**Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged 18-64 Years in the United States**

Guijing Wang, Zefeng Zhang, Carma Ayala, Diane Dunet and Jing Fang

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53499

## **1. Introduction**

Acute Myocardial Infarction (AMI) is both a common and deadly type of cardiac event in the United States. Although the age-adjusted hospitalization rate for AMI and its in-hospital case fatality rates have both declined since the mid-1990s, there were still 634,000 inpatient admissions in 2009 for which AMI was listed as the primary diagnosis [1, 2]. Moreover, Americans suffered an estimated 610,000 first-time AMIs and 325,000 recurrent attacks, and 133,958 deaths in 2008 [2]. Because the declines in hospitalization and in-hospital mortality rates have been associated with more aggressive therapeutic interventions [1], it is impor‐ tant to evaluate the cost-effectiveness of these interventions.

To evaluate specifically the cost-effectiveness of various interventions against AMI, direct cost estimates of AMI are required [3-5]. Surprisingly, however, these cost estimates have not been comprehensively examined in the U.S. Many studies have investigated the economic burden of AMI, but all had some limitations [6-17]. Furthermore, in part because of limitations in avail‐ able studies, the costs of coronary heart disease (CHD) were used in one study to represent the costs for AMI [6], albeit this is inappropriate. For example, a previous study of insured adults aged 18-64 years found that only about 30% of CHD cases represented AMI [9]. Moreover, the American Heart Association recently estimated that the total prevalence of CHD among per‐ sons aged ≥20 years was 7% but the AMI prevalence of AMI in this group was 3.1% [2]. In addi‐ tion, in 2005, hospitalization costs for AMI admissions among adults aged 18-64 years were about \$5000 more than those for CHD admissions of non-AMI [9]. Clearly, information on costs that does not clearly distinguish between AMI and non-AMI admissions is of little use in evaluating the cost-effectiveness of interventions to treat AMI [18].

© 2013 Wang et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In the present study we estimated AMI-specific costs by exploring the hospitalization costs of AMI while incorporating the impacts on costs of percutaneous coronary intervention (PCI), coronary artery bypass graft (CABG) surgery, comorbidities, complications, ST-eleva‐ tion status, and length of stay (LOS) while controlling for age, sex, geographic regions, and urban versus non-urban location. Because PCI, CABG surgery, and LOS are likely to be the most influential factors on the costs and relevant factors for evaluating cost-effectiveness of AMI interventions, we also conducted multivariate logistic regressions to identify the fac‐ tors predicting PCI, CABG surgery, and LOS.

**AMI, comorbidity, complication, or**

Hypertension 401.xx-405.xx

Stroke 430.xx-438.xx

Ventricular fibrillation 427.41, 427.42

Atrial fibrillation 427.31, 427.32

ICD-9: International classification of disease, 9th revision. CPT-4: Current procedural terminology, 4th revision.

Diabetes 250.xx Hyperlipidemia 272.xx

Cardiogenic shock 785.51 Ventricular tachycardia 427.1

Atrial tachycardia 427.0

AMI: Acute myocardial infarction.

STEMI: ST-elevated myocardial infarction.

PCI: Percutaneous coronary intervention. CABG: Coronary artery bypass graft.

**2.2. Statistical analysis**

NSTEMI: Non-ST-elevated myocardial infarction.

**ICD-9 or CPT-4 code**

Congestive heart failure 402.01, 402.11, 402.91, 404.01, 404.03,404.11, 404.13, 404.91, 404.93,428.xx

410.01, 410.11. 410.21. 410.31

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged...

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410.xx

410.71

Kidney disease 403.xx, 404.xx, 582.xx, 583.xx, 585.xx, 586.xx, 587.xx

PCI 92980-92982, 92984, 92995, 92996, 00.66, 36.01-36.09 CABG surgery 33510-33519, 33521-33523, 33533-33536, 36.10-36.19

**Table 1.** Diagnostic codes for acute myocardial infarction (AMI) and selected comorbidities and procedures

After deriving the sample means of the costs for different population groups, AMI types, co‐ morbidities, complications, and procedures, we specified various versions of multivariate re‐ gression models to examine the factors influencing the costs while controlling for demographic variables and Charlson comorbidity index (CCI) [25]. We used CCI as a compre‐ hensive measure of disease severity. It measures the likelihood of death or serious disability in the subsequent year by diagnosis codes of up to 18 different diseases. In addition to estimating the various versions of regression for the whole study sample, we ran a regression on the costs

**procedure**

AMI STEMI NSTEMI

## **2. Methods**

#### **2.1. Data source**

The 2006-2008 MarketScan Commercial Claims and Encounter inpatient database was used for this study; this database contains information on patients up to age 64 years from approxi‐ mately 40 privately insured employers, including state governments, with an average of near‐ ly 21 million covered lives per year. In 2006-2008 the database had more than 2.4 billion service records representing commercially insured employees, qualified retirees and dependents from over 100 geographically diverse health insurance plans in all 50 U.S. states and the Dis‐ trict of Columbia. The advantages of using the MarketScan database for economic studies in‐ clude the large sample, detailed diagnosis codes for medical services, and hospitalization costs that are based on payment to providers [19]. Many researchers have used the MarketScan data‐ base to investigate medical costs associated with cardiovascular disease [9, 20, 21]

Using the International Classification of Diseases, 9th revision (ICD-9) codes, we identified hospitalizations with a primary diagnosis of AMI among patients aged 18-64 years who were enrolled in non-capitated health insurance plans. We further separated the hospitaliza‐ tions into ST-elevated myocardial infarction (STEMI) and non-ST-elevated myocardial in‐ farction (NSTEMI) cases. Based on secondary diagnosis codes, we identified major comorbidities, complications, and procedures for these hospitalizations (Table 1).

We excluded patients younger than 18 years because AMI is very uncommon in that group. We did not include patients in capitated health insurance plans because their costs of hospital‐ ization would not reflect the medical services provided to them. We excluded hospitalizations with a LOS greater than 30 days because we determined that these hospitalizations (n=131, fig‐ ure 1) would skew our results. To further limit the influence of extreme values on the cost esti‐ mates, we excluded all hospitalizations with a cost in the lowest or highest 1% of values (Figure 1). The costs in our study included all those for physician services, diagnostic tests, therapeu‐ tics, supplies, and room fees during the hospitalizations. These costs, as noted above, repre‐ sented total payment to providers rather than hospital charges. Accordingly, we did not need to adjust charges into payments to reflect the true economic burden of hospitalizations, nor did we use unit cost per bed day or an expert panel's suggested cost as in many other studies [5, 11, 7, 22, 23]. We expressed the costs in 2008 dollars by adjusting the 2006 and 2007 value by the consumer price index (CPI) provided by the Bureau of Labor Statistics [24].


AMI: Acute myocardial infarction.

In the present study we estimated AMI-specific costs by exploring the hospitalization costs of AMI while incorporating the impacts on costs of percutaneous coronary intervention (PCI), coronary artery bypass graft (CABG) surgery, comorbidities, complications, ST-eleva‐ tion status, and length of stay (LOS) while controlling for age, sex, geographic regions, and urban versus non-urban location. Because PCI, CABG surgery, and LOS are likely to be the most influential factors on the costs and relevant factors for evaluating cost-effectiveness of AMI interventions, we also conducted multivariate logistic regressions to identify the fac‐

The 2006-2008 MarketScan Commercial Claims and Encounter inpatient database was used for this study; this database contains information on patients up to age 64 years from approxi‐ mately 40 privately insured employers, including state governments, with an average of near‐ ly 21 million covered lives per year. In 2006-2008 the database had more than 2.4 billion service records representing commercially insured employees, qualified retirees and dependents from over 100 geographically diverse health insurance plans in all 50 U.S. states and the Dis‐ trict of Columbia. The advantages of using the MarketScan database for economic studies in‐ clude the large sample, detailed diagnosis codes for medical services, and hospitalization costs that are based on payment to providers [19]. Many researchers have used the MarketScan data‐

Using the International Classification of Diseases, 9th revision (ICD-9) codes, we identified hospitalizations with a primary diagnosis of AMI among patients aged 18-64 years who were enrolled in non-capitated health insurance plans. We further separated the hospitaliza‐ tions into ST-elevated myocardial infarction (STEMI) and non-ST-elevated myocardial in‐ farction (NSTEMI) cases. Based on secondary diagnosis codes, we identified major

We excluded patients younger than 18 years because AMI is very uncommon in that group. We did not include patients in capitated health insurance plans because their costs of hospital‐ ization would not reflect the medical services provided to them. We excluded hospitalizations with a LOS greater than 30 days because we determined that these hospitalizations (n=131, fig‐ ure 1) would skew our results. To further limit the influence of extreme values on the cost esti‐ mates, we excluded all hospitalizations with a cost in the lowest or highest 1% of values (Figure 1). The costs in our study included all those for physician services, diagnostic tests, therapeu‐ tics, supplies, and room fees during the hospitalizations. These costs, as noted above, repre‐ sented total payment to providers rather than hospital charges. Accordingly, we did not need to adjust charges into payments to reflect the true economic burden of hospitalizations, nor did we use unit cost per bed day or an expert panel's suggested cost as in many other studies [5, 11, 7, 22, 23]. We expressed the costs in 2008 dollars by adjusting the 2006 and 2007 value by the

base to investigate medical costs associated with cardiovascular disease [9, 20, 21]

comorbidities, complications, and procedures for these hospitalizations (Table 1).

consumer price index (CPI) provided by the Bureau of Labor Statistics [24].

tors predicting PCI, CABG surgery, and LOS.

**2. Methods**

78 Ischemic Heart Disease

**2.1. Data source**

ICD-9: International classification of disease, 9th revision.

CPT-4: Current procedural terminology, 4th revision.

STEMI: ST-elevated myocardial infarction.

NSTEMI: Non-ST-elevated myocardial infarction.

PCI: Percutaneous coronary intervention.

CABG: Coronary artery bypass graft.

**Table 1.** Diagnostic codes for acute myocardial infarction (AMI) and selected comorbidities and procedures

#### **2.2. Statistical analysis**

After deriving the sample means of the costs for different population groups, AMI types, co‐ morbidities, complications, and procedures, we specified various versions of multivariate re‐ gression models to examine the factors influencing the costs while controlling for demographic variables and Charlson comorbidity index (CCI) [25]. We used CCI as a compre‐ hensive measure of disease severity. It measures the likelihood of death or serious disability in the subsequent year by diagnosis codes of up to 18 different diseases. In addition to estimating the various versions of regression for the whole study sample, we ran a regression on the costs for STEMI and NSTEMI patients separately. Because PCI, CABG surgery and LOS were major factors determining the costs, we used logistic regression to investigate the predictors of these three factors. For the regression estimation, we used mixed-effects models with a repeated measures approach to account for the fact that a single patient might have multiple admis‐ sions during the 3-year period. All tests of statistical significance were 2-tailed, and a p<0.001 was considered significant. All statistical analyses were performed using SAS version 9.1 [26].

**N Mean costs (± SD)**

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18-44 4671 27,537.1 ± 20,693.3 45-54 13,991 29,661.7 ± 22,073.7 55-64 22,884 30,419.4 ± 23,778.6

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged...

Female 10,874 27,102.7 ± 22,110.1 Male 30,672 30,810.7 ± 23,096.9

Yes 31,511 29,639.3 ± 22,661.9 No 10,035 30,471.0 ± 23,624.5

Northeast 3296 27,623.5 ± 22,012.1 North Central 13,051 29,452.9 ± 21,927.1

> South 20,992 29, 637.4 ± 23,020.8 West 4207 33,790.2 ± 25,373.3

STEMI 18,979 32,030.3 ± 22,282.8 NSTEMI 22,567 27,998.3 ± 23,248.8

Yes 16,020 29,403.5 ± 21,868.0 No 25,526 30,114.3 ± 23,521.8

Yes 4813 36,758.5 ± 29,163.4 No 36,733 28,933.7 ± 21,786.3

Yes 551 29,024.5 ± 23,356.1 No 40,995 29,851.2 ± 22,894.5

Yes 14,075 29,375.3 ± 20,655.4 No 27,471 30,078.4 ± 23,966.5

Yes 296 34,324.6 ± 26,393.2

Total sample 41,546 29,840.2 ± 22,900.6

Age group (year)

Sex

MSA

Region

AMI type

Hypertension

Cancer

Hyperlipidemia

Peripheral vascular disease

Congestive Heart Failure

**Figure 1.** Diagram showing how the study sample was selected from all patients with a primary diagnosis of AMI in the 2006-2008 MarketScan Commercial Claims and Encounters inpatient database. **Figure 1.** Diagram showing how the study sample was selected from all patients with a primary diagnosis of AMI in the 2006-2008 MarketScan Commercial Claims and Encounters inpatient database. STEMI: ST-elevated myocardial in‐ farction. NSTEMI: non-ST-elevated myocardial infarction.

STEMI: ST-elevated myocardial infarction. NSTEMI: non-ST-elevated myocardial infarction.

17

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged... http://dx.doi.org/10.5772/53499 81

for STEMI and NSTEMI patients separately. Because PCI, CABG surgery and LOS were major factors determining the costs, we used logistic regression to investigate the predictors of these three factors. For the regression estimation, we used mixed-effects models with a repeated measures approach to account for the fact that a single patient might have multiple admis‐ sions during the 3-year period. All tests of statistical significance were 2-tailed, and a p<0.001 was considered significant. All statistical analyses were performed using SAS version 9.1 [26].

Age<18 years: 12

7295

Capitated insurance plan:

Length of stay >30 days: 131

Cost <1% or >99%: 845

Total hospitalizations: 49,829

Age ≥18 years: 49,817

Non-capitated insurance plan:

Length of stay ≤30 days:

Study sample: 41,546

42,522

80 Ischemic Heart Disease

42,391

and Encounters inpatient database.

farction. NSTEMI: non-ST-elevated myocardial infarction.

STEMI: ST-elevated myocardial infarction. NSTEMI: non-ST-elevated myocardial infarction.

STEMI: 18,979

17

NSTEMI: 22,567

**Figure 1.** Diagram showing how the study sample was selected from all patients with a primary diagnosis of AMI in the 2006-2008 MarketScan Commercial Claims

**Figure 1.** Diagram showing how the study sample was selected from all patients with a primary diagnosis of AMI in the 2006-2008 MarketScan Commercial Claims and Encounters inpatient database. STEMI: ST-elevated myocardial in‐



**N Mean costs (± SD)**

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No 39,571 29,427.5 ± 22,475.5

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged...

Charlson comorbidity index 41,456 1.55 ± 1.39 Length of stay (days) 41,456 4.66 ± 3.16

**Table 2.** Sample characteristics and mean costs (ages 18-64 years), 2006-2008 MarketScan inpatient database

During 2006-2008, there were 41,546 hospitalizations with a primary diagnosis of AMI; their mean cost was \$29,840 (± 22,901) (Table 2). Mean cost increased with age, but just marginal‐ ly. Male patients cost more than female patients (\$30,811 vs. \$27,103, p<0.001), and the cost of STEMI exceeded that of NSTEMI (\$32,030 vs. 27,998, p<0.001). Major comorbidities that increased the cost were stroke, heart failure, peripheral vascular disease, kidney disease, and diabetes. All of the complications except atrial tachycardia increased the cost greatly. Hospi‐ talizations in which CABG surgery was performed cost a mean of \$63,106, more than twice as high as the mean of \$26,415 for those without CABG surgery. PCI increased the cost mar‐

The regression results indicated that age influenced the cost marginally after controlling for procedures, comorbidities, complications, LOS, and ST-elevation status, as well as other demographic variables (Model 6, Table 3). Hospitalizations of male patients had about \$3350-\$4000 higher costs than those of their female counterparts in Model 1-4, but the differ‐ ences by sex dropped to \$1437 when all the procedures and complications were considered (Model 6). The cost in the West was \$5608 to \$6530 higher than in any other regions in the fully adjusted model. The cost of hospitalization for STEMI was higher than that for NSTE‐ MI, but the difference decreased from about \$3776 (model 2) to \$1003 with adjustment for all of the comorbidities, LOS, procedures, and complication (Model 6). CCI increased the cost by \$2362 (Model 3), but this increase largely disappeared after adding the LOS, procedures, and complications (Model 6). Longer LOS increased the cost by about \$2941 (p<0.001) per day (Model 6). After controlling for all other factors, PCI increased the cost by about \$12,546, and CABG surgery increased the cost by about \$28,406. These two procedures were the big‐ gest factors influencing the cost of AMI hospitalizations. Complications increased the cost

MSA: Metropolitan statistical area (resided in).

by \$4669 in the fully adjusted model.

STEMI: ST-elevated myocardial infarction. NSTEMI: Non-ST-elevated myocardial infarction. PCI: Percutaneous coronary intervention. CABG: Coronary artery bypass graft.

AMI: Acute myocardial infarction.

**3. Results**

ginally.

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged... http://dx.doi.org/10.5772/53499 83


**Table 2.** Sample characteristics and mean costs (ages 18-64 years), 2006-2008 MarketScan inpatient database

### **3. Results**

**N Mean costs (± SD)**

No 41,250 29,808.0 ± 22,870.8

Yes 7367 31,917.7 ± 24,735.0 No 34,179 29,392.4 ± 22,460.8

Yes 2944 28,862.3 ± 21,845.5 No 38,602 29,914.8 ± 22,977.6

Yes 1739 42,133.5 ± 30,090.3 No 39,807 29,303.2 ± 22,381.4

Yes 1584 33,499.2 ± 27,595.5 No 39,962 29,695.2 ± 22,682.8

Yes 27,062 30,960.8 ± 19,564.6 No 14,484 27,746.5 ± 27,972.1

Yes 3879 63,105.9 ± 26,886.0 No 37,667 26,414.5 ± 19,450.5

Yes 1135 53,016.1 ± 32,754.6 No 40,411 29,189.3 ± 22,216.0

Yes 2170 37,306.5 ± 27,619.9 No 39,376 29,428.7 ± 22,540.5

Yes 299 29,365.2 ± 25,149.5 No 41,247 29,843.6 ± 22,883.8

Yes 1286 43,165.1 ± 29,468.8 No 40,260 29,414.6 ± 22,530.4

Yes 1975 38,109.7 ± 28,974.3

Diabetes

82 Ischemic Heart Disease

Obesity

Stroke

PCI

CABG

Cardiogenic shock

Ventricular tachycardia

Atrial tachycardia

Ventricular fibrillation

Atrial fibrillation

Kidney disease

During 2006-2008, there were 41,546 hospitalizations with a primary diagnosis of AMI; their mean cost was \$29,840 (± 22,901) (Table 2). Mean cost increased with age, but just marginal‐ ly. Male patients cost more than female patients (\$30,811 vs. \$27,103, p<0.001), and the cost of STEMI exceeded that of NSTEMI (\$32,030 vs. 27,998, p<0.001). Major comorbidities that increased the cost were stroke, heart failure, peripheral vascular disease, kidney disease, and diabetes. All of the complications except atrial tachycardia increased the cost greatly. Hospi‐ talizations in which CABG surgery was performed cost a mean of \$63,106, more than twice as high as the mean of \$26,415 for those without CABG surgery. PCI increased the cost mar‐ ginally.

The regression results indicated that age influenced the cost marginally after controlling for procedures, comorbidities, complications, LOS, and ST-elevation status, as well as other demographic variables (Model 6, Table 3). Hospitalizations of male patients had about \$3350-\$4000 higher costs than those of their female counterparts in Model 1-4, but the differ‐ ences by sex dropped to \$1437 when all the procedures and complications were considered (Model 6). The cost in the West was \$5608 to \$6530 higher than in any other regions in the fully adjusted model. The cost of hospitalization for STEMI was higher than that for NSTE‐ MI, but the difference decreased from about \$3776 (model 2) to \$1003 with adjustment for all of the comorbidities, LOS, procedures, and complication (Model 6). CCI increased the cost by \$2362 (Model 3), but this increase largely disappeared after adding the LOS, procedures, and complications (Model 6). Longer LOS increased the cost by about \$2941 (p<0.001) per day (Model 6). After controlling for all other factors, PCI increased the cost by about \$12,546, and CABG surgery increased the cost by about \$28,406. These two procedures were the big‐ gest factors influencing the cost of AMI hospitalizations. Complications increased the cost by \$4669 in the fully adjusted model.


