Article, Pulmonology

Ischemia-modified albumin in the diagnosis of pulmonary embolism: an experimental study

Original Contribution

ischemia-modified albumin in the diagnosis of pulmonary embolism: an experimental study

Suleyman Turedi MDa,?, Tevfik Patan MDa, Abdulkadir Gunduz MDa, Ahmet Menteseb, Celal Tekinbas MDc, Murat Topbas MDd, Suleyman Caner Karahan MDb,

Esin Yulug MDe, Suha Turkmen MDb, Utku Ucar MDb

aDepartment of Emergency Medicine, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey bDepartment of Biochemistry, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey cDepartment of Thoracic Surgery, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey dDepartment of Public Health, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey eDepartment of Histology, Karadeniz Technical University Faculty of Medicine, Trabzon, Turkey

Received 7 April 2008; revised 8 May 2008; accepted 14 May 2008

Abstract

Study Objective: We designed this experimental study to determine the value of ischemia-modified albumin in the diagnosis of pulmonary embolism.

Methods: Twenty-four mature female New Zealand rabbits were divided into 4 groups, each consisting of 6 animals. These were classified into group 1 ,the control group; group 2, the deep venous thrombosis group; group 3, the deep venous thrombosis with pulmonary embolism group; and group 4, the pulmonary embolism-alone group. Deep venous thrombosis was produced by ligation of the iliac vein. To establish pulmonary embolism, 2 clots were administered from the iliac vein. Blood samples were taken from all the groups at hours 0, 1, 3, and 6 for ischemia-modified albumin measurement.

Results: Pulmonary embolism was established in all the rabbits in groups 3 and 4, and this was confirmed by tomographic and histologic findings. Measurement of mean ischemia-modified Albumin levels for all rabbits at hours 0, 1, 3, and 6 revealed that mean ischemia-modified albumin levels in groups 3 and 4 were statistically significantly higher than those in groups 1 and 2. There was no difference between the mean ischemia-modified albumin levels in groups 1 and 2 nor between groups 3 and 4. The alteration in ischemia-modified albumin levels over time was statistically significant.

Conclusions: The results of our experimental study demonstrate that ischemia-modified albumin levels may be useful in the diagnosis of pulmonary embolism.

(C) 2009

* Corresponding author. Acil Tip AD, Karadeniz Teknik Universitesi, Tip Fakultesi Hastanesi, 61080 Trabzon, Turkey. Tel.: +90 0462 377 5715; fax: +90 0462 325 12 46.

E-mail address: [email protected] (S. Turedi).

0735-6757/$ - see front matter (C) 2009 doi:10.1016/j.ajem.2008.05.002

Introduction

Background

Pulmonary embolism (PE) is a life-threatening condition and is estimated to occur in 3.5 of every 1000 hospitalized patients [1]. There are an estimated 600000 cases of PE per year in the United States, resulting in 50000 to 100000 fatalities [2]. Objective testing for PE is crucial because clinical assessment alone has been found to be incorrect in up to 50% of cases, and the mortality rate accompanying untreated PE is high. Algorithms to rule out or diagnose PE often involve ventilation-perfusion scans, Spiral computed tomography (s-CT), or pulmonary angiography, which are invasive, complex, and time-consuming procedures [3]. In recent years, extensive research has been devoted to developing noninvasive and cost-effective diagnostic strate- gies for PE [4,5].

Importance

One of these techniques in use today, plasma D-dimer measurement, a degradation product of cross-linked fibrin, has been studied extensively as a first-line test to rule out PE [5,6]. However, D-dimer is a rather low specific marker with a poor positive predictive value. Instead, it is the negative predictive value that is useful in ruling out PE. Therefore, there is a need for more appropriate markers to rule out PE and to reduce the number of sophisticated Imaging techniques required [3].

Recently, evidence was provided by Turedi et al [7], showing that ischemia-modified albumin levels may be useful as a Discriminative marker to exclude PE.

Objectives of this investigation

Laboratory analysis“>We therefore designed this experimental study to establish the value of IMA in the diagnosis of PE.

