS

Work in the authors’ laboratory was supported by grants from the Netherlands HeartFoundation (D90.003, 95.189 and 98.063), the Ministry of Economic Affairs (StiPT/MTR88.002 and BTS 97.188), and the European Community (BMH1-CT93.1692 and CIPD-CT94.0273)

ABBREVIATIONS

ACS, Acute coronary syndrome(s); AMI, acute myocardial infarction; CK, creatinekinase; CK-MB, MB isoenzyme of CK; cTnI, cTnT, cardiac troponins I and T; ECG,electrocardiogram; ELISA, enzyme-linked immunosorbent assay; FABP, fatty acid bind-ing protein; H-FABP, heart FABP; I-FABP, intestinal FABP; LDH, lactate dehydroge-nase; L-FABP, liver FABP; PTCA, percutaneous transluminal coronary angioplasty;ROC, receiver operating characteristics; TpP, thrombus precursor protein

reti-FABP as Marker of Myocardial Ischemia 333

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FABP as Marker of Myocardial Ischemia 335

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338 Glatz et al.

Oxidized and MDA-Modified LDL in CAD 339

Paul Holvoet

INTRODUCTION

Increased low-density lipoprotein (LDL) oxidation is associated with coronary arterydisease (CAD) The predictive value of circulating oxidized LDL is additive to the GlobalRisk Assessment Score for cardiovascular risk prediction based on age, gender, total andhigh-density lipoprotein (HDL) cholesterol, diabetes, hypertension, and smoking Cir-culating oxidized LDL does not originate from extensive metal ion induced oxidation

in the blood but from mild oxidation in the arterial wall by cell-associated lipoxygenaseand/or myeloperoxidase The increase of circulating oxidized LDL is most probablyindependent of plaque instability Indeed, plasma levels of oxidized LDL are very simi-lar among patients with stable CAD and patients with acute coronary syndromes (ACS).Endothelial ischemia induces increased prostaglandin synthesis and platelet adhesion/activation These processes are associated with the release of aldehydes, which induceoxidative modification of the protein moiety of LDL in the absence of lipid oxidationand thus in the generation of malondialdehyde (MDA)-modified LDL Levels of MDA-modified LDL are higher among patients with ACS The release of MDA-modified LDL

is independent of necrosis of myocardial cells

Our data suggest that oxidized LDL is a marker of coronary atherosclerosis whereasMDA-modified LDL is a marker of plaque instability

OXIDATIVE MODIFICATION OF LDL

Figure 1 summarizes different possible mechanisms of oxidative modification of LDL.Endothelial cells, monocytes, macrophages, lymphocytes, and smooth muscle cells areall capable of enhancing the rate of oxidation of LDL (1) During inflammation, severalcell types synthesize and secrete phospholipase A2 Myeloperoxidase, a heme proteinsecreted by activated phagocytes, oxidizes L-tyrosine to a tyrosyl radical that is a physio-logical catalyst for the initiation of lipid oxidation in LDL In striking contrast to othercell-mediated mechanisms for LDL oxidation, the myeloperoxidase-catalyzed reaction

is independent of free metal ions (2) Lipid oxidation results in the generation of hydes that substitute lysine residues in the apolipoprotein B-100 moiety of LDL andcauses its fragmentation The resulting oxidatively modified LDL is generally referred

alde-340 Holvoet

to as oxidized LDL A monoclonal antibody (MAb)-4E6 based competition linked immunosorbent assay (ELISA) can be used for the measurement of oxidizedLDL in plasma (3) The C50 values, concentrations that are required to obtain 50% inhi-bition of antibody binding in the ELISA, are 25 mg/dL for native LDL and 0.25 mg/dLfor oxidized LDL with at least 60 aldehyde-substituted lysines per apolipoprotein B-100.Oxidative stress in endothelial cells and platelet activation are associated with the oxi-dation of arachidonic acid to aldehydes These interact with lysine residues in the apo-lipoprotein B-100 moiety of LDL resulting in oxidative modification of the protein part

enzyme-of LDL in the absence enzyme-of lipid oxidation (4–6) The resulting oxidatively modified LDL

is generally referred to as MDA-modified LDL A MAb-1H11-based competition ELISAmay be used for the measurement of MDA-modified LDL in plasma (7,8) The C50 valuesare 0.25 mg/dL for MDA-modified LDL with at least 60 aldehyde-substituted lysinesper apolipoprotein B-100 compared to 25 mg/dL for native LDL and oxidized LDL.OXIDIZED LDL IS A MARKER OF CAD