CABG --- --- --- --- 28189.4

Model 1: Age, sex, MSA, and region;

Model 3: Model 2 + comorbidities; Model 4: Model 3 + length of stay; Model 5: Model 4 + PCI, CABG surgery; Model 6: Model 5 + complications;

MSA: Metropolitan statistical area (resided in). STEMI: ST-elevated myocardial infarction.

NSTEMI: Non-ST-elevated myocardial infarction.

were far more likely to have a long LOS.

PCI: Percutaneous coronary intervention. CABG: Coronary artery bypass graft.

Model 2: Model 1 + STEMI;

Complications --- --- --- --- --- 4669.1

**Table 3.** Coefficient estimates of hospitalization costs for patients with acute myocardial infarction

PCI and CABG surgery increased the cost for both the STEMI and NSTEMI groups, with both procedures increasing the cost more for the NSTEMI group than for STEMI. LOS, in contrast, increased the cost more for the STEMI than the NSTEMI group, while living in an urban area (MSA in Table 3) decreased cost by \$1496 for STEMI and \$903 for NSTEMI.

Logistic regression indicated that patients aged 18-44 years were less likely than those aged 55-64 to undergo PCI or to have CABG surgery, and they were more likely to have a shorter LOS (i.e., <5 days) (Table 4). Patients in urban area were more likely to have PCI, but less likely to have CABG. Men were more likely to undergo PCI or to have CABG surgery than were women, but their odd of a short LOS was greater. Versus patients who did not live in urban areas, urban patients were more likely to have PCI, but they were less likely to under‐ go CABG surgery. Compared with patients in the West, patients in other regions were more likely to have a long LOS (i.e., ≥5 days), but they were usually less likely to have PCI and CABG surgery, with PCI in the North Central region the exception. STEMI patients were more likely than NSTEMI patients to undergo PCI and CABG surgery, and they were more likely to have a long LOS. Patients with comorbidities or complications were more likely to have a long LOS, but they were less likely to have PCI or CABG surgery. Patients undergo‐ ing PCI were more likely to have a short LOS, while patients undergoing CABG surgery

±352.7 (<0.0001)

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged...

28405.6 ±351.5 (<0.0001)

±252.5 (<0.0001)

26476.2 ±599.8 (<0.0001)

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4803.8 ±350.6 (<0.0001)

29395.9 ±431.8 (<0.0001) 85

4498.8 ±368.0 (<0.0001)


**Table 3.** Coefficient estimates of hospitalization costs for patients with acute myocardial infarction

**Independent variable Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 STEMI NSTEMI**

64.9 ±307.7 (0.8329)

998.0 ±205.6 (<0.0001)

3995.7 ±213.9 (<0.0001)





3654.9 ±188.9 (<0.0001)


±31.1 (<0.0001)

643.8 ±282.1 (0.0225)

722.3 ±188.5 (0.0001)

1574.6 ±198.0 (<0.0001)





1335.6 ±182.3 (<0.0001)

169.1 ±65.3 (0.0085)

3044.0 ±32.3 (<0.0001)

±204.5 (<0.0001)

808.3 ±281.0 (0.0040)

817.3 ±187.8 (<0.0001)

1437.1 ±197.3 (<0.0001)





1002.8 ±182.4 (<0.0001)

169.7 ±65.0 (0.0091)

2940.7 ±32.6 (<0.0001)

12546.1 ±203.7 (<0.0001)

554.9 ± 420.3 (0.1868)

502.8 ±283.7 (0.0763)

1046.8 ±316.9 (<0.0001)





361.6 ±11067 (0.0011)

3061.7 ±51.6 (<0.0001)

10169.0 ±366.5 (<0.0001)


955.6 ±376.9 (0.0112)

1069.4 ±249.6 (<0.0001)

1428.8 ±250.0 (<0.0001)





80.9 ±79.5 (0.3091)

2865.5 ± 41.8 (<0.0001)

13657.1 ±241.1 (<0.0001)

2278.4 ±362.4 (<0.0001)

564.5 ±242.1 (0.0197)

3804.2 ±252.4 (<0.0001)





4116.3 ±222.8 (<0.0001)

±80.3 (<0.0001)

Age

84 Ischemic Heart Disease

Region

18-44 vs. 55-64 -2895.2

45-54 vs. 55-64 -848.5

Male 3720.4

MSA -829.5

Northeast vs. West -6009.0

North Central vs. West -4235.0

South vs. West -3980.0

Charlson comorbidity

index

STEMI --- 3775.6

±366.3 (<. 0001)

±244.7 (0.0005)

±254.5 (<0.0001)

±262.0 (0.0015)

± 530.3 (<0.0001)

±404.0 (<0.0001)

±385.3 (<0.0001)



3356.3 ±254.5 (<0.0001)





±224.8 (<0.0001)


PCI --- --- --- --- 12490.0

Length of stay --- --- --- 3974.8

PCI and CABG surgery increased the cost for both the STEMI and NSTEMI groups, with both procedures increasing the cost more for the NSTEMI group than for STEMI. LOS, in contrast, increased the cost more for the STEMI than the NSTEMI group, while living in an urban area (MSA in Table 3) decreased cost by \$1496 for STEMI and \$903 for NSTEMI.

Logistic regression indicated that patients aged 18-44 years were less likely than those aged 55-64 to undergo PCI or to have CABG surgery, and they were more likely to have a shorter LOS (i.e., <5 days) (Table 4). Patients in urban area were more likely to have PCI, but less likely to have CABG. Men were more likely to undergo PCI or to have CABG surgery than were women, but their odd of a short LOS was greater. Versus patients who did not live in urban areas, urban patients were more likely to have PCI, but they were less likely to under‐ go CABG surgery. Compared with patients in the West, patients in other regions were more likely to have a long LOS (i.e., ≥5 days), but they were usually less likely to have PCI and CABG surgery, with PCI in the North Central region the exception. STEMI patients were more likely than NSTEMI patients to undergo PCI and CABG surgery, and they were more likely to have a long LOS. Patients with comorbidities or complications were more likely to have a long LOS, but they were less likely to have PCI or CABG surgery. Patients undergo‐ ing PCI were more likely to have a short LOS, while patients undergoing CABG surgery were far more likely to have a long LOS.


\$31,379 and \$63,909 [10]. Unfortunately, Zhao and Winget did not explore the effects of PCI and CABG on the costs of stay, as we did in our study. Such information is needed to evalu‐

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87

Two other significant drivers of cost in our study were complications and LOS. Having one or more complications increased the cost by over \$4600, and LOS increased the cost by over \$2900 per day. LOS was highly correlated with CABG surgery and with complications, as indicated in our logistic models (Table 4). Thus, interventions aiming to prevent or better manage the complications of AMI patients might be cost-effective in reducing the hospitali‐

Hospitalizations with STEMI had, on average, higher costs than NSTEMI hospitalizations, but after including PCI and CABG surgery as well as complications, comorbidities, and LOS in the regression model, the magnitude of the effect became much smaller. This may be be‐ cause of differences in treatment approaches and in complications between the two kinds of hospitalizations. For example, over 80% of STEMI hospitalizations had a PCI while only about 51% in the NSTEMI group did. However, the NSTEMI group had a higher rate of CABG surgery than did STEMI (12% vs. 8%) (not shown in tables). On the other hand, com‐ pared with NSTEMI cases, the STEMI group had a higher rate of cardiogenic shock, ventric‐ ular tachycardia, and ventricular fibrillation, but it had a lower rate of heart failure, atrial tachycardia, and atrial fibrillation. All of these factors would affect the cost differences be‐ tween STEMI and NSTEMI hospitalizations. The fact that STEMI cost more than NSTEMI was consistent with the literature; in Mexico, for example, STEMI cost nearly \$2800 more

The predictors of PCI, CABG surgery, and LOS that we set forward in this study provide important information for secondary cost-effectiveness analyses of AMI interventions. We found that male patients were more likely than females to have PCI and CABG surgery, but their odds of a shorter LOS (<5 days) were greater. STEMI status greatly increased the prob‐ ability of having PCI (coefficient estimate of 4.514) and significantly increased the probabili‐ ty of CABG surgery (coefficient estimate of 1.337), and it was associated with greater odds of a longer LOS (≥5 days). Patients with comorbidities and complications were relatively less likely to undergo PCI and CABG surgery, but they were more likely to have a longer LOS. All of these results could be used as inputs in cost-effectiveness evaluations of AMI inter‐

The numerous strengths of this study notwithstanding, several limitations should be consid‐ ered when interpreting our results. First, all of our patients were covered by non-capitated private insurance plans. Although the costs of these patients accurately reflect the true eco‐ nomic burden imposed by their hospitalizations, the special population may have limited the generalizability of our results to the broader U.S. population. Second, all of our patients were 18-64 years old. The elderly population (aged >64 years) has much higher incidence and prevalence of AMI and its related comorbidities and complications [1, 2]; as a conse‐ quence, the total costs of AMI should be higher in this population than among those 18-64. Although many studies have focused on the cost of AMI among the elderly [4, 5, 8], new estimation methods are needed along with high-quality data to develop better cost estimates

ate the cost-effectiveness of AMI interventions [4].

zation costs of this group.

than NSTEMI [11].

ventions.

NSTEMI: Non-ST-elevated myocardial infarction.

**Table 4.** Coefficient estimates of logistic regression of PCI, CABG, and length of stay

#### **4. Discussion**

The large number of hospitalizations in our economic study of inpatients who had suffered an AMI enabled us to explore a variety of factors that influenced their costs. The results sug‐ gest that CABG and PCI are the biggest drivers of hospital costs for AMI patients, adding, respectively, \$12,546 and \$28,406 to the cost of a stay. The cost effects of PCI and CABG in our study were comparable to the \$15,089 and \$28,974 additional costs, respectively, found in a Medicare population [7]. Another study reported similar costs for PCI and CABG [17]. In an earlier study using MarketScan data from 2003 to 2006, Zhao and Winget found that the total hospitalization costs of PCI and CABG surgery patient costs were, respectively, \$31,379 and \$63,909 [10]. Unfortunately, Zhao and Winget did not explore the effects of PCI and CABG on the costs of stay, as we did in our study. Such information is needed to evalu‐ ate the cost-effectiveness of AMI interventions [4].

**Independent variable PCI (yes vs. no) CABG (yes vs. no) Length of stay (<5 vs. ≥5)**

Northeast vs. West 0.792 (0.711, 0.884) 0.488 (0.391, 0.608) 1.560 (1.392, 1.747)

South vs. West 0.884 (0.815, 0.959) 0.934 (0.806, 1.081) 1.486 (1.364, 1.620)

North Central vs. West 1.153 (1.059, 1.256) 0.969 (0.830, 1.132) 1.267 (1.158, 1.387)

STEMI vs. NSTEMI 4.514 (4.293, 4.746) 1.337 (1.219, 1.467) 1.333 (1.267, 1.402) Charlson comorbidity index 0.890 (0.876, 0.905) 0.887 (0.862, 0.913) 1.432 (1.408, 1.457)

PCI --- 0.060 (0.053, 0.067) 0.819 (0.776, 0.866)

CABG 0.062 (0.056, 0.069) --- 47.992 (41.288, 55.785)

Complications 0.894 (0.834, 0.959) 0.863 (0.771, 0.966) 2.621 (2.460, 2.793)

The large number of hospitalizations in our economic study of inpatients who had suffered an AMI enabled us to explore a variety of factors that influenced their costs. The results sug‐ gest that CABG and PCI are the biggest drivers of hospital costs for AMI patients, adding, respectively, \$12,546 and \$28,406 to the cost of a stay. The cost effects of PCI and CABG in our study were comparable to the \$15,089 and \$28,974 additional costs, respectively, found in a Medicare population [7]. Another study reported similar costs for PCI and CABG [17]. In an earlier study using MarketScan data from 2003 to 2006, Zhao and Winget found that the total hospitalization costs of PCI and CABG surgery patient costs were, respectively,

Length of stay (days) 0.981 (0.973, 0.990) 1.405 (1.388, 1.422) ---

**Table 4.** Coefficient estimates of logistic regression of PCI, CABG, and length of stay

Age 18-44 vs. 55-64 years 0.877 (0.814, 0.944) 0.718 (0.616, 0.836) 0.706 (0.651, 0.765) Age 45-54 vs. 55-64 years 1.170 (1.112, 1.232) 1.010 (0.921, 1.107) 0.807 (0.767, 0.851) Male 1.813 (1.724, 1.907) 2.776 (2.502, 3.081) 0.760 (0.721, 0.801) MSA 1.249 (1.184, 1.317) 0.905 (0.824, 0.995) 1.044 (0.988, 1.104)

Region

86 Ischemic Heart Disease

PCI: Percutaneous coronary intervention. CABG: Coronary artery bypass graft.

**4. Discussion**

MSA: Metropolitan statistical area (resided in). STEMI: ST-elevated myocardial infarction. NSTEMI: Non-ST-elevated myocardial infarction. Two other significant drivers of cost in our study were complications and LOS. Having one or more complications increased the cost by over \$4600, and LOS increased the cost by over \$2900 per day. LOS was highly correlated with CABG surgery and with complications, as indicated in our logistic models (Table 4). Thus, interventions aiming to prevent or better manage the complications of AMI patients might be cost-effective in reducing the hospitali‐ zation costs of this group.

Hospitalizations with STEMI had, on average, higher costs than NSTEMI hospitalizations, but after including PCI and CABG surgery as well as complications, comorbidities, and LOS in the regression model, the magnitude of the effect became much smaller. This may be be‐ cause of differences in treatment approaches and in complications between the two kinds of hospitalizations. For example, over 80% of STEMI hospitalizations had a PCI while only about 51% in the NSTEMI group did. However, the NSTEMI group had a higher rate of CABG surgery than did STEMI (12% vs. 8%) (not shown in tables). On the other hand, com‐ pared with NSTEMI cases, the STEMI group had a higher rate of cardiogenic shock, ventric‐ ular tachycardia, and ventricular fibrillation, but it had a lower rate of heart failure, atrial tachycardia, and atrial fibrillation. All of these factors would affect the cost differences be‐ tween STEMI and NSTEMI hospitalizations. The fact that STEMI cost more than NSTEMI was consistent with the literature; in Mexico, for example, STEMI cost nearly \$2800 more than NSTEMI [11].

The predictors of PCI, CABG surgery, and LOS that we set forward in this study provide important information for secondary cost-effectiveness analyses of AMI interventions. We found that male patients were more likely than females to have PCI and CABG surgery, but their odds of a shorter LOS (<5 days) were greater. STEMI status greatly increased the prob‐ ability of having PCI (coefficient estimate of 4.514) and significantly increased the probabili‐ ty of CABG surgery (coefficient estimate of 1.337), and it was associated with greater odds of a longer LOS (≥5 days). Patients with comorbidities and complications were relatively less likely to undergo PCI and CABG surgery, but they were more likely to have a longer LOS. All of these results could be used as inputs in cost-effectiveness evaluations of AMI inter‐ ventions.

The numerous strengths of this study notwithstanding, several limitations should be consid‐ ered when interpreting our results. First, all of our patients were covered by non-capitated private insurance plans. Although the costs of these patients accurately reflect the true eco‐ nomic burden imposed by their hospitalizations, the special population may have limited the generalizability of our results to the broader U.S. population. Second, all of our patients were 18-64 years old. The elderly population (aged >64 years) has much higher incidence and prevalence of AMI and its related comorbidities and complications [1, 2]; as a conse‐ quence, the total costs of AMI should be higher in this population than among those 18-64. Although many studies have focused on the cost of AMI among the elderly [4, 5, 8], new estimation methods are needed along with high-quality data to develop better cost estimates for this population. Unfortunately, our data would not be appropriate for an analysis of costs among the elderly population for AMI hospitalization. A third limitation is that we es‐ timated the costs of hospitalizations only. With survival rates increasing because of advan‐ ces in technology [1], AMI patients are living longer. Correspondingly, the lifetime costs of outpatient care and medications for afflicted patients should be increasing. Additionally, productivity losses from the morbidity and premature mortality associated with AMI are al‐ so high [10] and should be considered in any comprehensive economic evaluations.

**References**

[1] Fang J, Alderman MH, Keenan NL, Ayala C. Acute myocardial infarction hospitali‐

Costs of Hospitalizations with a Primary Diagnosis of Acute Myocardial Infarction Among Patients Aged...

http://dx.doi.org/10.5772/53499

89

[2] American Heart Association. Heart disease and stroke statistics – 2012 update: a re‐

[3] Ioannides-Demos LL, Makarounas-Kirchmann K, Ashton E, Stoelwinder J, McNeil JJ. Cost of myocardial infarction to the Australian community: a prospective, mutlticen‐

[4] Sloss EM, Wickstrom SL, McCaffrey DF, et al. Direct medical costs attributable to acute myocardial infarction and ischemic stroke in cohorts with atherosclerotic con‐

[5] Krumholz HM, Chen J, Murillo JE, Cohen DJ, Radford MJ. Clinical correlates of inhospital costs for acute myocardial infarction in patients 65 years of age and older.

[6] Turpie AG. Burden of disease: medical and economic impact of acute coronary syn‐

[7] Kugelmass AD, Cohen DJ, Brown PP, Simon AW, Becker ER, Culler SD. Hospital re‐ sources consumed in treating complications associated with percutaneous coronary

[8] Tiemann O. Variations in hospitalisation costs for acute myocardial infarction – a

[9] Wang G, Zhang Z, Ayala C, Dunet D, Fang J. Inpatient costs associated with ischemic heart disease among adults aged 18-64 years in the United States. In: Lakshmanados U, Ed. Novel strategies in ischemic heart disease. Rijeka, Croatia: InTech 2012; pp.

[10] Zhao Z, Winget M. Economic burden of illness of acute coronary syndromes: medical and productivity costs. BMC Health Serv Res 2011; 11: 35. http://www.biomedcen‐

[11] Reynales-Shigematsu LM, Campuzano-Rincon JC, Sesma-Vasquez S, et al. Costs of medical care for acute myocardial infarction attributable to tobacco consumption.

[12] Eisenstein EL, Shaw LK, Anstrom KJ, et al. Assessing the clinical and economic bur‐

[13] Etemad LR, McCollam PL. Total first-year costs of acute coronary syndrome in a

den of coronary artery disease: 1986-1998. Med Care 2001; 39: 824-35.

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tre survey. Clin Drug Investig 2010; 30: 533-43.

ditions. Cerebrovasc Dis 2004; 18: 8-15.

dromes. Am J Manag Care 2006; 12: S430-4.

interventions. Am J Cardiol 2006; 97: 322-7.

comparison across Europe. Health Econ 2008; 17: S33-45.

Am Heart J 1998; 135: 523-31.

319-32.

tral.com/1472-6963/11/25.

Arch Med Res 2006; 37: 871-9.

port from the American Heart Association. Circulation 2012; 125: e2-e220.

Given all of these factors, the hospitalization costs presented in our report should be treated as a conservative estimate of the economic burden associated with AMI. Moreover, we should note the limitation that we analyzed the costs of hospitalizations with AMI as a *pri‐ mary* diagnosis. Although this decision let us cover the majority of AMI cases, there may be substantial additional hospitalizations in which AMI is a secondary diagnosis [9]. These hos‐ pitalizations should certainly be included in any complete analysis of the costs of hospitali‐ zations of AMI patients. Because examining the costs of AMI as a secondary diagnosis would require a different analytical framework from the one we used, it would have been beyond the scope of our analysis.

#### **5. Conclusion**

Using a large set of claims data, we estimated the hospitalization costs of patients with a pri‐ mary diagnosis of AMI and identified the main cost drivers of this important problem. Be‐ cause most previous studies did not provide any information on the predictors of the costs of AMI hospitalizations [27], we hope that the present study has to some degree filled this gap in the literature. The high costs of AMI could be an economic justification for policy makers to support efforts to prevent AMI. In addition, the detailed information presented herein about the impact of various factors on the costs, procedures, and LOS associated with hospitalizations having a primary diagnosis of AMI can be used to evaluate and support health economic research such as studies on the cost-effectiveness of interventions to control this problem.