Materials and methods

Study design

This was a randomized, controlled, nonblinded inter- ventional animal study. The study protocol was approved by the Karadeniz Technical University animal care and ethics committee.

Setting and selection of participants

Twenty-four mature female New Zealand rabbits weighing 2 to 2.5 kg were used. The animals were kept in steel cages until the day of the experiment at a Room temperature of 22?C

and were given water and standard rabbit chow. For the final 12 hours before the experiment, they were given only water.

Intervention

The rabbits were then divided into 4 groups, each consisting of 6 individuals. These were classified as group 1-the control group; group 2-the Deep venous thrombosis group; group 3-the DVT with PE group; and group 4-the PE-alone group.

To produce similar conditions, 5 mL of blood samples were taken from the rabbits in all the groups, but autologous clots were only produced from the blood of groups 3 and 4 rabbits. To obtain Blood clots, 5 mL of blood previously taken from the rabbits was allowed to clot in a beaker for 1 hour (until the clot had reached a gelatine-like consis- tency). All the subjects were anesthetized with 50 mg/kg ketamine and 5 mg/kg xylazine, applied intramuscularly.

All rabbits were first catheterized from the dorsal ear vein. The iliac vein was then visualized with an approximately 1.5-cm incision from the femoral region. Stasis-induced DVT was produced by ligation of the iliac vein. To create PE, a 12-gauge catheter was inserted into the iliac vein. Two clots approximately 4 mm in diameter and 5 mm in length were diluted with 2 mL of saline given through the iliac vein, followed by 2 mL of saline as a flush. Iliac veins of group 3 rabbits were first catheterized, after which, ligation was performed from below the catheter and the clot given through it. Groups 1 and 2 rabbits were injected with an equivalent volume of normal saline in the dorsal ear vein to produce similar conditions. No intubation was performed during the entire experiment, oxygen at a level of 4 L/min was supplied by mask until killing, and respiration rates and saturation were monitored. No other hemodynamic monitoring was performed.

Stasis-induced DVT models and the average diameter of the clots for PE were derived from the available literature regarding experimental PE and DVT [8-10].

Laboratory analysis

      1. IMA measurement

Two millimeters of blood samples were taken from all groups at hours 0, 1, 3, and 6. Reduced cobalt-albumin binding capacity (IMA level) was analyzed using the rapid and colorimetric method developed by Bar-Or et al [11]. This colorimetric method of measurement scanning is based on the principle of quantitative scanning of free cobalts present after cobalt binding has taken place. This means that high absorbance levels as a result of increased amounts of free cobalt in the environment can be determined.

      1. Tomographic examination

Spiral computed tomography was performed 6 hours after blood specimens were taken. A total volume of 2 mL/kg of contrast medium (Iemeron; Bracco, Milan, Italy) containing 300 mg of iodine per milliliter was injected intravenously at a

Fig. 1 Measured IMA levels for all groups at hours 0, 1, 3, and 6.

rate of 4 mL/s, and selective pulmonary tomographic angiography using a 16-detector s-CT scanner (SOMATOM Sensation; Siemens, Forchheim, Germany) was also per- formed. Computed tomographic scans were reviewed by a blinded radiologist experienced in reviewing s-CT data. The s-CT criterion used to diagnose PE consisted of visualization of nonocclusive endoluminal embolus or of complete occlusion by embolus.

      1. Histologic analysis and staining

All rabbits were killed 7 hours after the beginning of the experiment, and their lung tissues were fixed immediately in 10% buffered formaldehyde. These were dehydrated in a graded ethanol series, cleared in xylene, embedded in paraffin, and stained with hematoxylin and eosin. The lung tissues of the affected lung lobes were identified at pulmonary s-CT in groups 3 and 4. histologic examinations were carried out on 5-um slices and viewed under a light microscope. The tissue samples of each rat were examined in a blinded fashion by the same histologist. In addition, after the killing process, DVT was confirmed macroscopically with iliac vein incision in the rabbits in groups 2 and 4.

      1. Statistical analysis

Statistical analysis was performed using SPSS version

13.0 (SPSS, Chicago, Ill). Kruskal-Wallis variance analysis (the Mann-Whitney U test with Bonferroni correction as post hoc) was used to compare the groups, and Friedman test

(Wilcoxon test with Bonferroni correction as post hoc) was used to evaluate changes in IMA levels over time. Statistical significance was assumed at a level of P b .05.