The association of oxidative modification of LDL and stable angina and ACS has beenstudied (3) Table 1 shows characteristics of controls and patients with angiographicallyconfirmed CAD CAD patients were older; more often male and smokers; and had morefrequently hypertension, diabetes, and hypercholesterolemia CAD patients had higherlevels of total and LDL cholesterol and of triglycerides, lower levels of HDL choles-terol, and 2.6-fold higher levels of oxidized LDL Receiver operating characteristic(ROC) curve analysis revealed that oxidized LDL had a higher sensitivity for CAD than

Fig 1 Overview of possible mechanisms of oxidative modification of LDL

Oxidized and MDA-Modified LDL in CAD 341

the total cholesterol to HDL cholesterol (Tot-C/HDL-C) ratio The area under the curve(AUC) was 0.93 (95% CI: 0.91–0.94) for oxidized LDL compared to 0.68 (0.65–0.71)for the Tot-C/HDL-C ratio (p < 0.0001) Plasma levels of oxidized LDL were very simi-lar among patients with stable CAD and patients with ACS (3)

Major independent risk factors for CAD are advancing age, elevated blood pressure,elevated serum total and LDL cholesterol, low serum HDL cholesterol, diabetes melli-tus, and cigarette smoking (9–11) The Framingham Heart Study has elucidated thequantitative relationship between these risk factors and CAD It showed that the majorrisk factors are additive in predictive power Accordingly, the total risk of a person can beestimated by a summing of the risk imparted by each of the major risk factors Recently,the American Heart Association and the American College of Cardiology issued a sci-entific statement that assessed the Global Risk Assessment Scoring (GRAS) as a guide toprimary prevention (12) GRAS is based on age, total cholesterol, HDL cholesterol, sys-tolic blood pressure, diabetes mellitus, and smoking Predisposing factors such as obe-sity, physical inactivity, and family history of premature CAD are not included in GRAS

We have compared the diagnostic value of circulating oxidized LDL for CAD withthat of established risk factors in a subsequent and independent study (13) A total of 304subjects were included: 178 patients with angiographically proven CAD (mean age 59yr) and 126 age-matched (mean age 60 yr) subjects without clinical evidence of cardio-vascular disease CAD patients had higher levels of circulating oxidized LDL (p < 0.001),higher Tot-C/HDL-C ratio, and higher GRAS (p < 0.001) than controls (Table 2) GRASwas calculated on the basis of age, total and HDL cholesterol, blood pressure, diabetesmellitus, and smoking

ROC analysis revealed that oxidized LDL had a higher sensitivity for CAD than theTot-C/HDL-C ratio and GRAS, respectively The AUC was 0.91 (95% CI: 0.87–0.94)

Table 1

Characteristics of Controls and CAD Patients

Table 4 shows the relationship of CAD with age, sex, hypertension, diabetes type 2,hypercholesterolemia, dyslipidemia, smoking, body mass index, and circulating oxi-dized LDL Inclusion of circulating oxidized LDL in the multivariate model resulted in

an increase of r2 value from 0.22 to 0.67 Overall 72% of subjects were predicted rectly by the multivariate model containing established cardiovascular risk factors andoxidized LDL, compared to 40% by a model that did not include oxidized LDL (13).Recently, two other groups have developed and used assays for circulating oxidizedLDL to study the relationship between oxidation of LDL and CAD Nagai’s group (14,15) developed a test based on an antioxidized phosphatidylcholine monoclonal anti-body and an anti-human apolipoprotein B antibody Levels of oxidized LDL were 1.8-fold higher for CAD patients than for subjects without clinical evidence of CAD Thesensitivity of the assay for CAD was 79% with a specificity of 75% Ehara et al (16) used