## **Author details**

Guijing Wang\* , Zefeng Zhang, Carma Ayala, Diane Dunet and Jing Fang

\*Address all correspondence to: Gbw9@cdc.gov

Division for Heart Disease and Stroke Prevention, Centers for Disease Control and Preven‐ tion (CDC), Atlanta, GA, USA

The findings and conclusions of this article are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention (CDC).

## **References**

for this population. Unfortunately, our data would not be appropriate for an analysis of costs among the elderly population for AMI hospitalization. A third limitation is that we es‐ timated the costs of hospitalizations only. With survival rates increasing because of advan‐ ces in technology [1], AMI patients are living longer. Correspondingly, the lifetime costs of outpatient care and medications for afflicted patients should be increasing. Additionally, productivity losses from the morbidity and premature mortality associated with AMI are al‐

Given all of these factors, the hospitalization costs presented in our report should be treated as a conservative estimate of the economic burden associated with AMI. Moreover, we should note the limitation that we analyzed the costs of hospitalizations with AMI as a *pri‐ mary* diagnosis. Although this decision let us cover the majority of AMI cases, there may be substantial additional hospitalizations in which AMI is a secondary diagnosis [9]. These hos‐ pitalizations should certainly be included in any complete analysis of the costs of hospitali‐ zations of AMI patients. Because examining the costs of AMI as a secondary diagnosis would require a different analytical framework from the one we used, it would have been

Using a large set of claims data, we estimated the hospitalization costs of patients with a pri‐ mary diagnosis of AMI and identified the main cost drivers of this important problem. Be‐ cause most previous studies did not provide any information on the predictors of the costs of AMI hospitalizations [27], we hope that the present study has to some degree filled this gap in the literature. The high costs of AMI could be an economic justification for policy makers to support efforts to prevent AMI. In addition, the detailed information presented herein about the impact of various factors on the costs, procedures, and LOS associated with hospitalizations having a primary diagnosis of AMI can be used to evaluate and support health economic research such as studies on the cost-effectiveness of interventions to control

, Zefeng Zhang, Carma Ayala, Diane Dunet and Jing Fang

Division for Heart Disease and Stroke Prevention, Centers for Disease Control and Preven‐

The findings and conclusions of this article are those of the authors and do not necessarily represent the official position of the US Centers for Disease Control and Prevention (CDC).

so high [10] and should be considered in any comprehensive economic evaluations.

beyond the scope of our analysis.

**5. Conclusion**

88 Ischemic Heart Disease

this problem.

**Author details**

tion (CDC), Atlanta, GA, USA

\*Address all correspondence to: Gbw9@cdc.gov

Guijing Wang\*


[14] McCollam P, Etemad L. Cost of care for new-onset acute coronary syndrome patients who undergo coronary revascularization. J Invasive Cardiol 2005; 17: 307-11.

**Chapter 6**

**Biomarkers of Cardiac Ischemia**

Additional information is available at the end of the chapter

Ischemia (from the Greek ισχαιμία, *ischaimía*; *isch-* root denoting a restriction or thinning or to make or grow thin, *haema* blood) is the restriction of blood supply and thus the inadequate delivery of oxygen and removal of carbon dioxide from cellular tissue. This imbalance may

Cardiac ischemia occurs when there is a supply versus demand mismatch in coronary blood flow. In patients who present with unstable angina, ischemia occurs due to partial or total occlusion of a coronary artery due to plaque rupture. In stable angina however, there is progressive vascular occlusion resulting ultimately in a luminal stenosis of greater than 70%, impeding blood flow to the distal tissue. If the ischemia is reversible, no permanent myocardial damage occurs.If however the ischemic episode is prolonged; there will be cellular necrosis which will lead to acute myocardial infarction (AMI).The immediate clinical challenge is to be able to identify acutely impaired myocardial perfusion before the necrotic process starts. Currently, the only strategy for this is to detect ST-segment changes on the electrocardiogram (ECG), however the ECG is non-diagnostic in many cases. The sensitivity of the admission ECG for the diagnosis of AMI is typically around 50%. Reperfusion, be it pharmacological or surgical, is the essential life-saving intervention with the aim of salvaging myocardial tissue localised at the affected site. Many patients however who present with chest pain to the emergency department (ED) do not have a final diagnosis of AMI. There is therefore a need for a strategy which could detect cardiac ischemia before necrosis occurs and result in prompt revascularisation. Blood borne biomarkers for ischemia may be of diagnostic and prognostic

To date, a number of candidate biomarkers of ischemia are being researched. However, one, Ischemia modified albumin (IMA®), has been developed into a commercially available cardiac biomarker assay and licensed for routine clinical application both by CE marking in Europe

and reproduction in any medium, provided the original work is properly cited.

© 2013 Gaze; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

lead to dysfunctional or permanent damage to the affected tissue and organ.

David C. Gaze

**1. Introduction**

value.

http://dx.doi.org/10.5772/55250


## **Chapter 6**

## **Biomarkers of Cardiac Ischemia**

David C. Gaze

[14] McCollam P, Etemad L. Cost of care for new-onset acute coronary syndrome patients who undergo coronary revascularization. J Invasive Cardiol 2005; 17: 307-11.

[15] Menzin J, Wygant G, Hauch O,Jackel J, Friedman M. One-year costs of ischemic heart disease among patients with acute coronary syndromes: findings from a multi-em‐

[16] Russell MW, Huse DM, Drowns S, Hamel EC, Hartz SC. Direct medical costs of coro‐

[17] Kauf TL, Velazquez EJ, Crosslin DR, et al. The cost of acute myocardial infarction in the new millennium: evidence from a multinational registry. Am Heart J 2006; 151:

[18] Luengo-Fernandez R, Gray AM, Rothwell PM. Costs of stroke using patient-level da‐

[19] Adamson DM, Chang S, Hansen LG. Health research data from the real world: the MarketScan database (white paper). 2008. Available from http://thomsonreuters.com.

[20] Wang G, Zhang Z, Ayala C. Hospitalization costs associated with hypertension as a secondary diagnosis among insured patients aged 18-64 years. Am J Hypertens 2010;

[21] Kahende JW, Woollery TA, Lee CW. Assessing medical expenditures on 4 smoking-

[22] Ringborg A, Yin DD, Martinel M, Stalhammar J, Linggren P. The impact of acute my‐ ocardial infarction and stroke on health care costs in patients with type 2 diabetes in

[23] Wang G, Dietz WH. Economic burden of obesity in youths aged 6 to 17 years: 1979-1999. Pediatrics 2002; 109: e81. http://www.pediatrics.org/cgi/content/full/109/5/

[24] Bureau of Labor Statistics (BLS). Consumer price index (CPI). Available from ftp://

[25] Charlson ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying prog‐ nostic comorbidity in longitudinal studies: development and evaluation. J Chronic

[27] Tarride JE, Lim M, DesMeules M, et al. A review of the cost of cardiovascular dis‐

ftp.bls.gov/pub/special.requests/cpi/cpiai.txt. Accessed March 16, 2012.

nary artery disease in the United States. Am J Cardiol 1998; 81: 1110-5.

ployer claims database. Curr Med Res Opin 2008; 24): 461-8.

ta: a critical review of the literature. Stroke. 2009; 40: e18-23.

related diseases, 1996-2001. Am J Health Behav 2007; 31: 601-11.

Sweden. Eur J Cardiovasc Prev Rehabil 2009; 16: 576-82.

[26] SAS. SAS/STAT User's Guide. Cary NC: SAS Institute Inc.; 2007.

206-12.

90 Ischemic Heart Disease

Requested May 2010.

23: 275-81.

e81.

Dis 1987;40:373-383.

ease. Can J Cardiol 2009; 25: e195-202.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55250

## **1. Introduction**

Ischemia (from the Greek ισχαιμία, *ischaimía*; *isch-* root denoting a restriction or thinning or to make or grow thin, *haema* blood) is the restriction of blood supply and thus the inadequate delivery of oxygen and removal of carbon dioxide from cellular tissue. This imbalance may lead to dysfunctional or permanent damage to the affected tissue and organ.

Cardiac ischemia occurs when there is a supply versus demand mismatch in coronary blood flow. In patients who present with unstable angina, ischemia occurs due to partial or total occlusion of a coronary artery due to plaque rupture. In stable angina however, there is progressive vascular occlusion resulting ultimately in a luminal stenosis of greater than 70%, impeding blood flow to the distal tissue. If the ischemia is reversible, no permanent myocardial damage occurs.If however the ischemic episode is prolonged; there will be cellular necrosis which will lead to acute myocardial infarction (AMI).The immediate clinical challenge is to be able to identify acutely impaired myocardial perfusion before the necrotic process starts. Currently, the only strategy for this is to detect ST-segment changes on the electrocardiogram (ECG), however the ECG is non-diagnostic in many cases. The sensitivity of the admission ECG for the diagnosis of AMI is typically around 50%. Reperfusion, be it pharmacological or surgical, is the essential life-saving intervention with the aim of salvaging myocardial tissue localised at the affected site. Many patients however who present with chest pain to the emergency department (ED) do not have a final diagnosis of AMI. There is therefore a need for a strategy which could detect cardiac ischemia before necrosis occurs and result in prompt revascularisation. Blood borne biomarkers for ischemia may be of diagnostic and prognostic value.

To date, a number of candidate biomarkers of ischemia are being researched. However, one, Ischemia modified albumin (IMA®), has been developed into a commercially available cardiac biomarker assay and licensed for routine clinical application both by CE marking in Europe

© 2013 Gaze; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and Food and Drug Administration (FDA) approval in the United States. This chapter will explore the rational for the necessity of cardiac ischemia biomarker testing and detail the development of the IMA assay with emphasis on its clinical and prognostic utility.

presenting at any stage in the process may be diagnosed with acute coronary syndrome (ACS). The earlier in the disease continuum the presentation is; the greater the opportunity for successful myocardial tissue preservation. As there is no definitive biomarker for ischemia, current treatment focuses on the need for urgent therapeutic revascularisation in patients with

Local ischemia

Neurohormonal activation

Systolic dysfunction

Cardiac Failure

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 93

Ventricular remodelling

Metabolic Abnormalities

Deranged diastole

Deranged systole

ECG abnormalities

Chest Pain

The pathological process unless disrupted by therapeutic intervention results in the death of cardiac myocytes. Predisposing this terminal event, a vulnerable atherosclerotic plaque becomes disrupted exposing the thrombogenic lipid core and sub endothelium to the luminal milieu. Exposure results in platelet activation and aggregation and along with the coagulation cascade, an intracoronary thrombus forms. The thrombus may not obstruct the lumen and the patient is asymptomatic, however if the lumen is totally occluded, AMI will ensue. A partially occluded lumen and reduced oxygen supply both contribute to the development of ischemic

The clinical presentation of cardiac ischemia is difficult to definitively diagnose. Currently there is no gold standard test to detect ischemia however a number of reliable tests exist.

established cardiac necrosis, identified by the cardiac troponins.

Risk factors

Vascular injury

Coronary Artery Disease

myocardium.

Plaque rupture

**Figure 1.** Development of Ischemia in the ischemia cascade.

**3. Clinical detection of cardiac ischemia**

#### **1.1. The cardiovascular disease epidemic**

Cardiovascular disease (CVD) accounts for the majority of global deaths. CVD was responsible for 29% of all global deaths in 2004. According to the World Heart Federation, CVD is responsible for 17.1 million deaths globally each year. Surprisingly, 82% of these deaths occur in the developing world. Such numbers are often difficult to comprehend. CVD is responsible for one in every five deaths disease kills one person every 34 seconds in the USA alone. 35 people under the age of 65 die prematurely in the United Kingdom every day due to CVD. It is predicted that by 2030 23 million people will die annually from a cardiovascular related disease. Data from the USA suggests that CVD was responsible for 34% of all deaths in 2006 and over 151,000 Americans who died were under 65 years of age.

#### **1.2. Acute chest pain**

Patients with chest pain constitute the largest single category of patients admitted to hospitals in the UK [1]. In the USA, registry data recorded 11.2 million chest pain presentations to the ED in 2008 alone. The presentations are also diagnostically challenging. The majority of admissions have either stable ischemic heart disease (IHD) or no ischemic heart disease [2]. Such admission episodes are often short and clinically inappropriate. Conversely, it has been estimated that between 2 and 7% of patients with AMI are inappropriately discharged from the ED [3, 4] and suffer disproportionate morbidity and mortality. Attempts to improve diagnosis have included risk scoring systems [5], computerised decision support [6, 7] and automated ECG interpretation [8]. Although clinical assessment remains integral to the assessment of patients with chest pain, cardiac biomarker measurement has become an essential component in the diagnostic armamentarium.

## **2. Pathophysiology of cardiac ischemia**

The mechanisms involved in the development of cardiovascular disease are multifactorial and include abnormalities in cholesterol and lipid metabolism, inflammation and oxidative stress processes within the vascular wall, cellular disruption to the endothelium and intra-lumenal platelet activation/aggregation. The ischemia cascade from initiation of local ischemia to the development of symptomatic chest pain is depicted in figure 1.The pathological processes responsible for the development of atherosclerotic lesions and endothelial dysfunction are advanced far earlier than when patients typically become symptomatic and present with chest pain. The disease process does not occur in distinct episodes but rather is a continuum from asymptomatic vascular dysfunction thorough to angina in those with myocardial ischemia, which, without intervention can progress to non-ST segment elevation myocardial infarction (NSTEMI) or cumulate into ST segment elevation myocardial infarction (STEMI). Patients presenting at any stage in the process may be diagnosed with acute coronary syndrome (ACS). The earlier in the disease continuum the presentation is; the greater the opportunity for successful myocardial tissue preservation. As there is no definitive biomarker for ischemia, current treatment focuses on the need for urgent therapeutic revascularisation in patients with established cardiac necrosis, identified by the cardiac troponins.

**Figure 1.** Development of Ischemia in the ischemia cascade.

and Food and Drug Administration (FDA) approval in the United States. This chapter will explore the rational for the necessity of cardiac ischemia biomarker testing and detail the

Cardiovascular disease (CVD) accounts for the majority of global deaths. CVD was responsible for 29% of all global deaths in 2004. According to the World Heart Federation, CVD is responsible for 17.1 million deaths globally each year. Surprisingly, 82% of these deaths occur in the developing world. Such numbers are often difficult to comprehend. CVD is responsible for one in every five deaths disease kills one person every 34 seconds in the USA alone. 35 people under the age of 65 die prematurely in the United Kingdom every day due to CVD. It is predicted that by 2030 23 million people will die annually from a cardiovascular related disease. Data from the USA suggests that CVD was responsible for 34% of all deaths in 2006

Patients with chest pain constitute the largest single category of patients admitted to hospitals in the UK [1]. In the USA, registry data recorded 11.2 million chest pain presentations to the ED in 2008 alone. The presentations are also diagnostically challenging. The majority of admissions have either stable ischemic heart disease (IHD) or no ischemic heart disease [2]. Such admission episodes are often short and clinically inappropriate. Conversely, it has been estimated that between 2 and 7% of patients with AMI are inappropriately discharged from the ED [3, 4] and suffer disproportionate morbidity and mortality. Attempts to improve diagnosis have included risk scoring systems [5], computerised decision support [6, 7] and automated ECG interpretation [8]. Although clinical assessment remains integral to the assessment of patients with chest pain, cardiac biomarker measurement has become an

The mechanisms involved in the development of cardiovascular disease are multifactorial and include abnormalities in cholesterol and lipid metabolism, inflammation and oxidative stress processes within the vascular wall, cellular disruption to the endothelium and intra-lumenal platelet activation/aggregation. The ischemia cascade from initiation of local ischemia to the development of symptomatic chest pain is depicted in figure 1.The pathological processes responsible for the development of atherosclerotic lesions and endothelial dysfunction are advanced far earlier than when patients typically become symptomatic and present with chest pain. The disease process does not occur in distinct episodes but rather is a continuum from asymptomatic vascular dysfunction thorough to angina in those with myocardial ischemia, which, without intervention can progress to non-ST segment elevation myocardial infarction (NSTEMI) or cumulate into ST segment elevation myocardial infarction (STEMI). Patients

development of the IMA assay with emphasis on its clinical and prognostic utility.

and over 151,000 Americans who died were under 65 years of age.

essential component in the diagnostic armamentarium.

**2. Pathophysiology of cardiac ischemia**

**1.1. The cardiovascular disease epidemic**

**1.2. Acute chest pain**

92 Ischemic Heart Disease

The pathological process unless disrupted by therapeutic intervention results in the death of cardiac myocytes. Predisposing this terminal event, a vulnerable atherosclerotic plaque becomes disrupted exposing the thrombogenic lipid core and sub endothelium to the luminal milieu. Exposure results in platelet activation and aggregation and along with the coagulation cascade, an intracoronary thrombus forms. The thrombus may not obstruct the lumen and the patient is asymptomatic, however if the lumen is totally occluded, AMI will ensue. A partially occluded lumen and reduced oxygen supply both contribute to the development of ischemic myocardium.

## **3. Clinical detection of cardiac ischemia**

The clinical presentation of cardiac ischemia is difficult to definitively diagnose. Currently there is no gold standard test to detect ischemia however a number of reliable tests exist. Historically patients were admitted for monitoring or discharged on the basis of clinical interpretation by the ED physician. It is accepted that this is no longer acceptable clinical practice.

reviewed below. Of these, Ischemia Modified Albumin has been the most successful biomarker

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 95

Malondialdehyde low density lipoprotein (MDA-LDL) is a sensitive biomarker for ACS patients with unstable angina and AMI. MDA is a candidate compound which causes oxidative modification of LDL. MDA (propanedial, C3H4O2) is a reactive aldehyde produced by degra‐ dation of polyunsaturated lipids or released during prostanoid metabolism. This reactive oxygen species causes oxidative modification to LDL. MDA-LDL reacts with the charged amino group of B-100 protein lysyl residues. Plasma concentrations of MDA-LDL identify patients with coronary artery disease. Modified LDL may also instigate an immune response leading to autoantibody and LDL immune complex production. MDA-LDL not only serves as

Myeloperoxidase (MPO, EC 1.11.2.2, 1.11.2.2) is a 150 KDa protein dimer consisting of two 15 KDa light chains and two variable weight glycosylated heavy chains bound by a heme group responsible for the green colour when secreted in pus and mucus. There are three known isoforms which differ in the size of the heavy chain [9]. It is encoded by the MPO gene located on chromosome 17 [10]. MPO is most abundant in neutrophil granulocytes. It is a lysosomal enzyme stored in azurophilic granules of polymorphonucleocytes and macrophages. MPO catalyses the conversion of chloride and hydrogen peroxide into hypochlorite (hypochlorous acid). Furthermore, MPO oxidises tyrosine to the tyrosyl radical using hydrogen peroxide as an oxidising agent. Both hypochlorite and the tyrosyl radical are cytotoxic and are produce to kill pathogens in response to infection. Elevation in MPO is therefore not indicative of cardiac ischemia, as increases occur in infection, inflammation and infiltrative disease processes, thus

MPO may contribute to the pathophysiology of ACS, as the hypochlorite end product is an oxidizing agent of low density lipoprotein (LDL) and may play a key role in the degradation of collagen and contributing to the destabilisation of the plaque. Patients with ACS who have elevated MPO are at risk of short and long-term adverse outcomes. In a case-control study from the USA, Zhang and colleagues demonstrate that MPO concentrations are significantly greater in patients (n=158) with coronary artery disease compared to controls (n=175) who do not demonstrate angiographically significant coronary disease [11]. Plasma MPO concentra‐ tions identify patients at risk of major adverse cardiac events in the absence of necrosis. In 604 sequential chest pain admissions, MPO predicted adverse cardiac events (AMI, need for revascularisation or death)at 30 days (odds ratio 2.2, 95%CI 1.1-4.6) and 6 months (odds ratio

and greater attention is given to this marker.

**5. Myeloperoxidase**

4.1, 95%CI 2.0-8.4) [12].

reducing the specificity for cardiac ischemia.

**4. Malondialdehyde low density lipoprotein**

an oxidative stress marker but as a marker of plaque destabilisation.