Results

All rabbits (n = 24) were still alive at hour 6. Measured IMA levels for all rabbits at hours 0, 1, 3, and 6 are shown in Fig. 1. Mean IMA levels for all groups are 0.239 +- 0.0047,

Fig. 2 Alterations in IMA levels over time.

0.219 +- 0.0201, 0.231 +- 0.0101, and 0.213 +- 0.0140 absor-

bance unit (ABSU) for group 1; 0.239 +- 0.0047, 0.178 +- 0.0328, 0.254 +- 0.0328, and 0.223 +- 0.0152 ABSU for group

2; 0.239 +- 0.0047, 0.416 +- 0.0795, 0.451 +- 0.0300, and

0.519 +- 0.0629 ABSU for group 3; 0.239 +- 0.0047, 0.304 +-

0.1193, 0.460 +- 0.0323, and 0.510 +- 0.0555 ABSU for

group 4, respectively.

According to these results, mean IMA levels in groups 3 and 4 were statistically significantly higher than those in groups 1 and 2 (for hour 1, P b .0005; for hour 3, P b .0005; and for hour 6, P = .001). There was no difference between mean IMA levels in groups 1 and 2 nor in groups 3 and 4 (for all timings, P N .05).

For all groups, the alteration in IMA levels over time is shown in Fig. 2. This alteration was statistically significant (P = .013 for group 1; P = .002 for group 2; P = 0.004 for

group 3; and P = 0.001 for group 4).

Pulmonary embolism was created in all the rabbits of groups 3 and 4 and shown at Figs. 3 and 4.

At histologic examination and light microscopic exam- ination in all the rabbits in the PE-alone group (group 4), we observed changes in the alveolar epithelium, widespread hemorrhage, inflammatory cell infiltration, and localized intra-alveolar edema.

In all the rabbits in the DVT with PE group (group 3), the changes in the alveolar structure were more evident. We observed irregularities in the alveolar structure and localized degeneration in the epithelium. In addition, widespread hemorrhage, inflammatory cell infiltration, and intra-alveolar and periarterial edema were observed.

There were no histologic or tomographic findings of PE in any of the rabbits in groups 1 and 2.

Limitations

There are some limitations of this study. First, IMA is a new biomarker. Ischemia-modified albumin levels are

Fig. 3 Pulmonary embolism under s-CT (black arrow).

Fig. 4 Macroscopic image of PE (black arrow).

influenced significantly by a wide array of physiologic variables including exercise and hydration. However, we were not able to control all variables that could possibly influence IMA levels. Second, we did not investigate the comparison of other biochemical markers and IMA in the diagnosis of PE. The other limitation is that we investigated the levels of IMA for the first 6 hours only. The results of this study show that IMA levels started to increase after the first hour and continued to rise up to the sixth hour. Because the IMA levels after 6 hours were not investigated, it is not possible to state the peak time of IMA and how it proceeds afterward. Therefore, it should be noted that further studies investigating the comparison of other markers with IMA as well as studies in which the course of IMA levels of PE patients are under longer examination are needed.

In addition, our study had some limitations in terms of the model used. Our study was controlled but may not mimic typical PE cases seen in practice. Most patients with DVT do not have complete occlusion of the iliac vessel, so although DVT was established in our model, it may not be the same in practice. In addition, the literature we used for the stasis-related DVT model contains an approximately 48 waiting period after ligation. In our study, blood specimens were taken at hours 0, 1, 3, and 6. Although DVT formation was observed macroscopically, stasis-related DVT may not have manifested itself because of the limited waiting time, and IMA levels may therefore have been low in the isolated DVT group.