cor-an ELISA based on cor-another cor-antioxidized phosphatidylcholine MAb Compared to trols, levels of circulating oxidized LDL were 1.5- to 3.4-fold higher in CAD patientsindependent of differences in serum levels of total and LDL and HDL cholesterol.MDA-MODIFIED LDL IS A MARKER OF ACS

con-We have collected plasma samples of 64 patients with angiographically confirmedstable CAD, 42 patients with unstable angina pectoris, and 62 patients with acute myo-cardial infarction (AMI) (Table 5) (8) Plasma levels of MDA-modified LDL were sim-ilar for controls and patients with stable angina pectoris, were 3.6-fold higher (p < 0.001)for patients with unstable angina pectoris, and were 3.1-fold higher (p < 0.001) for AMIpatients C-reactive protein (CRP) levels were similar for controls and patients withstable CAD, were 3.7-fold higher for unstable angina patients, and were 6.5-fold higher

Table 2

Comparison of Tot-C to HDL-C Ratio, GRAS, and Circulating

Oxidized LDL (OxLDL) in Controls and CAD Patients

Oxidized and MDA-Modified LDL in CAD 343

Fig 2 Comparison of the diagnostic value of oxidized LDL for CAD with that of the Tot-C

to HDL-C (A) or the GRAS (B) The AUC was 0.91 (95% CI: 0.87–0.94) for oxidized LDLcompared to 0.71 (0.66–0.76) for the Tot-C to HDL-C (p < 0.0001) and 0.82 (0.77–0.86) forthe GRAS (p < 0.0001)

Table 3

Prediction of CAD with GRAS and Circulating Oxidized LDL

344 Holvoet

Table 4

Logistic Regression Analysis of the Relationship

Between CAD and Potential Cardiovascular Risk Factors

The multivariate model 1 contained age, sex, hypertension, diabetes type 2,

hypercholesterolemia, dyslipidemia, smoking, and body mass index as covariates.

The r 2 value of this model was 0.22 Overall 40% of patients were predicted

cor-rectly at a classification cutoff of 0.9 The multivariate model 2 contained levels of

circulating oxidized LDL and all other covariants included in the first model The

r 2 value of this model was 0.67 Overall 72% of patients were predicted correctly

at a classification cutoff of 0.9.

for AMI patients (p < 0.001) Plasma levels of CRP were higher (p < 0.05) for AMI patientsthan for patients with unstable angina Cardiac troponin I levels were similar for controlsand patients with stable CAD, were 5.4-fold higher for patients with unstable angina(p < 0.001), and were 19-fold higher for AMI patients (p < 0.001) than for controls AMIpatients had 3.6-fold higher plasma cTnI level than patients with unstable angina (p <0.001) D-dimer levels were similar for controls and for patients with chronic stableangina or unstable angina pectoris and were 4.4-fold higher for AMI patients (8).Logistic regression analysis revealed an association of clinically diagnosed ACS withCRP (p < 0.0001), cTnI (p < 0.0001), and MDA-modified LDL (p = 0.0003) ROCcurve analysis revealed that MDA-modified LDL (p = 0.0014), but not cTnI or CRP, coulddiscriminate between stable CAD and unstable angina In contrast, cTnI (p = 0.0007),but neither MDA-modified LDL nor CRP, discriminated between unstable angina andAMI Both MDA-modified LDL (p = 0.0001) and cTnI (p = 0.021), but not CRP, discrim-inated between stable CAD and AMI At a cutoff value of 10 mg/dL (value exceeding the95th percentile of distribution for patients with stable angina), the sensitivity of CRP was19% for unstable angina and 42% for AMI, whereas the specificity was 95% At a cut-off value of 0.05 ng/mL (value exceeding the 95th percentile of distribution for patientswith stable angina), the sensitivity of cTnI was 38% for unstable angina and 90% forAMI, whereas the specificity was 95% At a cutoff value of 0.70 mg/dL (value exceedingthe 95th percentile of distribution for patients with stable angina), the sensitivity of MDA-modified LDL was 95% for unstable angina and 95% for AMI, whereas the specificitywas 95%