The typical presentation is exertional or stress induced central chest pain. These episodes usually last from a few minutes to hours and can resolve upon rest. Common descriptions by the patient include tightness, crushing stabbing or burning pain. Patients may also have nausea and vomiting, dyspnoea, palpitations. Typical symptoms increase the likelihood of an AMI however atypical presentations cannot be used to exclude AMI. Women, the elderly and those with diabetes mellitus often present with atypical chest pain.

The clinical history and physical examination will assess the presence of risk factors for AMI, however alone; the initial clinical examination is insensitive and unspecific for diagnosis. It may however give insight to differential or alternative diagnoses in those patients who, upon further investigation do not have an AMI. The 12 lead ECG is additive to the physical exami‐ nation. The majority of ECG traces performed at admission are non-diagnostic with approxi‐ mately 5% of suspected AMI patients having a diagnostic trace indicative of AMI. Although the ECG is relatively insensitive, the presence of ST segment elevation however is 100% diagnostic for AMI and serves as the criterion for immediate induction of fibrinolytic therapy or emergency interventional revascularization.

#### **3.1. Cardiac imaging**

Recently cardiac imaging has played an important role in the detection of ischemia. Perfusion abnormalities can be detected by single-photon emission computer tomography (SPECT) myocardial perfusion imaging (MPI) and mechanical dysfunction can be detected by echo‐ cardiography or gated MPI. Gated SPECT MPI can identify regional and global dysfunction of the left ventricle as ischemia impairs myocellular contractility. SPECT requires uptake of an isotope by active membrane transport mechanisms and caution should be advised in those patients with impaired renal clearance. Both echocardiography and SPECT are sensitive and specific and have a high negative predictive value for the diagnosis and prognosis of patients with suspected ACS. These diagnostic modalities however are grossly expensive, time consuming, technically more challenging and are not as widely available as compared to the simple ECG or a blood borne biomarker. The use cardiac imaging in the ED on a 24 hour, 7 day a week basis is therefore compromised.

#### **3.2. Cardiac biomarkers of ischemia**

There have been progressive developments within basic and clinical research to identify candidate biomarkers of ischemia and to develop simple to use assays. Any such assay needs to have similar analytical (limit of detection, precision, reference intervals) and clinical performance (sensitivity, specificity, risk stratification and predictive value) compared to that of markers of necrosis, such as high sensitivity cardiac troponin assays. A number of candidate biomarkers have been identified. However very few make it from a research grade assay to a fully licenced automated assay for clinical use. The most promising biomarkers to date are reviewed below. Of these, Ischemia Modified Albumin has been the most successful biomarker and greater attention is given to this marker.

## **4. Malondialdehyde low density lipoprotein**

Malondialdehyde low density lipoprotein (MDA-LDL) is a sensitive biomarker for ACS patients with unstable angina and AMI. MDA is a candidate compound which causes oxidative modification of LDL. MDA (propanedial, C3H4O2) is a reactive aldehyde produced by degra‐ dation of polyunsaturated lipids or released during prostanoid metabolism. This reactive oxygen species causes oxidative modification to LDL. MDA-LDL reacts with the charged amino group of B-100 protein lysyl residues. Plasma concentrations of MDA-LDL identify patients with coronary artery disease. Modified LDL may also instigate an immune response leading to autoantibody and LDL immune complex production. MDA-LDL not only serves as an oxidative stress marker but as a marker of plaque destabilisation.

## **5. Myeloperoxidase**

Historically patients were admitted for monitoring or discharged on the basis of clinical interpretation by the ED physician. It is accepted that this is no longer acceptable clinical

The typical presentation is exertional or stress induced central chest pain. These episodes usually last from a few minutes to hours and can resolve upon rest. Common descriptions by the patient include tightness, crushing stabbing or burning pain. Patients may also have nausea and vomiting, dyspnoea, palpitations. Typical symptoms increase the likelihood of an AMI however atypical presentations cannot be used to exclude AMI. Women, the elderly and those

The clinical history and physical examination will assess the presence of risk factors for AMI, however alone; the initial clinical examination is insensitive and unspecific for diagnosis. It may however give insight to differential or alternative diagnoses in those patients who, upon further investigation do not have an AMI. The 12 lead ECG is additive to the physical exami‐ nation. The majority of ECG traces performed at admission are non-diagnostic with approxi‐ mately 5% of suspected AMI patients having a diagnostic trace indicative of AMI. Although the ECG is relatively insensitive, the presence of ST segment elevation however is 100% diagnostic for AMI and serves as the criterion for immediate induction of fibrinolytic therapy

Recently cardiac imaging has played an important role in the detection of ischemia. Perfusion abnormalities can be detected by single-photon emission computer tomography (SPECT) myocardial perfusion imaging (MPI) and mechanical dysfunction can be detected by echo‐ cardiography or gated MPI. Gated SPECT MPI can identify regional and global dysfunction of the left ventricle as ischemia impairs myocellular contractility. SPECT requires uptake of an isotope by active membrane transport mechanisms and caution should be advised in those patients with impaired renal clearance. Both echocardiography and SPECT are sensitive and specific and have a high negative predictive value for the diagnosis and prognosis of patients with suspected ACS. These diagnostic modalities however are grossly expensive, time consuming, technically more challenging and are not as widely available as compared to the simple ECG or a blood borne biomarker. The use cardiac imaging in the ED on a 24 hour, 7

There have been progressive developments within basic and clinical research to identify candidate biomarkers of ischemia and to develop simple to use assays. Any such assay needs to have similar analytical (limit of detection, precision, reference intervals) and clinical performance (sensitivity, specificity, risk stratification and predictive value) compared to that of markers of necrosis, such as high sensitivity cardiac troponin assays. A number of candidate biomarkers have been identified. However very few make it from a research grade assay to a fully licenced automated assay for clinical use. The most promising biomarkers to date are

with diabetes mellitus often present with atypical chest pain.

or emergency interventional revascularization.

day a week basis is therefore compromised.

**3.2. Cardiac biomarkers of ischemia**

**3.1. Cardiac imaging**

practice.

94 Ischemic Heart Disease

Myeloperoxidase (MPO, EC 1.11.2.2, 1.11.2.2) is a 150 KDa protein dimer consisting of two 15 KDa light chains and two variable weight glycosylated heavy chains bound by a heme group responsible for the green colour when secreted in pus and mucus. There are three known isoforms which differ in the size of the heavy chain [9]. It is encoded by the MPO gene located on chromosome 17 [10]. MPO is most abundant in neutrophil granulocytes. It is a lysosomal enzyme stored in azurophilic granules of polymorphonucleocytes and macrophages. MPO catalyses the conversion of chloride and hydrogen peroxide into hypochlorite (hypochlorous acid). Furthermore, MPO oxidises tyrosine to the tyrosyl radical using hydrogen peroxide as an oxidising agent. Both hypochlorite and the tyrosyl radical are cytotoxic and are produce to kill pathogens in response to infection. Elevation in MPO is therefore not indicative of cardiac ischemia, as increases occur in infection, inflammation and infiltrative disease processes, thus reducing the specificity for cardiac ischemia.

MPO may contribute to the pathophysiology of ACS, as the hypochlorite end product is an oxidizing agent of low density lipoprotein (LDL) and may play a key role in the degradation of collagen and contributing to the destabilisation of the plaque. Patients with ACS who have elevated MPO are at risk of short and long-term adverse outcomes. In a case-control study from the USA, Zhang and colleagues demonstrate that MPO concentrations are significantly greater in patients (n=158) with coronary artery disease compared to controls (n=175) who do not demonstrate angiographically significant coronary disease [11]. Plasma MPO concentra‐ tions identify patients at risk of major adverse cardiac events in the absence of necrosis. In 604 sequential chest pain admissions, MPO predicted adverse cardiac events (AMI, need for revascularisation or death)at 30 days (odds ratio 2.2, 95%CI 1.1-4.6) and 6 months (odds ratio 4.1, 95%CI 2.0-8.4) [12].

## **6. Whole blood choline**

Choline (2-hydroxy-N,N,N-trimethylethanaminium, C5H11NO) is a water soluble essential nutrient. It is a product of phosphodiesteric cleavage of membrane phospholipids such as phosphatidylcholine and sphingomyelin; catalysed by phospholipase D (EC 3.1.4.4). Choline is the precursor to actylcholine production.

**8. Ischemia modified albumin**

Zn++ binds site A and Au+

cardiac ischemia.

N-terminal alpha-amino group.

The NH2-terminal of human serum albumin (HSA, 66.5 kDa, 585 amino acids) is known to be a binding site for transition metal ions such as cobalt, copper and nickel [18]. Using one and two dimensional 1H-NMR studies, Sadler and colleagues demonstrated binding of Ni++, Cu++, Co++, Cd++ and Al+++ to bovine and human serum albumin. Strong binding was associated with three N-terminal amino acid residues (Asp-Thr-His in bovine albumin and Asp-Ala-His in human albumin). A Lysine residue designated Lys4 is also involved in the binding site. The authors demonstrated for the fist time selective reduction in the intensities of resonances to the εCH<sup>2</sup> resonance of Lys4 on the addition of Co++ to HSA. There are in fact, four metal-binding sites with differentspecificitiesinHSA.InadditiontotheNH2-terminal,threeothersitesoccurat(i)reduced cysteine at residue Cys34, (ii) site A, including histidine at His67 as a ligand and (iii) the nonlocalized site B. Cu++ and Ni++ preferentially bind the NH2-terminus site. Cd++ bind sites A and B,

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 97

and Pt++ bind at residue Cys34.

A reduction in oxygen supply causes localized acidosis and the generation of free radicals. Copper and zinc ions, normally bound to proteins in the plasma are released from protein binding sites to circulate in the free form [19-21]. The N-terminus of albumin binds transition metals. The N-terminus however, is susceptible to biochemical alteration [22]. The altered form is referred to as ischemia modified albumin (IMA). Following a period of ischemia, a reduction in the ability of albumin to bind cobalt is apparent. This is the basis of the albumin cobaltbinding test (ACB® test) for IMA. IMA has been extensively studied in the basic science and clinical research settings and is an FDA cleared CE marked clinical assay for the detection of

It is currently not known if there are any significant changes in total human serum albumin between ischemic and non ischemic patients in the general chest pain population. Many divalent metals bind HSA in the circulation but in concentrations far lower than that required to impact albumin directly. The N-terminal portion of HSA is susceptible to biochemical degradation and is less stable than the albumin of other species [22] including bovine, dog, goat, horse, pig rabbit, rat and sheep but not chicken. Using electrospray-mass spectrometry and N-terminal sequencing, Chan and colleagues have demonstrated degradation corre‐ sponding to the first two resides (Asp-Ala) which is dependent both on temperature and the

IMA however is a form of HSA where the N-terminal amino acids are unable to bind transition metal ions. Myocardial ischemia is known to generate free radicals [21;23], induce localised acidosis [20] and the release of free iron and copper ions bound to enzymes and proteins. [19;24]. Direct evidence of Cu/Fe mobilization in the coronary flow following prolonged (25-60 minute) ischemia but not short (15-21 minute) ischemia has been demonstrated [24]. Both copper and iron concentrations in the first coronary flow fraction were 50-fold and 15-fold higher respectively following prolonged ischemia, compared to pre-ischemic concentrations. This suggests that both copper and iron play a causative role in ischemic cardiac injury by their ability to catalyse the production of free radicals and could be the target of therapeutic intervention to salvage tissue damage [19]. It was therefore postulated that following a period

Physiologically choline provides cell structural integrity, is the precursor for acetylcholine production and a source of methyl groups that participate in the S-adenosylmethionine synthesis pathway.

Whole blood (WBCHO) and plasma choline concentrations increase after stimulation of phospholipase D and the activation of coronary plaque cell surface receptors or ischemia. Phospholipase D activation in coronary plaques causes stimulation of macrophage by oxidised LDL, secretion of matrix metalloproteinase enzymes and activation of platelets. WBCHO can be measured by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In a study of over 300 patients with suspected ACS, WBCHO measured at admission was a significant predictor of cardiac death, cardiac arrest, arrhythmia, heart failure or the need for percutaneous coronary intervention (PCI) at 30 day follow up [13]. The predictive power was enhanced by the addition of either cTnT or cTnI and served not as a marker of myocardial cell necrosis but identified patients at high risk with unstable angina. WBCHO is therefore a better predictive tool than plasma choline for early risk stratification in patients who are cardiac troponin negative on admission. The current detection methodology using HPLC-MS is not suitable for urgent clinical use.

## **7. Free fatty acids**

Fatty acids are carboxylic acid molecules with a long aliphatic tail known as a chain, which are either saturated or unsaturated. Most naturally occurring fatty acids have an even number of carbon atoms (4 to 28) in the tail region. Fatty acids are produced from the breakdown of triglyceride or phospholipid. The majority fatty acids circulate bound to albumin with a very small percentage appearing as the unbound free fatty acid (FFAu) form [14]. The circulating level of FFA is limited to the availability of the albumin binding sites.

The mechanism of FFAu release is not fully understood however increased catecholamines following cardiac ischemia may activate FFAu release following lipolysis in adipocytes. FFAu are 14-fold higher post-PCI, compared to pre procedural concentrations and were higher in those with associated ischemic ST segment changes [15]. A recombinant fatty acid binding protein bound to a fluorescent tag (ADIFAB) [16;17] has been developed and a second generation assay using a fluorescent molecular probe (ADIFAB2] and a portable reader makes this a potential early marker for the point of care setting. Whilst this marker shows promise in the early phase of ischemia induced ACS, further trials are required to evaluate the diagnosis and prognostic value of FFAu in the chest pain population.

## **8. Ischemia modified albumin**

**6. Whole blood choline**

96 Ischemic Heart Disease

synthesis pathway.

**7. Free fatty acids**

is the precursor to actylcholine production.

using HPLC-MS is not suitable for urgent clinical use.

level of FFA is limited to the availability of the albumin binding sites.

and prognostic value of FFAu in the chest pain population.

Choline (2-hydroxy-N,N,N-trimethylethanaminium, C5H11NO) is a water soluble essential nutrient. It is a product of phosphodiesteric cleavage of membrane phospholipids such as phosphatidylcholine and sphingomyelin; catalysed by phospholipase D (EC 3.1.4.4). Choline

Physiologically choline provides cell structural integrity, is the precursor for acetylcholine production and a source of methyl groups that participate in the S-adenosylmethionine

Whole blood (WBCHO) and plasma choline concentrations increase after stimulation of phospholipase D and the activation of coronary plaque cell surface receptors or ischemia. Phospholipase D activation in coronary plaques causes stimulation of macrophage by oxidised LDL, secretion of matrix metalloproteinase enzymes and activation of platelets. WBCHO can be measured by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In a study of over 300 patients with suspected ACS, WBCHO measured at admission was a significant predictor of cardiac death, cardiac arrest, arrhythmia, heart failure or the need for percutaneous coronary intervention (PCI) at 30 day follow up [13]. The predictive power was enhanced by the addition of either cTnT or cTnI and served not as a marker of myocardial cell necrosis but identified patients at high risk with unstable angina. WBCHO is therefore a better predictive tool than plasma choline for early risk stratification in patients who are cardiac troponin negative on admission. The current detection methodology

Fatty acids are carboxylic acid molecules with a long aliphatic tail known as a chain, which are either saturated or unsaturated. Most naturally occurring fatty acids have an even number of carbon atoms (4 to 28) in the tail region. Fatty acids are produced from the breakdown of triglyceride or phospholipid. The majority fatty acids circulate bound to albumin with a very small percentage appearing as the unbound free fatty acid (FFAu) form [14]. The circulating

The mechanism of FFAu release is not fully understood however increased catecholamines following cardiac ischemia may activate FFAu release following lipolysis in adipocytes. FFAu are 14-fold higher post-PCI, compared to pre procedural concentrations and were higher in those with associated ischemic ST segment changes [15]. A recombinant fatty acid binding protein bound to a fluorescent tag (ADIFAB) [16;17] has been developed and a second generation assay using a fluorescent molecular probe (ADIFAB2] and a portable reader makes this a potential early marker for the point of care setting. Whilst this marker shows promise in the early phase of ischemia induced ACS, further trials are required to evaluate the diagnosis The NH2-terminal of human serum albumin (HSA, 66.5 kDa, 585 amino acids) is known to be a binding site for transition metal ions such as cobalt, copper and nickel [18]. Using one and two dimensional 1H-NMR studies, Sadler and colleagues demonstrated binding of Ni++, Cu++, Co++, Cd++ and Al+++ to bovine and human serum albumin. Strong binding was associated with three N-terminal amino acid residues (Asp-Thr-His in bovine albumin and Asp-Ala-His in human albumin). A Lysine residue designated Lys4 is also involved in the binding site. The authors demonstrated for the fist time selective reduction in the intensities of resonances to the εCH<sup>2</sup> resonance of Lys4 on the addition of Co++ to HSA. There are in fact, four metal-binding sites with differentspecificitiesinHSA.InadditiontotheNH2-terminal,threeothersitesoccurat(i)reduced cysteine at residue Cys34, (ii) site A, including histidine at His67 as a ligand and (iii) the nonlocalized site B. Cu++ and Ni++ preferentially bind the NH2-terminus site. Cd++ bind sites A and B, Zn++ binds site A and Au+ and Pt++ bind at residue Cys34.

A reduction in oxygen supply causes localized acidosis and the generation of free radicals. Copper and zinc ions, normally bound to proteins in the plasma are released from protein binding sites to circulate in the free form [19-21]. The N-terminus of albumin binds transition metals. The N-terminus however, is susceptible to biochemical alteration [22]. The altered form is referred to as ischemia modified albumin (IMA). Following a period of ischemia, a reduction in the ability of albumin to bind cobalt is apparent. This is the basis of the albumin cobaltbinding test (ACB® test) for IMA. IMA has been extensively studied in the basic science and clinical research settings and is an FDA cleared CE marked clinical assay for the detection of cardiac ischemia.

It is currently not known if there are any significant changes in total human serum albumin between ischemic and non ischemic patients in the general chest pain population. Many divalent metals bind HSA in the circulation but in concentrations far lower than that required to impact albumin directly. The N-terminal portion of HSA is susceptible to biochemical degradation and is less stable than the albumin of other species [22] including bovine, dog, goat, horse, pig rabbit, rat and sheep but not chicken. Using electrospray-mass spectrometry and N-terminal sequencing, Chan and colleagues have demonstrated degradation corre‐ sponding to the first two resides (Asp-Ala) which is dependent both on temperature and the N-terminal alpha-amino group.

IMA however is a form of HSA where the N-terminal amino acids are unable to bind transition metal ions. Myocardial ischemia is known to generate free radicals [21;23], induce localised acidosis [20] and the release of free iron and copper ions bound to enzymes and proteins. [19;24]. Direct evidence of Cu/Fe mobilization in the coronary flow following prolonged (25-60 minute) ischemia but not short (15-21 minute) ischemia has been demonstrated [24]. Both copper and iron concentrations in the first coronary flow fraction were 50-fold and 15-fold higher respectively following prolonged ischemia, compared to pre-ischemic concentrations. This suggests that both copper and iron play a causative role in ischemic cardiac injury by their ability to catalyse the production of free radicals and could be the target of therapeutic intervention to salvage tissue damage [19]. It was therefore postulated that following a period of cardiac ischemia, these processes would result in a change in the ability of the N-terminus of HSA to bind transition metal ions. The release of these ions likely initiates one potential pathway for IMA generation, rather than be considered an interference that may negatively affect IMA. In support of this suggestion, decreased albumin cobalt binding was reported in 99 acute chest pain patients with myocardial ischemia [25] compared to 44 chest pain patients with no evidence of myocardial ischemia. Albumin cobalt binding was also assessed in 41 patients undergoing elective coronary artery angioplasty. Samples were tested using the Albumin Cobalt Binding (ACB) assay before, immediately after, 6 and 24 hours post procedure and compared to results from 13 patients undergoing cardiac catheterization without balloon angioplasty, thus serving as the control group. ACB concentrations were significantly elevated immediately post procedure, compared to the control population and ACB concentrations returned to baseline after six hours [26]; suggesting that HSA undergoes a significant reduction in the capacity to bind exogenous Co++ immediately after coronary artery occlusion induced during elective angioplasty. Modification of the Asp-Ala-His-Lys site by N-terminal acetyla‐ tion or deletion of one or more residues abolishes this cobalt binding [27].