Discussion

venous thromboembolism including DVT and PE afflicts an estimated 71 people of every 100000 annually. Approximately one third of patients with VTE also have PE, whereas two thirds have DVT alone [12,13]. Although studies using various biochemical markers in the diagnosis of Venous thromboembolic diseases have been performed,

D-dimer is the best known of these markers and is still used today. Several high-quality systematic reviews have evalu- ated the use of D-dimer testing for diagnosis or exclusion of VTE. The evidence obtained supports the use of a negative D- dimer assay to exclude VTE, although test performance varies significantly depending on population and type of assay [14]. However, D-dimer is a rather low specific marker with a low positive predictive value. Therefore, there is a need for more appropriate markers for use in PE. With that aim in mind, we evaluated the experimental value of IMA in the diagnosis of PE.

Human serum albumin consists of 585 amino acids. The first 3 amino acids in the N-terminus, Asp-Ala-His, constitute a specific binding site for transition metals such as cobalt, copper, and nickel and are the most susceptible region for degradation compared with other albumin regions [15]. During acute ischemia conditions, the metal-binding capacity of the albumin N-terminus is modified and reduces transition metal binding, generating a metabolic variant of the protein. This change is quantifiable and commonly known as IMA [16].

In recent years, IMA measurement has been proposed as a sensitive marker for the diagnosis of myocardial ischemia presenting with typical acute chest pain [17]. However, chest pain is the most frequent symptom reported in the emergency department and may be caused by both myocardial ischemia and PE [18]. Although the literature contains many studies of IMA levels in myocardial ischemia, we encountered only 1 report about IMA levels in the diagnosis of PE. In that interesting study, Turedi et al [7] demonstrated that IMA levels may be useful as a discriminative marker to exclude PE. Those authors examined the serum IMA levels of 60 individuals, consisting of 30 PE patients who had been definitively diagnosed via s-CT angiography and 30 healthy volunteers, and suggested that IMA levels may be useful as a discriminative marker to exclude PE. In this study, serum IMA levels were significantly high in 97.7% of PE patients, in which an IMA upper limit of Reference interval value for the exclusion of PE of 0.540 ABSU was determined. The finding that only 1 PE patient had an IMA level than this value is striking. In addition, no healthy individuals had IMA levels greater than the upper limit of reference interval level. Ours is also the first experimental study to show that IMA levels are significantly elevated in PE. According to our results, mean IMA levels in groups 3 and 4 were statistically significantly higher than those in groups 1 and 2. These results are correlated with the study of Turedi et al [7].

Pulmonary embolism frequently initially accompanies DVT. As a fibrin destruction product, D-dimer is high in both diseases. Therefore, the use of D-dimer to determine whether PE is present in DVT patients is not practical. The effect of DVT on arterial circulation and, therefore, on the develop- ment of ischemia is nothing out of the ordinary. For that reason, high IMA, specific to ischemia in DVT patients, may suggest PE. The fact that IMA levels in group 4, in which DVT accompanied PE, were higher than those in group 2, the

isolated DVT group, supports this hypothesis. However, stasis-related DVT may not have manifested itself because of the limited waiting time, and IMA levels may therefore have been low in the isolated DVT group.

Ischemia-modified albumin is also a rapid and low-cost technique. The measurement process took around 12 minutes when blood samples from 16 patients were manually and simultaneously analyzed. Compared with D-dimer, the costs involved are some 100 to 200 times lower. To reduce patient exposure to radiation and lengthy and high-cost Radiologic tests, there is a need for economical and high-accuracy markers. Although it cannot be said, on the basis of the results of our study, that IMA is superior to D-dimer for that purpose, it may still be regarded as an alternative to D-dimer in terms of cost and the results determined. Further clinical studies are required to evaluate this issue.

An increasing number of studies have shown that IMA levels rise in various acute ischemic conditions, such as cerebral ischemia, myocardial ischemia, mesenteric ische- mia, skeletal ischemia, and pulmonary ischemia, for which reason it can be used as a diagnostic marker [19-21]. There is also some evidence to suggest that IMA increases end-stage renal disease, liver disease, and some neoplasms, even in marathon runners [22,23]. Ischemia-modified albumin tests measure unbound cobalt after incubation with patient serum. The amount of unbound cobalt is directly correlated to the concentration of IMA, but it is also inversely correlated to the concentration of unmodified albumin. Therefore, changes in serum albumin concentration during periods of muscle ischemia, such as decreased intravascular volume during a marathon race or increased intravascular volume due to intravenous fluid infusion, will affect cobalt binding independent of changes in the concentration of IMA [24]. With the data available, we are unable to say whether the diagnostic value of IMA decreases to a significant extent.