Oxidized and MDA-Modified LDL in CAD 345

CONCLUSIONS

Our studies show that plasma levels of oxidized LDL are significantly elevated inCAD patients These levels are very similar for patients with stable CAD and patientswith ACS, suggesting that their increase is independent of plaque instability The simi-lar ROC values of oxidized LDL for CAD in two independent studies (0.93 and 0.91,respectively) demonstrate that the assay for circulating oxidized LDL is indeed validfor studying the relationship between oxidation of LDL and CAD

The sensitivity of oxidized LDL for CAD was higher than that of the total to HDLcholesterol ratio and of the GRAS Logistic regression analysis revealed that the predic-tive value of oxidized LDL was additive to that of GRAS (p < 0.001) Ninety-four per-cent of subjects with high (exceeding the 90th percentile of distribution in controls)circulating oxidized LDL and high GRAS had CAD (94% of men and 100% of women)CAD Thus, circulating oxidized LDL is a sensitive marker of CAD

Our prospective study in heart transplant patients showed that baseline levels of dized LDL predicted the development of transplant CAD independent of levels of LDLand HDL cholesterol and of pretransplant history of ischemic heart disease (17) Thus,the level of oxidized LDL in the blood is an independent predictor of the development

of transplant CAD that was associated with a further increase of plasma levels of dized LDL Although the study identifies oxidized LDL as a prognostic marker of trans-plant CAD it does not prove that oxidized LDL has an active role in the development ofCAD Our recent finding of increased plasma levels of oxidized LDL in obese and type

oxi-2 diabetes patients, who are at increased risk for CAD even before there is any clinical

Table 5

Characteristics of Controls and CAD Patients

Stable angina Unstable angina AMI

a p < 0.05.

b p < 0.01.

c p < 0.001.

346 Holvoet

evidence of CAD, suggests that this may be the case Oxidized LDL may contribute tothe progression of atherosclerosis by enhancing endothelial injury, by inducing foamcell generation and smooth muscle proliferation

Intervention trials are required to evaluate the active role of oxidized LDL in the opment of CAD in general Interventions may aim at a further decrease of LDL choles-terol, at an increase of levels of antioxidants in LDL, and/or at an increase of HDL thatcontain enzymes such as paraoxonase and platelet-activating factor acetylhydrolasethat may prevent the oxidation of LDL or may degrade oxidized phospholipids in LDL.Levels of MDA-modified LDL are dependent on the ischemic syndromes for patientswith unstable angina pectoris or AMI The association between MDA-modified LDLand cTnI, a marker of ischemic syndromes, further supports this hypothesis

devel-In conclusion, oxidized LDL is a marker of coronary atherosclerosis whereas modified LDL is a marker of plaque instability

MDA-ACKNOWLEDGMENTS

This work was supported in part by a grant from the Fonds voor Geneeskundig schappelijk Onderzoek-Vlaanderen (FWO) (Projects 7.0022.98 and 7.0033.98) and bythe Interuniversitaire Attractiepolen (Program 4/34) The patient studies were performed

Weten-at the University Hospital of the KWeten-atholieke Universiteit Leuven, Belgium, in tion with Prof Dr Désiré Collen of the Center for Molecular and Vascular Biology, Prof

collabora-Dr Erik Muls of the Department of Endocrinology, and Prof collabora-Dr Frans Van de Werf ofthe Department of Cardiology

ABBREVIATIONS

ACS, Acute coronary syndrome(s); AMI, acute myocardial infarction; AUC, areaunder the curve; C, cholesterol; CAD, coronary artery disease; CRP, C-reactive protein;cTnI, cardiac troponin I; ELISA, enzyme-linked immunosorbent assay; GRAS, GlobalRisk Assessment Scoring; HDL, high-density lipoprotein; LDL, low-density lipopro-tein; MAb, monoclonal antibody; MDA, malondialdehyde; ROC, receiver operatingcharacteristic; Tot-C, total cholesterol

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