The postulated mechanism (figure 2) of IMA generation is that localised ischemia results in acidosis. The localised acidotic environment stimulates the release of Cu++ ions from weak binding sites on circulating proteins such as caeruloplasmin. Caeruloplasmin (EC 1.16.3.1, 151kDa) is a ferroxidase enzyme encoded by the *CP* gene located on Chromosome 3. The enzyme is synthesised in the liver and carries approximately 70% of the total copper in human plasma (a further 15% carried by HSA and the remainder by macroglobulins). Each enzyme molecule contains 6 atoms of copper within its structure.

In the presence of a reducing agent such as ascorbic acid, free copper II is converted to copper I which can react with oxygen to form copper II and generate superoxide free radicals (O2 •–). Superoxide dismutase (EC 1.15.1.1) converts the superoxide free radical to hydro‐ gen peroxide which is then degraded by catalase. The copper II ions released are immediate‐ ly scavenged by human serum albumin but they are tightly bound to the N-terminus. Copper bound albumin is then damaged by hydroxyl free radicals (OH •), causing removal of the three N terminal amino acids and release of the copper II ion to repeat the process in a chain reaction [28].Marx and Chevion demonstrated by SDS/polyacrylaminde gel electropho‐ resis the site specific alteration of HSA in the presence of 50 μM Cu++ and increasing portions of 0.2 m Mascorbate; where after the addition of 5 portions, bands at 3, 18, 22, 47 and 50 kDa were observed. The authors also demonstrated that degradation does not occur in the absence of Cu++ or in the addition of 1 m Methylenediaminetetraacetic acid (EDTA) or citrate chelating agents [28].

**Subject NH2-terminal HSA Sequence**

**Figure 2. Mechanism of Ischemia Modified Albumin generation.** [1]Tissue hypoxia from anaerobic metabolism re‐ duces ATP and causes a [2]lower localized pH inducing acidosis. [3]Cu++ ions are released from plasma proteins such as caeruloplasmin. In the presence of ascorbic acid, [4]Cu++ is converted to Cu+. Cu+ reacts with O2 to form [5]O2 •–. Super‐ oxide dismutase dismutates the O2 •– to [6]H2O2, which in presence of Cu++ or Fe+, undergoes the Fenton reaction

cals alter the amino acid N-terminus of [9]HSA rendering it incapable of binding Cu++. These two altered forms are

hydroxyl radicals. Free Cu++ is scavenged by [8]HSA, where it binds tightly to the N-terminus. OH •

radi‐

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 99

**Table 1.** Amino (NH2) terminal sequence analysis of human serum albumin (HAS) from 6 ischemic patients, a control (wild type) and one non ischemic patient with a high serum IMA concentration. (Source: Adapted from Bhagavan et

The *in vivo* half-life of HSA is 19-20 days. HSA with a truncated NH2-terminus would pre‐ sumably have similar *in vivo* half life properties and yet IMA returns to baseline rapidly after an ischaemic cardiac event. This indicates that the alteration to albumin to create IMA is transient and reversible, rather than a finite chemical alteration. Recent physicochemical

Control (wild type) DAHKSEVAHRF Non-ischemic patient, high serum IMA ----HKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF Ischemic patient, high serum IMA DAHKSEVAHRF

al, ClinChem 2003;49:581-585)

forming [7]OH •

known as IMA.

This postulated mechanism, although theoretically attractive has not been borne out in practice. In a study of patients with increased IMA, the N-terminal portion of albumin was sequenced in 8 cases [29] by cleavage of the 11 amino acid resides at the NH2 terminus, by rapid liquid-phase Edman degradation. The N-terminal amino acid sequence showed normal residues for 6 of 7 patient samples with elevated IMA and one non-ischemic sample (table 1). The remaining patient sample with high IMA demonstrated two missing amino acids at the N-terminus. Clinically this patient did not have an ischemic cardiac event.

of cardiac ischemia, these processes would result in a change in the ability of the N-terminus of HSA to bind transition metal ions. The release of these ions likely initiates one potential pathway for IMA generation, rather than be considered an interference that may negatively affect IMA. In support of this suggestion, decreased albumin cobalt binding was reported in 99 acute chest pain patients with myocardial ischemia [25] compared to 44 chest pain patients with no evidence of myocardial ischemia. Albumin cobalt binding was also assessed in 41 patients undergoing elective coronary artery angioplasty. Samples were tested using the Albumin Cobalt Binding (ACB) assay before, immediately after, 6 and 24 hours post procedure and compared to results from 13 patients undergoing cardiac catheterization without balloon angioplasty, thus serving as the control group. ACB concentrations were significantly elevated immediately post procedure, compared to the control population and ACB concentrations returned to baseline after six hours [26]; suggesting that HSA undergoes a significant reduction in the capacity to bind exogenous Co++ immediately after coronary artery occlusion induced during elective angioplasty. Modification of the Asp-Ala-His-Lys site by N-terminal acetyla‐

The postulated mechanism (figure 2) of IMA generation is that localised ischemia results in acidosis. The localised acidotic environment stimulates the release of Cu++ ions from weak binding sites on circulating proteins such as caeruloplasmin. Caeruloplasmin (EC 1.16.3.1, 151kDa) is a ferroxidase enzyme encoded by the *CP* gene located on Chromosome 3. The enzyme is synthesised in the liver and carries approximately 70% of the total copper in human plasma (a further 15% carried by HSA and the remainder by macroglobulins). Each enzyme

In the presence of a reducing agent such as ascorbic acid, free copper II is converted to copper I which can react with oxygen to form copper II and generate superoxide free radicals (O2 •–). Superoxide dismutase (EC 1.15.1.1) converts the superoxide free radical to hydro‐ gen peroxide which is then degraded by catalase. The copper II ions released are immediate‐ ly scavenged by human serum albumin but they are tightly bound to the N-terminus. Copper bound albumin is then damaged by hydroxyl free radicals (OH •), causing removal of the three N terminal amino acids and release of the copper II ion to repeat the process in a chain reaction [28].Marx and Chevion demonstrated by SDS/polyacrylaminde gel electropho‐ resis the site specific alteration of HSA in the presence of 50 μM Cu++ and increasing portions of 0.2 m Mascorbate; where after the addition of 5 portions, bands at 3, 18, 22, 47 and 50 kDa were observed. The authors also demonstrated that degradation does not occur in the absence of Cu++ or in the addition of 1 m Methylenediaminetetraacetic acid (EDTA) or citrate

This postulated mechanism, although theoretically attractive has not been borne out in practice. In a study of patients with increased IMA, the N-terminal portion of albumin was sequenced in 8 cases [29] by cleavage of the 11 amino acid resides at the NH2 terminus, by rapid liquid-phase Edman degradation. The N-terminal amino acid sequence showed normal residues for 6 of 7 patient samples with elevated IMA and one non-ischemic sample (table 1). The remaining patient sample with high IMA demonstrated two missing amino acids at the

N-terminus. Clinically this patient did not have an ischemic cardiac event.

tion or deletion of one or more residues abolishes this cobalt binding [27].

molecule contains 6 atoms of copper within its structure.

chelating agents [28].

98 Ischemic Heart Disease

**Figure 2. Mechanism of Ischemia Modified Albumin generation.** [1]Tissue hypoxia from anaerobic metabolism re‐ duces ATP and causes a [2]lower localized pH inducing acidosis. [3]Cu++ ions are released from plasma proteins such as caeruloplasmin. In the presence of ascorbic acid, [4]Cu++ is converted to Cu+. Cu+ reacts with O2 to form [5]O2 •–. Super‐ oxide dismutase dismutates the O2 •– to [6]H2O2, which in presence of Cu++ or Fe+, undergoes the Fenton reaction forming [7]OH • hydroxyl radicals. Free Cu++ is scavenged by [8]HSA, where it binds tightly to the N-terminus. OH • radi‐ cals alter the amino acid N-terminus of [9]HSA rendering it incapable of binding Cu++. These two altered forms are known as IMA.


**Table 1.** Amino (NH2) terminal sequence analysis of human serum albumin (HAS) from 6 ischemic patients, a control (wild type) and one non ischemic patient with a high serum IMA concentration. (Source: Adapted from Bhagavan et al, ClinChem 2003;49:581-585)

The *in vivo* half-life of HSA is 19-20 days. HSA with a truncated NH2-terminus would pre‐ sumably have similar *in vivo* half life properties and yet IMA returns to baseline rapidly after an ischaemic cardiac event. This indicates that the alteration to albumin to create IMA is transient and reversible, rather than a finite chemical alteration. Recent physicochemical studies using electronic absorption EPR and NMR spectroscopy of Co-binding to HSA under anaerobic conditions to prevent Co++ oxidation have suggested a different explanation. Using competition experiments with cadmium (Cd++) which binds sites A and B and Cu++ which binds the NH2-terminus, three binding sites for Co++ were identified on HSA. Sites A and B showed greater avidity for Co++ binding than the NH2-terminal binding site [30]. Fatty acid binding to albumin occurs at one of the additional cobalt binding sites with a negative allosteric interac‐ tion. It is hypothesised, that in myocardial ischemia the release of fatty acids results in binding of fatty acids to albumin. This would then reduce the ability of albumin to take up cobalt hence account for the presence of IMA [30]. If this also produced a conformational change in the albumin affecting the N terminal site, this would also reduce cobalt binding.

routine care for elective single vessel PCI for the management of stable angina pectoris. 44% of patients received 1-4 balloon inflations whilst, 56% received >5 inflations. IMA concentra‐ tions were higher in those with more balloon inflations, higher pressure load of the balloon and the longer the duration of the inflation. IMA is thus not only a marker of the occurrence

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 101

IMA concentrations are lower in patients who demonstrate angiographic evidence of collateral vessels present in the coronary circulation, according to Rentrop's classification [34]. IMA levels post-PCI are higher than baseline, however post-PCI values are lower compared to post-PCI values in those patients without a collateral circulation; irrespective of the extent of coronary artery disease or those who underwent a large number of balloon inflations for longer duration [35]. The lower IMA concentrations in patients with a collateral circulation likely represent a cardioprotective effect against PCI-induced ischemia. IMA elevation is also correlated to the need for subsequent revascularization [36]. Elevated IMA greater than 130 KU/L was associated with a higher frequency of target lesion revascularization at 4-years follow-up in 60 patients who underwent a successful elective single vessel PCI for stable angina pectoris at baseline. The accepted gold standard blood marker for myocardial ischemia is myocardial lactate extraction. Simultaneous IMA and lactate was measured in 10 patients undergoing PCI for chronic stable angina. Post-PCI IMA concentrations paralleled that of

Elevation in serum IMA has been recorded following coronary vasospasm [37]. Twenty six patients with variant angina underwent intracoronary ergonovine spasm provocation testing. Arterial IMA concentrations were measured pre and post procedure and compared to 18 patients undergoing elective PCI and 10 patients with normal coronary angiography. IMA was significantly elevated following drug induced coronary vasospasm compared to baseline and elevated values detected coronary vasospasm with an area under the curve (AUC) of the receiver operating characteristic (ROC) curve of 0.98 (95%CI 0.92-1.00). Other studies involving invasive cardiac procedures have shown rises in IMA where ischemia might occur, occurring concurrently with ECG changes in cardioversion[38], but show a variable picture when there

The original biochemical test for IMA was known as the albumin cobalt binding (ACB®) assay. This was developed by Ischemia Technologies Inc, Colorado, USA). The assay measures the cobalt binding capacity of albumin in a sample of serum. A known amount of cobalt is added to the patient serum sample. Dithiothreitol (DTT) is added which binds any remaining unbound cobalt and the colorimetric change is measured spectrophotometrically. In serum from non-ischaemic patients, cobalt binds to the N-terminus of HSA, leaving little free cobalt to react with DTT and form a coloured product. Conversely, in serum of patients with ischemia, cobalt does not bind to the N-terminus of modified HSA, leaving more free cobalt to react with DTT and form a darker colour. As normal albumin will bind cobalt, the amount of free cobalt,

hence the absorbance will be proportional to the amount of IMA present (figure 4).

is non-ischemic myocardial damage as in cardiac ablation [39;40].

**8.2. Measurement of ischemia modified albumin**

of ischemia but is also an indicator of the severity of the ischemic episode.

transmyocardial lactate [32].

#### **8.1. Kinetic release of ischemia modified albumin**

Studies in patients receiving angioplasty where ischemia is induced in a controlled manner, have defined the kinetics of IMA production. There is a rapid rise in IMA values after balloon inflation with a subsequent fall at 6 hours and return to normal by 24 hours [31;32]. The rise in IMA occurs earlier than the rise in cardiac troponin and natriuretic peptides (figure 3) and occurs early after the onset of plaque rupture.

**Figure 3. Kinetic release of Ischemia modified albumin** (IMA, dotted line) and other cardiac markers, cardiac tropo‐ nin (cTn, solid line) and natriuretic peptide (NTproBNP, dashed line) [bottom panel], in relation to extent and timing of tissue damage [top panel]

The magnitude of IMA elevation has been found to correlate with the number and frequency of transluminal balloon inflations during the PCI procedure [33]. 34 patients received standard routine care for elective single vessel PCI for the management of stable angina pectoris. 44% of patients received 1-4 balloon inflations whilst, 56% received >5 inflations. IMA concentra‐ tions were higher in those with more balloon inflations, higher pressure load of the balloon and the longer the duration of the inflation. IMA is thus not only a marker of the occurrence of ischemia but is also an indicator of the severity of the ischemic episode.

IMA concentrations are lower in patients who demonstrate angiographic evidence of collateral vessels present in the coronary circulation, according to Rentrop's classification [34]. IMA levels post-PCI are higher than baseline, however post-PCI values are lower compared to post-PCI values in those patients without a collateral circulation; irrespective of the extent of coronary artery disease or those who underwent a large number of balloon inflations for longer duration [35]. The lower IMA concentrations in patients with a collateral circulation likely represent a cardioprotective effect against PCI-induced ischemia. IMA elevation is also correlated to the need for subsequent revascularization [36]. Elevated IMA greater than 130 KU/L was associated with a higher frequency of target lesion revascularization at 4-years follow-up in 60 patients who underwent a successful elective single vessel PCI for stable angina pectoris at baseline. The accepted gold standard blood marker for myocardial ischemia is myocardial lactate extraction. Simultaneous IMA and lactate was measured in 10 patients undergoing PCI for chronic stable angina. Post-PCI IMA concentrations paralleled that of transmyocardial lactate [32].

Elevation in serum IMA has been recorded following coronary vasospasm [37]. Twenty six patients with variant angina underwent intracoronary ergonovine spasm provocation testing. Arterial IMA concentrations were measured pre and post procedure and compared to 18 patients undergoing elective PCI and 10 patients with normal coronary angiography. IMA was significantly elevated following drug induced coronary vasospasm compared to baseline and elevated values detected coronary vasospasm with an area under the curve (AUC) of the receiver operating characteristic (ROC) curve of 0.98 (95%CI 0.92-1.00). Other studies involving invasive cardiac procedures have shown rises in IMA where ischemia might occur, occurring concurrently with ECG changes in cardioversion[38], but show a variable picture when there is non-ischemic myocardial damage as in cardiac ablation [39;40].

#### **8.2. Measurement of ischemia modified albumin**

studies using electronic absorption EPR and NMR spectroscopy of Co-binding to HSA under anaerobic conditions to prevent Co++ oxidation have suggested a different explanation. Using competition experiments with cadmium (Cd++) which binds sites A and B and Cu++ which binds the NH2-terminus, three binding sites for Co++ were identified on HSA. Sites A and B showed greater avidity for Co++ binding than the NH2-terminal binding site [30]. Fatty acid binding to albumin occurs at one of the additional cobalt binding sites with a negative allosteric interac‐ tion. It is hypothesised, that in myocardial ischemia the release of fatty acids results in binding of fatty acids to albumin. This would then reduce the ability of albumin to take up cobalt hence account for the presence of IMA [30]. If this also produced a conformational change in the

Studies in patients receiving angioplasty where ischemia is induced in a controlled manner, have defined the kinetics of IMA production. There is a rapid rise in IMA values after balloon inflation with a subsequent fall at 6 hours and return to normal by 24 hours [31;32]. The rise in IMA occurs earlier than the rise in cardiac troponin and natriuretic peptides (figure 3) and

**All Ischemia Some Ischemia, some Necrosis All Necrosis**


**Figure 3. Kinetic release of Ischemia modified albumin** (IMA, dotted line) and other cardiac markers, cardiac tropo‐ nin (cTn, solid line) and natriuretic peptide (NTproBNP, dashed line) [bottom panel], in relation to extent and timing of

The magnitude of IMA elevation has been found to correlate with the number and frequency of transluminal balloon inflations during the PCI procedure [33]. 34 patients received standard

**cTn**

**NT-Pro-BNP**

albumin affecting the N terminal site, this would also reduce cobalt binding.

**8.1. Kinetic release of ischemia modified albumin**

occurs early after the onset of plaque rupture.

**Plaque Rupture Onset of Pain**

Amount of Tissue

100 Ischemic Heart Disease

Concentration of marker

tissue damage [top panel]

**ED Presentation**

**IMA**

The original biochemical test for IMA was known as the albumin cobalt binding (ACB®) assay. This was developed by Ischemia Technologies Inc, Colorado, USA). The assay measures the cobalt binding capacity of albumin in a sample of serum. A known amount of cobalt is added to the patient serum sample. Dithiothreitol (DTT) is added which binds any remaining unbound cobalt and the colorimetric change is measured spectrophotometrically. In serum from non-ischaemic patients, cobalt binds to the N-terminus of HSA, leaving little free cobalt to react with DTT and form a coloured product. Conversely, in serum of patients with ischemia, cobalt does not bind to the N-terminus of modified HSA, leaving more free cobalt to react with DTT and form a darker colour. As normal albumin will bind cobalt, the amount of free cobalt, hence the absorbance will be proportional to the amount of IMA present (figure 4).

The nickel binding assay correlated well to the ACB assay (r=0.5387, p<0.001) however the AUC of the ROC curve was higher for the nickel binding assay (0.7582) compared to the ACB assay (0.7289) suggesting nickel binding has a superior ability to discriminate be‐ tween ACS and non-ACS compared to the ACB assay. There are rumours of an ELISA assay for IMA. This assay is not validated for clinical diagnostic or therapeutic use and an independent performance validation and comparison to the ACB assay does not appear in the current literature. The development of an ELISA however is probably not valid given the rapid alteration and return to baseline of IMA following ischemia, suggesting the alteration is transient and not a permanent change to a specific epitope which could be

values increase significantly after four hours irrespective of the storage temperature [33].It is likely that the changes are due to *in vitro* pH changes altering the metal binding capacity of human serum albumin. Samples frozen at -20°C are stable although values have been reported to be slightly higher once thawed, compared to freshly analysed samples [47]. The assay incubation temperature can also affect cobalt binding to HSA and thus influence the IMA

A study of 109 subjects (55 men and 54 women; age range, 20 to 85 years) to determine the 95th percentile reference range for IMA has been performed [49]. The concentrations ranged from 25.7 to 84.5 KU/L with an upper 95th percentile of 80.2 KU/L. This study used the first generation of the assay utilising the pre-handling sample preparation step. Further studies of healthy subjects have reported higher IMA ranges. Abadie and colleagues demonstrated a mean IMA value of 89 KU/L from 69 subjects with a mean age of 49 years [50] whilst Maguire and colleagues demonstrated a 97.5th percentile of 110 KU/L [42] from a population of 81 healthy volunteers (28 men and 53 women aged 22-86 years). Values ranged from 82.0 to 110 KU/L and values were similar between males and females (99.1 vs 100.7 KU/L, p=0.12). The biological variation of IMA has been studied [51].In a population of 17 apparently healthy individuals (7 male, 10 female, aged 26-61 years), the within subject coefficient of variation was 2.89% and the between subject coefficient of variation was 6.76%, calculated from weekly blood draws performed at the same time by the same phlebotomist for 5 consecutive weeks. Again there was no specific gender difference in IMA concentrations however the authors reported statistically different IMA concentrations between Caucasian and Black populations, with higher IMA concentrations in Black males and females compared to Caucasian counter‐

Total serum albumin concentrations might be expected to affect the performance of IMA measurement. There is a relationship between IMA values and serum albumin concentration, although this is much less marked across the reference interval for albumin [52]. The use of an albumin adjusted correction has been proposed [53] although a reference interval study found albumin correction to have little impact compared to other analytical factors [42]. It has been reported that the changes in IMA observed in patients with chest pain was attributable only

to changes in the serum albumin concentration [54].

C or 20°C, but

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 103

The *in vitro* stability of the IMA has been shown to be two hours at either 4o

detected by an antibody.

concentration [48].

parts.

**Figure 4. Measurement of Ischemia Modified Albumin by the Albumin Cobalt Binding (ACB) assay.** A known amount of CoCl2 is added to a serum sample. DTT is added which binds unbound Co++ causing a colorimetric change read spectrophotometrically.

The first generation assay was semi-automated and required a sample pre-treatment step where 500μL of serum sample was added to an Eppendorf tube containing 0.45g CaCl2. The sample was inverted twice and centrifuged at 1200g for 10 minutes. 300μL of supernatant was removed for assay of IMA. For the assay, powdered DTT was provided which required reconstitution in the ratio 15mg DTT: 10 mL diluent. The reconstituted reagent and power required storage at 2-8o C and the working solution had a shelf life of 3-5 days. The second generation of the assay used 7.5mg DTT to 10mL diluent. The third generation assay which became commercially available did not contain the sample pre treatment step and the assay kit contained a concentrated liquid form of DTT which was reconstituted in buffer with a fixed volume 200 μL pipette.

The assay can be performed manually with a spectrophotometer, however it was also initially automated on the Cobas MIRA Plus (Roche Diagnostics) automated spectrophotometer [41]. The assay has since been adapted for other automated clinical chemistry platforms includ‐ ing the LX-20 (Beckman Coulter, Brea, CO, USA) [42], Hitachi 911 (Hitachi, Japan) [43] Hitachi 7600 (Hitachi, Japan) [44] and the Konelab 20 (Thermo Scientific, United Kingdom) [45]. To date a commercialised point of care device for IMA remains to be developed however a precommercial portable spectrophotometer and IMA assay has been developed by (Micro‐ wells Biotechnology Co. Ltd, Shanghai, China). In the Microwells assay, the DTT has been replaced with a stable azo dye chromogen. An automated method to measure ischemia induced alterations of the binding capacity of HSA for nickel [27] has been described [46]. The nickel binding assay correlated well to the ACB assay (r=0.5387, p<0.001) however the AUC of the ROC curve was higher for the nickel binding assay (0.7582) compared to the ACB assay (0.7289) suggesting nickel binding has a superior ability to discriminate be‐ tween ACS and non-ACS compared to the ACB assay. There are rumours of an ELISA assay for IMA. This assay is not validated for clinical diagnostic or therapeutic use and an independent performance validation and comparison to the ACB assay does not appear in the current literature. The development of an ELISA however is probably not valid given the rapid alteration and return to baseline of IMA following ischemia, suggesting the alteration is transient and not a permanent change to a specific epitope which could be detected by an antibody.

**Non ischemic Sample Ischemic Sample**

**IMA** Cu++

**Figure 4. Measurement of Ischemia Modified Albumin by the Albumin Cobalt Binding (ACB) assay.** A known amount of CoCl2 is added to a serum sample. DTT is added which binds unbound Co++ causing a colorimetric change

The first generation assay was semi-automated and required a sample pre-treatment step where 500μL of serum sample was added to an Eppendorf tube containing 0.45g CaCl2. The sample was inverted twice and centrifuged at 1200g for 10 minutes. 300μL of supernatant was removed for assay of IMA. For the assay, powdered DTT was provided which required reconstitution in the ratio 15mg DTT: 10 mL diluent. The reconstituted reagent and power

generation of the assay used 7.5mg DTT to 10mL diluent. The third generation assay which became commercially available did not contain the sample pre treatment step and the assay kit contained a concentrated liquid form of DTT which was reconstituted in buffer with a fixed

The assay can be performed manually with a spectrophotometer, however it was also initially automated on the Cobas MIRA Plus (Roche Diagnostics) automated spectrophotometer [41]. The assay has since been adapted for other automated clinical chemistry platforms includ‐ ing the LX-20 (Beckman Coulter, Brea, CO, USA) [42], Hitachi 911 (Hitachi, Japan) [43] Hitachi 7600 (Hitachi, Japan) [44] and the Konelab 20 (Thermo Scientific, United Kingdom) [45]. To date a commercialised point of care device for IMA remains to be developed however a precommercial portable spectrophotometer and IMA assay has been developed by (Micro‐ wells Biotechnology Co. Ltd, Shanghai, China). In the Microwells assay, the DTT has been replaced with a stable azo dye chromogen. An automated method to measure ischemia induced alterations of the binding capacity of HSA for nickel [27] has been described [46].

HSA with Co++ at the N-terminus

102 Ischemic Heart Disease

read spectrophotometrically.

required storage at 2-8o

volume 200 μL pipette.

Unbound Cobalt Ischemia modified Albumin

C and the working solution had a shelf life of 3-5 days. The second

The *in vitro* stability of the IMA has been shown to be two hours at either 4o C or 20°C, but values increase significantly after four hours irrespective of the storage temperature [33].It is likely that the changes are due to *in vitro* pH changes altering the metal binding capacity of human serum albumin. Samples frozen at -20°C are stable although values have been reported to be slightly higher once thawed, compared to freshly analysed samples [47]. The assay incubation temperature can also affect cobalt binding to HSA and thus influence the IMA concentration [48].

A study of 109 subjects (55 men and 54 women; age range, 20 to 85 years) to determine the 95th percentile reference range for IMA has been performed [49]. The concentrations ranged from 25.7 to 84.5 KU/L with an upper 95th percentile of 80.2 KU/L. This study used the first generation of the assay utilising the pre-handling sample preparation step. Further studies of healthy subjects have reported higher IMA ranges. Abadie and colleagues demonstrated a mean IMA value of 89 KU/L from 69 subjects with a mean age of 49 years [50] whilst Maguire and colleagues demonstrated a 97.5th percentile of 110 KU/L [42] from a population of 81 healthy volunteers (28 men and 53 women aged 22-86 years). Values ranged from 82.0 to 110 KU/L and values were similar between males and females (99.1 vs 100.7 KU/L, p=0.12). The biological variation of IMA has been studied [51].In a population of 17 apparently healthy individuals (7 male, 10 female, aged 26-61 years), the within subject coefficient of variation was 2.89% and the between subject coefficient of variation was 6.76%, calculated from weekly blood draws performed at the same time by the same phlebotomist for 5 consecutive weeks. Again there was no specific gender difference in IMA concentrations however the authors reported statistically different IMA concentrations between Caucasian and Black populations, with higher IMA concentrations in Black males and females compared to Caucasian counter‐ parts.

Total serum albumin concentrations might be expected to affect the performance of IMA measurement. There is a relationship between IMA values and serum albumin concentration, although this is much less marked across the reference interval for albumin [52]. The use of an albumin adjusted correction has been proposed [53] although a reference interval study found albumin correction to have little impact compared to other analytical factors [42]. It has been reported that the changes in IMA observed in patients with chest pain was attributable only to changes in the serum albumin concentration [54].

#### **8.3. Clinical utility of ischemia modified albumin in chest pain patients**

Clinical validation of any test for ischemia is difficult as there is currently no accepted diagnostic gold standard, although blood lactate has been used previously.In addition, there is no predicate test which can be used against which to perform an initial validation. The initial studies using IMA were based on the ability of an early measurement to predict the final diagnosis of AMI as defined by the elevation of cardiac troponin at 6-12 hours post chest pain. Two studies utilised the first generation pre-release ACB test and a third study manufactured an in-house method. The first study examined acute coronary syndrome (ACS) patients and utilised serial sampling on admission and two subsequent samples [55]. Diagnostic sensitivity of the admission sample for a final diagnosis of AMI was 23.9% for cardiac troponin I (cTnI) alone, 39.1% for IMA alone and 55.9% for the two combined. The second study examined enrolled 256 ACS patients [49]. AUC of the ROC curve for the ACB test was 0.78 with a sensitivity and specificity of 83% and 69% respectively at the opti‐ mised decision threshold for AMI. The third study enrolled 75 patients with ischemia and 92 non-ischemic patients [29]. IMA had poor predictive power in discriminating between AMI and non-AMI in patients with underlying ischemic heart disease (AUC of 0.66). However, the test gave good discrimination between patients with or without ischemia. The AUC for the ROC curve for diagnosis of ischemia was 0.95 with sensitivity of 94% and specificity of 88%. In these initial studies there were significant problems with sample stability and the assay involved in addition of calcium chloride and centrifugation as part of the routine method. This made the method unsuitable from routine analysis and the assay was reformulated.

the definitive test found only a 97.6% sensitivity with 97% negative predictive value. The investigators did not consider this to be adequate when compared with troponin but did not provide any follow-up data [58]. A second large study prospectively enrolling 189 patients presenting to the ED with chest pain and found an elevated IMA was a poor predictor of cardiac events within the next 72 hours [59]. Conversely, another study found elevated IMA predicted long-term cardiac events [60]. The most consistent finding across all studies of IMA is of a high negative predictive value. This has been highlighted in a meta-analysis specifically examining the role of IMA as a rule out test [61]. The summar‐ ised data of over 1800 patients demonstrated a triple negative prediction test (non-diagnos‐ tic ECG, negative cTn and negative IMA) with a sensitivity and negative predictive value

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 105

The prognostic value of IMA in the ACS setting has been investigated [57;60;62;63]. Using a ROC derived cut off of 477 KU/L, Aparci and colleagues found significantly higher mortality at one year in those who had serum IMA >477 KU/L, compared to those with IMA <477 KU/L [60]. Furthermore, using cox regression modelling, IMA was related to mortality, independ‐ ently of the presence of hypertension, diabetes or advanced age. In a larger cohort of 245 consecutive attendances to the ED, in which there were 31 composite endpoint (cardiac death, AMI or recurrent angina) at 30-days from presentation and 16 deaths at one year; the short and long term ability of IMA to predict outcome was assessed. Short term survival was significantly compromised in those with IMA > 93.3 KU/L compared to those with lower IMA concentrations at both 30 days and 1 year [57]. Using the cohort of the French Nationwide OPERA study IMA, cTn, CRP and BNP were measured within 24hours from admission in 471 patients hospitalized with AMI. Using a primary end point of death, resuscitated cardiac arrest, recurrent AMI or ischemia, heart failure or stroke, 75 in-hospital events and 144 events at 1 year were recorded. Using quartile analysis, 40% of patients reached the end point with IMA concentrations in the highest quartile (>104 KU/L), compared to only 20% of patients in the lowest quartile of 83 KU/L [63]. In those STEMI patients who are treated with primary PCI, IMA is a powerful predictor of 30-day mortality however it does not add to the validated

for ACS of 94.4% and 97.1% respectively.

Thrombolysis In Myocardial Infarction (TIMI) risk score [64].

studied most are explained in more detail below.

**8.4. Clinical utility of ischemia modified albumin in non–chest pain patients**

Any marker associated with pathological processes upstream of cardiac necrosis will invari‐ ably suffer from a lack of specificity; unlike the cardiac troponins for cardiac necrosis. The further upstream in the ischemic continuum the more likely is the lack of cardiac specificity of the biomarker. Elevations in circulating IMA concentrations are not specific for myocardial ischemia. Mechanistically, IMA can be generated during any ischemic process within the body. A comprehensive review of IMA elevations in non-cardiac conditions is beyond the scope of this chapter but an in-depth summary is given in table 2.Those conditions that have been

The majority of patients who present to hospital with chest pain and suspected ACS are eventually ruled out for acute myocardial infarction and active unstable coronary disease. The ideal role of an ischemia marker would therefore be as rule out test.The most logical place to use such a test is therefore in the ED.A study of ED presentations examined 208 patients the diagnostic sensitivity of IMA measurement alone was 82% at 46% specificity in samples taken within the first 3 hours. The combination of ECG, cardiac troponin T (cTnT) and IMA showed 95% sensitivity for diagnosis of ACS at presentation [56]. One year follow up performed on this population demonstrated a survival disadvantage in patients with IMA greater than the median concentration of the study group [57]. A subsequent study of 538 patients admitted to a chest pain evaluation unit found admission measurement of IMA plus cTnT had 100% sensitivity for prediction of a final diagnosis of AMI [1]. The pres‐ ence of an elevated IMA and an elevated cTnT on admission predicted 21% risk of major adverse cardiac events (MACE) compared to patients where both were not elevated, even in patients where the final diagnosis excluded AMI by troponin based criteria. IMA measurement appears to work best as part of a panel of other tests or a test sequence [50]. Admission measurement of IMA has been found to be superior to biomarkers of necrosis and to show 97% sensitivity when combined with them. Not all investigators have consid‐ ered the diagnostic performance of IMA either alone or in combination with cardiac troponin, or other biomarkers of necrosis, to be adequate. A prospective ED study enrolling 277 patients and using a positive IMA or troponin as the index test and an 8 hour troponin as the definitive test found only a 97.6% sensitivity with 97% negative predictive value. The investigators did not consider this to be adequate when compared with troponin but did not provide any follow-up data [58]. A second large study prospectively enrolling 189 patients presenting to the ED with chest pain and found an elevated IMA was a poor predictor of cardiac events within the next 72 hours [59]. Conversely, another study found elevated IMA predicted long-term cardiac events [60]. The most consistent finding across all studies of IMA is of a high negative predictive value. This has been highlighted in a meta-analysis specifically examining the role of IMA as a rule out test [61]. The summar‐ ised data of over 1800 patients demonstrated a triple negative prediction test (non-diagnos‐ tic ECG, negative cTn and negative IMA) with a sensitivity and negative predictive value for ACS of 94.4% and 97.1% respectively.

**8.3. Clinical utility of ischemia modified albumin in chest pain patients**

reformulated.

104 Ischemic Heart Disease

Clinical validation of any test for ischemia is difficult as there is currently no accepted diagnostic gold standard, although blood lactate has been used previously.In addition, there is no predicate test which can be used against which to perform an initial validation. The initial studies using IMA were based on the ability of an early measurement to predict the final diagnosis of AMI as defined by the elevation of cardiac troponin at 6-12 hours post chest pain. Two studies utilised the first generation pre-release ACB test and a third study manufactured an in-house method. The first study examined acute coronary syndrome (ACS) patients and utilised serial sampling on admission and two subsequent samples [55]. Diagnostic sensitivity of the admission sample for a final diagnosis of AMI was 23.9% for cardiac troponin I (cTnI) alone, 39.1% for IMA alone and 55.9% for the two combined. The second study examined enrolled 256 ACS patients [49]. AUC of the ROC curve for the ACB test was 0.78 with a sensitivity and specificity of 83% and 69% respectively at the opti‐ mised decision threshold for AMI. The third study enrolled 75 patients with ischemia and 92 non-ischemic patients [29]. IMA had poor predictive power in discriminating between AMI and non-AMI in patients with underlying ischemic heart disease (AUC of 0.66). However, the test gave good discrimination between patients with or without ischemia. The AUC for the ROC curve for diagnosis of ischemia was 0.95 with sensitivity of 94% and specificity of 88%. In these initial studies there were significant problems with sample stability and the assay involved in addition of calcium chloride and centrifugation as part of the routine method. This made the method unsuitable from routine analysis and the assay was

The majority of patients who present to hospital with chest pain and suspected ACS are eventually ruled out for acute myocardial infarction and active unstable coronary disease. The ideal role of an ischemia marker would therefore be as rule out test.The most logical place to use such a test is therefore in the ED.A study of ED presentations examined 208 patients the diagnostic sensitivity of IMA measurement alone was 82% at 46% specificity in samples taken within the first 3 hours. The combination of ECG, cardiac troponin T (cTnT) and IMA showed 95% sensitivity for diagnosis of ACS at presentation [56]. One year follow up performed on this population demonstrated a survival disadvantage in patients with IMA greater than the median concentration of the study group [57]. A subsequent study of 538 patients admitted to a chest pain evaluation unit found admission measurement of IMA plus cTnT had 100% sensitivity for prediction of a final diagnosis of AMI [1]. The pres‐ ence of an elevated IMA and an elevated cTnT on admission predicted 21% risk of major adverse cardiac events (MACE) compared to patients where both were not elevated, even in patients where the final diagnosis excluded AMI by troponin based criteria. IMA measurement appears to work best as part of a panel of other tests or a test sequence [50]. Admission measurement of IMA has been found to be superior to biomarkers of necrosis and to show 97% sensitivity when combined with them. Not all investigators have consid‐ ered the diagnostic performance of IMA either alone or in combination with cardiac troponin, or other biomarkers of necrosis, to be adequate. A prospective ED study enrolling 277 patients and using a positive IMA or troponin as the index test and an 8 hour troponin as The prognostic value of IMA in the ACS setting has been investigated [57;60;62;63]. Using a ROC derived cut off of 477 KU/L, Aparci and colleagues found significantly higher mortality at one year in those who had serum IMA >477 KU/L, compared to those with IMA <477 KU/L [60]. Furthermore, using cox regression modelling, IMA was related to mortality, independ‐ ently of the presence of hypertension, diabetes or advanced age. In a larger cohort of 245 consecutive attendances to the ED, in which there were 31 composite endpoint (cardiac death, AMI or recurrent angina) at 30-days from presentation and 16 deaths at one year; the short and long term ability of IMA to predict outcome was assessed. Short term survival was significantly compromised in those with IMA > 93.3 KU/L compared to those with lower IMA concentrations at both 30 days and 1 year [57]. Using the cohort of the French Nationwide OPERA study IMA, cTn, CRP and BNP were measured within 24hours from admission in 471 patients hospitalized with AMI. Using a primary end point of death, resuscitated cardiac arrest, recurrent AMI or ischemia, heart failure or stroke, 75 in-hospital events and 144 events at 1 year were recorded. Using quartile analysis, 40% of patients reached the end point with IMA concentrations in the highest quartile (>104 KU/L), compared to only 20% of patients in the lowest quartile of 83 KU/L [63]. In those STEMI patients who are treated with primary PCI, IMA is a powerful predictor of 30-day mortality however it does not add to the validated Thrombolysis In Myocardial Infarction (TIMI) risk score [64].

#### **8.4. Clinical utility of ischemia modified albumin in non–chest pain patients**

Any marker associated with pathological processes upstream of cardiac necrosis will invari‐ ably suffer from a lack of specificity; unlike the cardiac troponins for cardiac necrosis. The further upstream in the ischemic continuum the more likely is the lack of cardiac specificity of the biomarker. Elevations in circulating IMA concentrations are not specific for myocardial ischemia. Mechanistically, IMA can be generated during any ischemic process within the body. A comprehensive review of IMA elevations in non-cardiac conditions is beyond the scope of this chapter but an in-depth summary is given in table 2.Those conditions that have been studied most are explained in more detail below.


IMA and myoglobin and decreased albumin were observed following release of the cuff [71]. In patients with peripheral vascular disease (PVD) undergoing a treadmill walk test, a decrease in serum IMA immediately post-test has been documented [72;73]. In 40 consecutive patients undergoing exercise electrocardiography, a significant decrease in IMA at peak exercise then a subsequent rise in IMA has been observed, however there was no difference in IMA concentrations between those patients with positive and negative stress test results [74]. Revascularisation for PVD is accompanied by a post procedural rise in IMA [72;73;75]. In skeletal muscle ischemia, an initial fall with subsequent rise appears to be a consistent finding without adequate explanation. Smooth muscle ischemia does not appear to be associated with a rise in IMA [76]. The effect of skeletal muscle on ischemia will limit the application of IMA measurement after cardiac stress testing for detection of myocardial ischemia and may explain

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 107

Patients with acute ischemic stroke demonstrate abnormalities in a number of biomarkers of nitrosative and oxidative stress. In 41 patients with ischemic stroke, Senes and colleagues demonstrate that nitrate, IMA and thiobarbituric acid-reactive substances (TBARS) concen‐ trations are significantly increased compared to 37 age and gender matched controls [79]. In a larger cohort of 118 patients presenting within 3 hours of neurological deficit, IMA was elevated in those with cerebral infarction and intracranial haemorrhage (ICH) but normal reference values were observed in those with transient ischemic attacks (TIA) lasting less than 1 hour or those with epileptic seizures [80]. Within 24 hours of injury IMA increased during cerebral infarction but not in intracranial haemorrhage and may offer diagnostic utility in the differential diagnosis of neurological deficit. IMA also correlated with National Institutes of Health Stroke Scale (NIHSS) Score in both cerebral infarction and ICH. Conversely, Herisson and colleagues did not demonstrate a causal relationship between IMA or heart type fatty acid binding protein and NIHSS score or stroke volume [81]. Ahn and colleagues have utilised an albumin-adjusted IMA index for the early detection of ischemic stroke [82]. In 52 patients, 28 (54%) with Ischemic stroke, 24 (46%) non-stroke, the AUC of ROC curve analysis was 0.928 for IMA but 0.99 for albumin-adjusted IMA index. The sensitivity and specificity of the IMA index

Pulmonary embolus (PE) is an acute medical emergency estimated to occur in 3.5/1000 hospitalized patients. Patients experience sudden onset dyspnoea, tachypnoea, pleuritictype chest pain, cyanosis and haemoptysis. PE has an associated mortality of 26%. Diagno‐ sis is primarily based on typical clinical presentation using the Wells and Geneva clinical probability scores. D-dimer measurement and pulmonary angiography are often clinically useful. The ECG can demonstrate acute *corpulmonale* in large PE's but lacks specificity. IMA has been measured in a number of studies of PE patients. Turedi and colleagues [83] have demonstrated that IMA was significantly elevated in 30 PE patients compared to 30 healthy controls and adequately discriminated between the presence and absence of PE. The positive

the inconsistent findings[54;74;77;78].

was superior to IMA concentration alone.

*8.4.3. Pulmonary embolus*

*8.4.2. Ischemic stroke*

**Table 2.** Increased serum IMA concentrations in conditions other than acute coronary syndrome.

#### *8.4.1. Skeletal muscle ischemia*

Studies of subjects with skeletal muscle ischemia have produced contradictory results. In healthy subjects undergoing arduous physical exertion, IMA has been reported to fall imme‐ diately post exercise and then subsequently rise [65-67] or return to normal [68]. Subjects undergoing a forearm ischemia test when the forearm muscles are exercised for 1 minute with the external compression of the arm blood supply showed a fall in IMA, maximal at 3 minutes from the test, returning to baseline by 30 minutes [69]. A similar rise in serum lactate occurred. Conversely during standardized exercise in a plantar flexion pedal combined with inflation of a femoral blood pressure cuff (at 0, 60, 90, 120 and 150 mmHg) to induce calf muscle ischemia an increase in IMA was observed after release of the cuff and returned to baseline within 30 minutes [70]. Peri-operative skeletal muscle ischemia induced by femoral blood pressure cuff being inflated to 300 mmHg in 23 patients undergoing arthroscopic knee surgery. Increased IMA and myoglobin and decreased albumin were observed following release of the cuff [71]. In patients with peripheral vascular disease (PVD) undergoing a treadmill walk test, a decrease in serum IMA immediately post-test has been documented [72;73]. In 40 consecutive patients undergoing exercise electrocardiography, a significant decrease in IMA at peak exercise then a subsequent rise in IMA has been observed, however there was no difference in IMA concentrations between those patients with positive and negative stress test results [74]. Revascularisation for PVD is accompanied by a post procedural rise in IMA [72;73;75]. In skeletal muscle ischemia, an initial fall with subsequent rise appears to be a consistent finding without adequate explanation. Smooth muscle ischemia does not appear to be associated with a rise in IMA [76]. The effect of skeletal muscle on ischemia will limit the application of IMA measurement after cardiac stress testing for detection of myocardial ischemia and may explain the inconsistent findings[54;74;77;78].

#### *8.4.2. Ischemic stroke*

Condition

106 Ischemic Heart Disease

Ischemic bowel Liver cirrhosis Neural tube defects

Pleural effusion

Polycythemia vera Preeclampsia

Pulmonary embolism Skeletal muscle ischemia

Uterine artery embolisation for fibroids

*8.4.1. Skeletal muscle ischemia*

**Table 2.** Increased serum IMA concentrations in conditions other than acute coronary syndrome.

Studies of subjects with skeletal muscle ischemia have produced contradictory results. In healthy subjects undergoing arduous physical exertion, IMA has been reported to fall imme‐ diately post exercise and then subsequently rise [65-67] or return to normal [68]. Subjects undergoing a forearm ischemia test when the forearm muscles are exercised for 1 minute with the external compression of the arm blood supply showed a fall in IMA, maximal at 3 minutes from the test, returning to baseline by 30 minutes [69]. A similar rise in serum lactate occurred. Conversely during standardized exercise in a plantar flexion pedal combined with inflation of a femoral blood pressure cuff (at 0, 60, 90, 120 and 150 mmHg) to induce calf muscle ischemia an increase in IMA was observed after release of the cuff and returned to baseline within 30 minutes [70]. Peri-operative skeletal muscle ischemia induced by femoral blood pressure cuff being inflated to 300 mmHg in 23 patients undergoing arthroscopic knee surgery. Increased

Polycystic ovary syndrome

Obesity

Stroke

b-thalassemia Testicular torsion

Carbon monoxide poisoning Congestive cardiac failure Chronic kidney disease Deep vein thrombosis Diabetes Mellitus Hypercholesterolaemia Intermittent claudication

> Patients with acute ischemic stroke demonstrate abnormalities in a number of biomarkers of nitrosative and oxidative stress. In 41 patients with ischemic stroke, Senes and colleagues demonstrate that nitrate, IMA and thiobarbituric acid-reactive substances (TBARS) concen‐ trations are significantly increased compared to 37 age and gender matched controls [79]. In a larger cohort of 118 patients presenting within 3 hours of neurological deficit, IMA was elevated in those with cerebral infarction and intracranial haemorrhage (ICH) but normal reference values were observed in those with transient ischemic attacks (TIA) lasting less than 1 hour or those with epileptic seizures [80]. Within 24 hours of injury IMA increased during cerebral infarction but not in intracranial haemorrhage and may offer diagnostic utility in the differential diagnosis of neurological deficit. IMA also correlated with National Institutes of Health Stroke Scale (NIHSS) Score in both cerebral infarction and ICH. Conversely, Herisson and colleagues did not demonstrate a causal relationship between IMA or heart type fatty acid binding protein and NIHSS score or stroke volume [81]. Ahn and colleagues have utilised an albumin-adjusted IMA index for the early detection of ischemic stroke [82]. In 52 patients, 28 (54%) with Ischemic stroke, 24 (46%) non-stroke, the AUC of ROC curve analysis was 0.928 for IMA but 0.99 for albumin-adjusted IMA index. The sensitivity and specificity of the IMA index was superior to IMA concentration alone.

#### *8.4.3. Pulmonary embolus*

Pulmonary embolus (PE) is an acute medical emergency estimated to occur in 3.5/1000 hospitalized patients. Patients experience sudden onset dyspnoea, tachypnoea, pleuritictype chest pain, cyanosis and haemoptysis. PE has an associated mortality of 26%. Diagno‐ sis is primarily based on typical clinical presentation using the Wells and Geneva clinical probability scores. D-dimer measurement and pulmonary angiography are often clinically useful. The ECG can demonstrate acute *corpulmonale* in large PE's but lacks specificity. IMA has been measured in a number of studies of PE patients. Turedi and colleagues [83] have demonstrated that IMA was significantly elevated in 30 PE patients compared to 30 healthy controls and adequately discriminated between the presence and absence of PE. The positive predictive value of IMA for PE is higher than that for D-dimer (79.4% compared to 69.4%) and in combination with the Wells and Geneva criteria, IMA offers an alternative to Ddimer testing [84].

*8.4.5. Hyperlipidaemia and obesity*

in hypercholesterolaemia.

26-32 kg/m2

model score [98].

*8.4.6. Diabetes mellitus*

*8.4.7. Bowel ischemia*

those subjects with BMI >30 kg/m2

IMA measurement may be of benefit in hypercholesterolaemic patients. IMA is correlated to cholesterol, low density lipoprotein (LDL) and antibodies to oxidised LDL (ox-LDL) [24]. In a study of 37 subjects with hypercholesterolaemia compared to 37 controls, Duarte and collea‐ gues [95] confirm these findings observing IMA correlations to cholesterol, LDL ox-LDL antibodies and to high sensitivity C-reactive protein, suggesting that hypercholesterolaemia is associated with inflammatory and oxidative stress processes, contributing to the advance‐ ment of atherosclerosis. IMA is related to the presence of metabolic syndrome independently of age, gender, presence of diabetes or hypercholesterolaemia[96]. Furthermore, the use of 10 mg/day ezetimibe immunotherapy for a duration of 12 weeks in 31 hypercholesterolaemic patients reduced both LDL cholesterol and IMA [97]. The reduction of IMA was independent of the reduction in LDL suggesting that ezetimibe may reduce the burden of oxidative stress

IMA concentrations are higher in obese subjects, with a positive correlation between IMA and body mass index (BMI).In a large study of 148 volunteers in Brazil; subjects were classified as normal, overweight or obese, defined as BMI of 18.5-24.9, 25.0-29.9 and >30 kg/m2 respectively. IMA concentrations increased exponentially between the three groups, the highest being in

menopausal women where IMA and IMA: Albumin ratio are higher in those subjects with BMI

to those with documented coronary artery disease but normal BMI. In the obese women IMA was positively correlated to BMI, hs-CRP, insulin concentrations and homeostasis assessment

Patients with type 2 diabetes mellitus who demonstrate poor glycaemic control have higher IMA concentrations than those with good glycaemic control. IMA was significantly higher in 76 diabetic patients compared to 25 control subjects and IMA concentrations are correlated to HbA1c [99], glucose and hs-CRP [100]. Conversely, Dahiya and colleagues suggest no significant changes in IMA occur in 60 newly diagnosed type 2 diabetics, compared to 30 control subjects [101]. Diabetic patients who undertake chronic exercise for three months demonstrate lower post exercise IMA concentrations suggesting that exercise alleviates some

Bowel (mesenteric) ischemia occurs infrequently however if not recognised early, carries a devastatingly high mortality. The presentation is often characterised by generalised abdominal pain, fever, diarrhoea or constipation, tachycardia, hematochezia (blood per rectum), nausea and vomiting. Diagnosis is difficult due to non specific signs and symptoms, plain x-ray or laboratory tests (increased white blood cell count and serum lactic acid). Mesenteric angiog‐ raphy is considered to be the gold standard test which can differentiate between embolic,

compared to those with BMI 21-25 kg/m2

of the oxidative stress associated with diabetes mellitus [102].

. Similar findings have been demonstrated in obese post‐

. The obese concentrations were similar

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 109

#### *8.4.4. Chronic kidney disease*

Patients with chronic kidney disease (CKD) have a reduced life span compared to those without renal disease. Mortality rates are highest in those receiving haemodialysis as renal replacement therapy (RRT). Cardiovascular mortality accounts for the majority of renal deaths. Between 2001 and 2006, 24% of deaths in UK RRT patients were due to ischemic heart disease [85]. This rate is consistent with data from other countries. Cardiovascular morbidity is also increased. 55% of patients receiving haemodialysis RRT also have concomitant congestive cardiac failure. [86]. IMA levels have been determined in patients with CKD [87-89] and in patients receiving haemodialysis (HD) [90-94].

In 2006, Sharma and colleagues demonstrated that patients with elevated IMA have a significantly large left ventricle, decreased systolic function and greater estimated left ventricular filling pressure [88]. Further, in multivariate analysis, a positive dobutamine stress echocardiogram (DSE) combined with elevated IMA and cTnT and E/Ea ratio were independent prognostic factors for death. IMA values increase significantly in those patients with a positive DSE compared to those with no ischemic response [87]. In a modestly small study of 17 anaemic CKD patients and 19 controls, Cichota and colleagues demonstrated that IMA increased in patients compared to the control group. IMA correlated to lactate, haemoglobin and creatinine [89].

Pre and post-HD IMA concentrations are significantly correlated [90], however in this study IMA concentrations were not significantly different between those CKD patients with or without ischemic heart disease, diabetes mellitus or peripheral vascular disease. Fast intravenous iron administration during HD is associated with oxidative stress and inflamma‐ tion. In a study of 20 HD patients receiving slow intravenous iron administration, IMA concentrations were significantly increased across three HD sessions independently of slow i.v. iron administration [91].Following adjustment of albumin by two methods, post dialysis IMA levels remain significantly increased following HD [92]. Paroxonase-1 (PON-1) is a calcium dependent esterase (arylesterase, aromatic esterase 1, serum aryldialkylpohospha‐ tase 1, EC 3.1.8.1) is a major anti-atherosclerotic component of HDL cholesterol. PON-1 concentrations are lower in CKD patients with and without haemodialysis RRT compared to controls suggesting chronic oxidative stress and accelerated atherosclerosis are a feature of CKD. In a pilot study of CKD patients receiving HD, PON-1 concentrations were significantly and inversely correlated to IMA suggesting an oxidative stress and ischemic process occurs during HD [93]. Recently Albarello and colleagues have evaluated the effect of IMA and protein carbonyl groups as markers of protein oxidation in 23 CKD patients receiving HD. The authors confirm previous reports of higher IMA post-HD than pre-HD and observed a significant correlation between IMA and protein carbonyl groups, attribut‐ ed to oxidative stress associated with HD [94].

#### *8.4.5. Hyperlipidaemia and obesity*

predictive value of IMA for PE is higher than that for D-dimer (79.4% compared to 69.4%) and in combination with the Wells and Geneva criteria, IMA offers an alternative to D-

Patients with chronic kidney disease (CKD) have a reduced life span compared to those without renal disease. Mortality rates are highest in those receiving haemodialysis as renal replacement therapy (RRT). Cardiovascular mortality accounts for the majority of renal deaths. Between 2001 and 2006, 24% of deaths in UK RRT patients were due to ischemic heart disease [85]. This rate is consistent with data from other countries. Cardiovascular morbidity is also increased. 55% of patients receiving haemodialysis RRT also have concomitant congestive cardiac failure. [86]. IMA levels have been determined in patients with CKD [87-89] and in

In 2006, Sharma and colleagues demonstrated that patients with elevated IMA have a significantly large left ventricle, decreased systolic function and greater estimated left ventricular filling pressure [88]. Further, in multivariate analysis, a positive dobutamine stress echocardiogram (DSE) combined with elevated IMA and cTnT and E/Ea ratio were independent prognostic factors for death. IMA values increase significantly in those patients with a positive DSE compared to those with no ischemic response [87]. In a modestly small study of 17 anaemic CKD patients and 19 controls, Cichota and colleagues demonstrated that IMA increased in patients compared to the control group. IMA correlated to lactate,

Pre and post-HD IMA concentrations are significantly correlated [90], however in this study IMA concentrations were not significantly different between those CKD patients with or without ischemic heart disease, diabetes mellitus or peripheral vascular disease. Fast intravenous iron administration during HD is associated with oxidative stress and inflamma‐ tion. In a study of 20 HD patients receiving slow intravenous iron administration, IMA concentrations were significantly increased across three HD sessions independently of slow i.v. iron administration [91].Following adjustment of albumin by two methods, post dialysis IMA levels remain significantly increased following HD [92]. Paroxonase-1 (PON-1) is a calcium dependent esterase (arylesterase, aromatic esterase 1, serum aryldialkylpohospha‐ tase 1, EC 3.1.8.1) is a major anti-atherosclerotic component of HDL cholesterol. PON-1 concentrations are lower in CKD patients with and without haemodialysis RRT compared to controls suggesting chronic oxidative stress and accelerated atherosclerosis are a feature of CKD. In a pilot study of CKD patients receiving HD, PON-1 concentrations were significantly and inversely correlated to IMA suggesting an oxidative stress and ischemic process occurs during HD [93]. Recently Albarello and colleagues have evaluated the effect of IMA and protein carbonyl groups as markers of protein oxidation in 23 CKD patients receiving HD. The authors confirm previous reports of higher IMA post-HD than pre-HD and observed a significant correlation between IMA and protein carbonyl groups, attribut‐

dimer testing [84].

108 Ischemic Heart Disease

*8.4.4. Chronic kidney disease*

patients receiving haemodialysis (HD) [90-94].

ed to oxidative stress associated with HD [94].

haemoglobin and creatinine [89].

IMA measurement may be of benefit in hypercholesterolaemic patients. IMA is correlated to cholesterol, low density lipoprotein (LDL) and antibodies to oxidised LDL (ox-LDL) [24]. In a study of 37 subjects with hypercholesterolaemia compared to 37 controls, Duarte and collea‐ gues [95] confirm these findings observing IMA correlations to cholesterol, LDL ox-LDL antibodies and to high sensitivity C-reactive protein, suggesting that hypercholesterolaemia is associated with inflammatory and oxidative stress processes, contributing to the advance‐ ment of atherosclerosis. IMA is related to the presence of metabolic syndrome independently of age, gender, presence of diabetes or hypercholesterolaemia[96]. Furthermore, the use of 10 mg/day ezetimibe immunotherapy for a duration of 12 weeks in 31 hypercholesterolaemic patients reduced both LDL cholesterol and IMA [97]. The reduction of IMA was independent of the reduction in LDL suggesting that ezetimibe may reduce the burden of oxidative stress in hypercholesterolaemia.

IMA concentrations are higher in obese subjects, with a positive correlation between IMA and body mass index (BMI).In a large study of 148 volunteers in Brazil; subjects were classified as normal, overweight or obese, defined as BMI of 18.5-24.9, 25.0-29.9 and >30 kg/m2 respectively. IMA concentrations increased exponentially between the three groups, the highest being in those subjects with BMI >30 kg/m2 . Similar findings have been demonstrated in obese post‐ menopausal women where IMA and IMA: Albumin ratio are higher in those subjects with BMI 26-32 kg/m2 compared to those with BMI 21-25 kg/m2 . The obese concentrations were similar to those with documented coronary artery disease but normal BMI. In the obese women IMA was positively correlated to BMI, hs-CRP, insulin concentrations and homeostasis assessment model score [98].

#### *8.4.6. Diabetes mellitus*

Patients with type 2 diabetes mellitus who demonstrate poor glycaemic control have higher IMA concentrations than those with good glycaemic control. IMA was significantly higher in 76 diabetic patients compared to 25 control subjects and IMA concentrations are correlated to HbA1c [99], glucose and hs-CRP [100]. Conversely, Dahiya and colleagues suggest no significant changes in IMA occur in 60 newly diagnosed type 2 diabetics, compared to 30 control subjects [101]. Diabetic patients who undertake chronic exercise for three months demonstrate lower post exercise IMA concentrations suggesting that exercise alleviates some of the oxidative stress associated with diabetes mellitus [102].

#### *8.4.7. Bowel ischemia*

Bowel (mesenteric) ischemia occurs infrequently however if not recognised early, carries a devastatingly high mortality. The presentation is often characterised by generalised abdominal pain, fever, diarrhoea or constipation, tachycardia, hematochezia (blood per rectum), nausea and vomiting. Diagnosis is difficult due to non specific signs and symptoms, plain x-ray or laboratory tests (increased white blood cell count and serum lactic acid). Mesenteric angiog‐ raphy is considered to be the gold standard test which can differentiate between embolic, thrombotic or nonocclusive ischemia. In a preliminary study of 26 patients presenting with symptoms of internal ischemia, Polk and colleagues [103] identified 12 with a positive clinical diagnosis. Positive patients had higher IMA concentrations than those without intestinal ischemia. IMA detected bowel ischemia with a sensitivity of 100% and a specificity of 86%. In a case-controlled study from Turkey, Gunduz and colleagues [104] demonstrated that preoperative IMA concentrations were significantly higher in patients with thromboembolic occlusion of the superior mesenteric artery (SMA) compared to an age-matched control group of healthy volunteers. A number of animal studies of mesenteric ischemia have provided conflicting results. In a Wistar rat model [105] a time dependent response in IMA in mesenteric ischemia has been demonstrated. 36 mature female rats underwent either simple laparotomy in the control groups or laparotomy followed by clamping of the SMA in the subject group. IMA concentrations were highest 6 hours from ischemic onset, however IMA at 30 minutes and 2 hours were also significantly higher in the clamped group compared to the control group. Elevations of IMA tracked changes in both lactate and malondialdehyde. A similar time dependent change in IMA was demonstrated in New Zealand rabbits undergoing ligation of the SMA compared to either a control group or those undergoing a sham procedure [106]with elevation of IMA at 2 and 6 hours significantly higher than baseline and higher than IMA concentrations in the control rabbits. IMA concentrations mimicked elevations in serum IL-6 with elevated IL-6 in the ischemia group at 1, 3 and 6 hours, but no elevations in the sham operated or control group. In a further study of mesenteric ischemia in a Wistar rat model, Uygun and colleagues [107] demonstrated similar IMA concentrations in control, sham, 2-hour and 6-hour post-SMA ischemia refuting the previous animal studies. It seems likely that IMA may offer additional diagnostic value in the early presentation of mesenteric ischemia. Further prospective studies are required to assess both the diagnostic and prognostic ability of IMA in conjunction with mesenteric angiography to detect bowel ischemia.

an overall net decrease in plasma protein concentration by 10-12 g/L which is reached around week 28 of gestation. The predominant cause of lowered albumin is dilutional, oestrogen is known to affect albumin. The alteration to plasma albumin concentrations throughout the pregnancy period is shown in table 3. The lower concentration of albumin also results in an

The HSA reference interval in the full term healthy neonate between term and day 4 is 28-44 g/L. Albumin concentrations increase a little from birth to puberty where the adolescent

Current experimental studies suggest that foetal development occurs in a hypoxic intrauterine environment and the presence of reperfusion and oxidative stress is believed to be crucial for trophoblast development [108]. Trophoblast invasion of the maternal spiral arteries allows the increase of uterine blood supply necessary to maintain the pregnant state. Serum IMA during normal pregnancy is elevated compared to non-pregnant controls [109-112]. Prefumo and colleagues [109] demonstrate supra-physiological IMA concentrations in early normal pregnancy (11-13 weeks of gestation) suggesting that trophoblast development occurs in a hypoxic uterus. In a large population of 117 pregnant women compared to non-pregnant healthy women, Guven and colleagues demonstrated a cross-sectional elevation in IMA in pregnant women. IMA increased significantly through each trimester. Further, there the authors demonstrated a significant negative correlation between IMA and HSA, suggesting

that elevated IMA in pregnancy represents a physiologic state of oxidative stress.

Increased intrauterine hypoxia predisposes to defective endovascular trophoblast invasion of the maternal spiral arteries which may possibly lead to the development of pre-eclampsia; a hypertensive state (>140/90 mmHg) associated with significant proteinuria (≥300mg/dL). Pre-

**Reference interval (g/L)**

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 111

apparent decrease in substances normally bound to this protein.

**Time point Mean albumin concentration (g/L)**

**Table 3.** Alteration to plasma HSA concentrations during the gestational period.

reference interval (day 4 to 14 years) is 38-54 g/L.

Non pregnant control 41 36-46 12 weeks 38 33-43 18 weeks 35 30-39 24 weeks 33 29-37 28 weeks 32 28-37 32 weeks 32 38-36 36 weeks 32 38-36 Full term 32 26-38 1 day post partum 29 23-38 6 weeks post partum 42 37-47

#### *8.4.8. Obstetric and gynaecological use of IMA*

The care of women and their unborn child during pregnancy is greatly challenging for obstetricians. The adult can interact and provide a history of signs and symptoms whereas the unborn child can only be examined indirectly by means of imaging, foetal heart monitors and a limited number of direct interventions. Women achieving spontaneous preterm (<37 weeks) labour account for 10% of all births and are attributable to 75% of neonatal deaths. The foetus relies entirely on the maternal placenta for O2/CO2 exchange. This delicate dependence, between the placenta and the foetus is crucial to normal healthy growth. Any malfunction or disruption to the adequate supply of oxygen can cause hypoxia and potentially fatal acidosis. A limited degree of acidosis is well tolerated by the foetus; however chronic acidosis or hypoxia may lead to a significant mortality and morbidity with potential log-term sequelae. Currently the mechanism of foetal hypoxia and acidosis is unclear, and physiological consequence of foetal acidosis is believed to target the cell energy availability and /or cell poisoning.

During pregnancy plasma proteins change markedly due to increased plasma volume, increased renal blood flow and altered protein synthesis in response to hormonal changes. Plasma volume expansion of up to 45% [1300mL) compared to the non-pregnant state causes an overall net decrease in plasma protein concentration by 10-12 g/L which is reached around week 28 of gestation. The predominant cause of lowered albumin is dilutional, oestrogen is known to affect albumin. The alteration to plasma albumin concentrations throughout the pregnancy period is shown in table 3. The lower concentration of albumin also results in an apparent decrease in substances normally bound to this protein.


**Table 3.** Alteration to plasma HSA concentrations during the gestational period.

thrombotic or nonocclusive ischemia. In a preliminary study of 26 patients presenting with symptoms of internal ischemia, Polk and colleagues [103] identified 12 with a positive clinical diagnosis. Positive patients had higher IMA concentrations than those without intestinal ischemia. IMA detected bowel ischemia with a sensitivity of 100% and a specificity of 86%. In a case-controlled study from Turkey, Gunduz and colleagues [104] demonstrated that preoperative IMA concentrations were significantly higher in patients with thromboembolic occlusion of the superior mesenteric artery (SMA) compared to an age-matched control group of healthy volunteers. A number of animal studies of mesenteric ischemia have provided conflicting results. In a Wistar rat model [105] a time dependent response in IMA in mesenteric ischemia has been demonstrated. 36 mature female rats underwent either simple laparotomy in the control groups or laparotomy followed by clamping of the SMA in the subject group. IMA concentrations were highest 6 hours from ischemic onset, however IMA at 30 minutes and 2 hours were also significantly higher in the clamped group compared to the control group. Elevations of IMA tracked changes in both lactate and malondialdehyde. A similar time dependent change in IMA was demonstrated in New Zealand rabbits undergoing ligation of the SMA compared to either a control group or those undergoing a sham procedure [106]with elevation of IMA at 2 and 6 hours significantly higher than baseline and higher than IMA concentrations in the control rabbits. IMA concentrations mimicked elevations in serum IL-6 with elevated IL-6 in the ischemia group at 1, 3 and 6 hours, but no elevations in the sham operated or control group. In a further study of mesenteric ischemia in a Wistar rat model, Uygun and colleagues [107] demonstrated similar IMA concentrations in control, sham, 2-hour and 6-hour post-SMA ischemia refuting the previous animal studies. It seems likely that IMA may offer additional diagnostic value in the early presentation of mesenteric ischemia. Further prospective studies are required to assess both the diagnostic and prognostic ability of IMA

in conjunction with mesenteric angiography to detect bowel ischemia.

The care of women and their unborn child during pregnancy is greatly challenging for obstetricians. The adult can interact and provide a history of signs and symptoms whereas the unborn child can only be examined indirectly by means of imaging, foetal heart monitors and a limited number of direct interventions. Women achieving spontaneous preterm (<37 weeks) labour account for 10% of all births and are attributable to 75% of neonatal deaths. The foetus relies entirely on the maternal placenta for O2/CO2 exchange. This delicate dependence, between the placenta and the foetus is crucial to normal healthy growth. Any malfunction or disruption to the adequate supply of oxygen can cause hypoxia and potentially fatal acidosis. A limited degree of acidosis is well tolerated by the foetus; however chronic acidosis or hypoxia may lead to a significant mortality and morbidity with potential log-term sequelae. Currently the mechanism of foetal hypoxia and acidosis is unclear, and physiological consequence of

foetal acidosis is believed to target the cell energy availability and /or cell poisoning.

During pregnancy plasma proteins change markedly due to increased plasma volume, increased renal blood flow and altered protein synthesis in response to hormonal changes. Plasma volume expansion of up to 45% [1300mL) compared to the non-pregnant state causes

*8.4.8. Obstetric and gynaecological use of IMA*

110 Ischemic Heart Disease

The HSA reference interval in the full term healthy neonate between term and day 4 is 28-44 g/L. Albumin concentrations increase a little from birth to puberty where the adolescent reference interval (day 4 to 14 years) is 38-54 g/L.

Current experimental studies suggest that foetal development occurs in a hypoxic intrauterine environment and the presence of reperfusion and oxidative stress is believed to be crucial for trophoblast development [108]. Trophoblast invasion of the maternal spiral arteries allows the increase of uterine blood supply necessary to maintain the pregnant state. Serum IMA during normal pregnancy is elevated compared to non-pregnant controls [109-112]. Prefumo and colleagues [109] demonstrate supra-physiological IMA concentrations in early normal pregnancy (11-13 weeks of gestation) suggesting that trophoblast development occurs in a hypoxic uterus. In a large population of 117 pregnant women compared to non-pregnant healthy women, Guven and colleagues demonstrated a cross-sectional elevation in IMA in pregnant women. IMA increased significantly through each trimester. Further, there the authors demonstrated a significant negative correlation between IMA and HSA, suggesting that elevated IMA in pregnancy represents a physiologic state of oxidative stress.

Increased intrauterine hypoxia predisposes to defective endovascular trophoblast invasion of the maternal spiral arteries which may possibly lead to the development of pre-eclampsia; a hypertensive state (>140/90 mmHg) associated with significant proteinuria (≥300mg/dL). Preeclampsia affects 6-8% of pregnancies worldwide. Papageorghiou and colleagues have demonstrated that first trimester serum IMA are significantly higher in women who develop pre-eclampsia compared to those with a normal pregnancy [113]. Both IMA and normalised IMA (IMA: Albumin ratio) were higher in 20 pre-eclamptic women compared to 22 normal pregnancies [112]. These data suggest IMA could be a biological marker of pre-eclampsia however larger studies are required to fully characterise the supra-normal IMA and normal‐ ised IMA reference interval in normal pregnancy.

shortlived with post even values returning to baseline normally within a 24 hour window. Although the mechanisms have not been elucidated postulates include, physiological cardio‐ myocyte turnover, cellular release of proteolytic degradation products, alteration in plasma membrane permeability and the formation of membranous secretion vesicles containing

Biomarkers of Cardiac Ischemia http://dx.doi.org/10.5772/55250 113

Although there are a number of candidate biomarkers for the detection of cardiac ischemia in the research and development world; biomarkers upstream of cardiac cell necrosis lack specificity. They are therefore, at best additive to the diagnostic and prognostic utility of cTn in the early investigation of patients presenting with ischemic type symptoms. The clinical utility of novel biomarkers of ischemia lies in their negative predictive value rather than their ability to adequately rule-in ACS. Given the development of sensitive cTn methods, further

Dept of Chemical Pathology Clinical Blood Sciences,St George's Healthcare NHS Trust, Lon‐

[1] Collinson PO, Gaze DC, Bainbridge K et al. Utility of admission cardiac troponin and "Ischemia Modified Albumin" measurements for rapid evaluation and rule out of suspected acute myocardial infarction in the emergency department. *Emerg Med J*.

[2] Collinson PO, Gaze DC. Ischaemia-modified albumin: clinical utility and pitfalls in

[3] Pope JH, Aufderheide TP, Ruthazer R et al. Missed diagnoses of acute cardiac ische‐

[4] Collinson PO, Premachandram S, Hashemi K. Prospective audit of incidence of prog‐ nostically important myocardial damage in patients discharged from emergency de‐

mia in the emergency department. *N Engl J Med*. 2000;342:1163-1170.

work is needed to characterise the release mechanisms of cTn from cardiomyocytes.

intracellular derived cTn.

**10. Conclusions**

**Author details**

David C. Gaze

**References**

2006;23:256-261.

measurement. *J ClinPathol*. 2008;61:1025-1028.

partment. *BMJ*. 2000;320:1702-1705.

don, UK

Maternal IMA and normalised IMA concentrations are also increased in women with recurrent pregnancy loss (two or more unexplained miscarriages in the first trimester) compared to healthy pregnancy [114], suggesting that an increase of intrauterine oxidative stress and hypoxia contribute to placental deficiency and subsequently recurrent early miscarriage.

The use of umbilical cord blood for IMA has also been examined. Neonatal cord blood IMA concentrations are higher than IMA concentrations in healthy adults [115] but is not attribut‐ able to changes in HSA concentration. Elevated fetal IMA may reflect transient localised ischemia from external forces exerted on the foetus during labour. In a case-control study of 26 newborns, 12 delivered at normal term and 14 with complicated labour or delivered preterm; cord blood IMA concentrations were significantly higher (50%) than those with un‐ eventful deliveries, suggesting IMA is a marker of fetal distress. Doubly-clamped cord blood IMA concentrations are similar in intrauterine growth restriction, compared to those delivered with appropriate for gestational age full-term pregnancies [116]. The similar IMA concentra‐ tions may be due to the 'brain sparing effect' accompanied by oligohydraminos (deficiency in amniotic fluid), which is characterised by rerouting the blood supply and the nutrient to vital organs such as the heart, brain and adrenal glands. IMA concentrations in cord blood are higher following caesarean section compared to vaginal delivery and in multigravida compared to primigravida [116] and may be attributable to higher oxidative stress on both accounts. Cigarette smoking during the gestational period alters the oxidant/antioxidant balance in favour of oxidative stress. In response, IMA and MDA concentrations in pregnant smokers are significantly higher and vitamins A and E, SOD and total antioxidant capacityare significantly lower, compared to non-smoking pregnant women [117].

## **9. Cardiac troponin: Ischemia or necrosis?**

The release of cTn was previously thought to occur only in the presence of cell necrosis. The recent development of high sensitivity cTn (hs-cTn) assays has lead to a) the ability to define a true 99th percentile and near-Gaussian distribution in the healthy population and b) earlier diagnosis of AMI with increased sensitivity but at the cost of specificity [118]. A number of clinical and physiological situations have arisen which suggests cTn is released during ischemia in the absence of overt necrosis [119]. These include patients who present with superventricular tachycardia [120] without electrocardiographic changes; in patients with pulmonary embolism [121] where cTn release may indicate a reversible release and under physiological strain following endurance exercise [122]. In all cases the kinetic release is shortlived with post even values returning to baseline normally within a 24 hour window. Although the mechanisms have not been elucidated postulates include, physiological cardio‐ myocyte turnover, cellular release of proteolytic degradation products, alteration in plasma membrane permeability and the formation of membranous secretion vesicles containing intracellular derived cTn.

## **10. Conclusions**

eclampsia affects 6-8% of pregnancies worldwide. Papageorghiou and colleagues have demonstrated that first trimester serum IMA are significantly higher in women who develop pre-eclampsia compared to those with a normal pregnancy [113]. Both IMA and normalised IMA (IMA: Albumin ratio) were higher in 20 pre-eclamptic women compared to 22 normal pregnancies [112]. These data suggest IMA could be a biological marker of pre-eclampsia however larger studies are required to fully characterise the supra-normal IMA and normal‐

Maternal IMA and normalised IMA concentrations are also increased in women with recurrent pregnancy loss (two or more unexplained miscarriages in the first trimester) compared to healthy pregnancy [114], suggesting that an increase of intrauterine oxidative stress and hypoxia contribute to placental deficiency and subsequently recurrent early miscarriage.

The use of umbilical cord blood for IMA has also been examined. Neonatal cord blood IMA concentrations are higher than IMA concentrations in healthy adults [115] but is not attribut‐ able to changes in HSA concentration. Elevated fetal IMA may reflect transient localised ischemia from external forces exerted on the foetus during labour. In a case-control study of 26 newborns, 12 delivered at normal term and 14 with complicated labour or delivered preterm; cord blood IMA concentrations were significantly higher (50%) than those with un‐ eventful deliveries, suggesting IMA is a marker of fetal distress. Doubly-clamped cord blood IMA concentrations are similar in intrauterine growth restriction, compared to those delivered with appropriate for gestational age full-term pregnancies [116]. The similar IMA concentra‐ tions may be due to the 'brain sparing effect' accompanied by oligohydraminos (deficiency in amniotic fluid), which is characterised by rerouting the blood supply and the nutrient to vital organs such as the heart, brain and adrenal glands. IMA concentrations in cord blood are higher following caesarean section compared to vaginal delivery and in multigravida compared to primigravida [116] and may be attributable to higher oxidative stress on both accounts. Cigarette smoking during the gestational period alters the oxidant/antioxidant balance in favour of oxidative stress. In response, IMA and MDA concentrations in pregnant smokers are significantly higher and vitamins A and E, SOD and total antioxidant capacityare significantly

The release of cTn was previously thought to occur only in the presence of cell necrosis. The recent development of high sensitivity cTn (hs-cTn) assays has lead to a) the ability to define a true 99th percentile and near-Gaussian distribution in the healthy population and b) earlier diagnosis of AMI with increased sensitivity but at the cost of specificity [118]. A number of clinical and physiological situations have arisen which suggests cTn is released during ischemia in the absence of overt necrosis [119]. These include patients who present with superventricular tachycardia [120] without electrocardiographic changes; in patients with pulmonary embolism [121] where cTn release may indicate a reversible release and under physiological strain following endurance exercise [122]. In all cases the kinetic release is

ised IMA reference interval in normal pregnancy.

112 Ischemic Heart Disease

lower, compared to non-smoking pregnant women [117].

**9. Cardiac troponin: Ischemia or necrosis?**

Although there are a number of candidate biomarkers for the detection of cardiac ischemia in the research and development world; biomarkers upstream of cardiac cell necrosis lack specificity. They are therefore, at best additive to the diagnostic and prognostic utility of cTn in the early investigation of patients presenting with ischemic type symptoms. The clinical utility of novel biomarkers of ischemia lies in their negative predictive value rather than their ability to adequately rule-in ACS. Given the development of sensitive cTn methods, further work is needed to characterise the release mechanisms of cTn from cardiomyocytes.

## **Author details**

David C. Gaze

Dept of Chemical Pathology Clinical Blood Sciences,St George's Healthcare NHS Trust, Lon‐ don, UK

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**Chapter 7**

**Is Hyperuricemia a Risk Factor to**

Additional information is available at the end of the chapter

overproduction of endogenous uric acid (Fam, 2002).

Uric acid is a weak acid distributed throughout the extracellular fluid compartments (Emmerson, 1996). The normal blood uric acid level in humans is approximately 4 mg/dl (0.24 mmol/l) (Ganong, 2005). Uric acid is the end product of purine degradation. Purines are degraded ultimately to uric acid through the action of the enzyme xanthine oxidase that converts xanthine to urate (Mc Lean, 2003). In most mammals, the liver enzyme uricase (urate oxidase) is responsible for further metabolism of uric acid to allantoin, which is more soluble waste product. However, humans lack the enzyme uricase, resulting in higher blood uric acid levels (Hediger et al., 2005). They might provide humans a survival advantage over the other primates because of the function of uric acid as antioxidant (Mc Lean, 2003). ). For an individ‐ ual, urate concentration is determined by the balance between the rate of purine metaboloism, both endogenous and exogenous, and the efficiency of renal clearance. Alteration in this balance may account for hyperuricemia. In the majority (90%) of patients with primary gout, hyperuricemia results from relative renal undersecretion, whereas in 10% of patients, there is

Elevated serum uric acid, besides its documented link to gouty arthritis, has been reported to be closely-associated with the metabolic syndrome and, as well, to be a correlate of the development and progression of cardiovascular diseases (Baker et al., 2005), though the role of uric acid in this respect is still unclear. Several possible pathological mechanisms linking hyperuricemia to cardiovascular disease were suggested; including the deleterious effects of elevated uric acid on endothelial dysfunction, oxidative metabolism, as well as platelet adhesiveness, hemorheology and aggregation (Hoieggen et al., 2003). However, no enough or definite experimental data exist concerning the association of hyperuricemia with the different cellular elements of blood. The aim of this work was to investigate the effects of elevated serum

> © 2013 Youssef; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Cardiovascular Disease?**

Magda H M Youssef

**1. Introduction**

http://dx.doi.org/10.5772/54871


**Chapter 7**