In summary; although IMA levels vary in many conditions like these, the first study, that by Turedi et al [7], and the results of our own experimental study demonstrate that IMA levels may be useful for the diagnosis of PE, and additional studies are now required to evaluate the value of this marker in PE.

References

  1. Silverstein MD, Heit JA, Mohr DN, Petterson TM, O’Fallon WM, Melton LJ. Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25 year population-based study. Arch Intern Med 1998;158:585-93.
  2. Aksay E, Yanturali S, Kiyan S. Can elevated Troponin I levels predict complicated clinical course and Inhospital mortality in patients with acute pulmonary embolism. Am J Emerg Med 2007;25(2):138-43.
  3. Steeghs N, Goeoop RJ, Niessen RWLM, Jonkers GJPM, Dik H, Huisman MV. C-reactive protein and D-dimer with clinical probability score in the exclusion of pulmonary embolism. British Journal of Haematology; 130: 614-9.
  4. Perrier A, Roy PM, Aujesky D, Chagnon I, Howarth N, Gourdier AL, et al. Diagnosing pulmonary embolism in outpatients with clinical

assessment, D-dimer measurement, venous ultrasound, and helical computed tomography: a multicenter management study. Am J Med 2004;116(5):291-9.

  1. Perrier A, Nendaz MR, Sarasin FP, Howarth N, Bounameaux H. Cost- effectiveness analysis of Diagnostic strategies for suspected pulmonary embolism including helical computed tomography. Am J Respir Crit Care Med 2003;167(1):39-44.
  2. Bates SM, Grand’Maison A, Johnston M, Naguit I, Kovacs MJ, Ginsberg JS. A latex D-dimer reliably excludes venous thromboem- bolism. Arch Intern Med 2001;161(3):447-53.
  3. Turedi S, Gunduz A, Mentese A, Karahan SC, Yilmaz SE, Eroglu O, et al. Value of ischemia-modified albumin in the diagnosis of pulmonary embolism. Am J Emerg Med 2007;25(7):770-3.
  4. Rectenwald JE, Deatrick KB, Sukheepod P, Lynch EM, Moore AJ, Moaveni DM, et al. Experimental pulmonary embolism: effects of the thrombus and attenuation of pulmonary artery injury by low- molecular-weight heparin. J Vasc Surg 2006;43(4):800-8.
  5. Trejo HE, Urich D, Pezzulo AA, Caraballo JC, Gutierrez J, Castro IJ, et al. Beneficial effects of hydrocortisone and papaverine on a model of pulmonary embolism induced by autologous blood clots in isolated and perfused rabbit lungs. Respirology 2007;12(6):799-806.
  6. Rhodes JM, Cho JS, Gloviczki P, Mozes G, Rolle R, Miller VM. Thrombolysis for experimental deep venous thrombosis maintains valvular competence and vasoreactivity. J Vasc Surg 2000;31(6): 1193-205.
  7. Bar-Or D, Winkler JV, Vanbenthuysen K, Harris L, Lau E, Hetzel FW. Reduced albumin-cobalt binding with transient myocardial ischemia after elective percutaneous transluminal coronary angioplasty: a preliminary comparison to creatine kinase-MB, myoglobin, and troponin I. Am Heart J 2001;141:985-91.
  8. Heit JA, Melton LJ, Lohse CM, Petterson TM, Silverstein MD, Mohr DN, et al. Incidence of venous thromboembolism in hospitalized patients vs community residents. Mayo Clin Proc 2001;76(11): 1102-10.
  9. White RH. The epidemiology of venous thromboembolism. Circula- tion 2003;107(23 Suppl 1):I4-8.
  10. Segal JB, Eng J, Tamariz LJ, Bass EB. Review of the evidence on diagnosis of deep venous thrombosis and pulmonary embolism. Ann Fam Med 2007;5(1):63-73.
  11. Cho DK, Choi JO, Kim SH, Choi J, Rhee I, Ki CS, et al. Ischemia- modified albumin is a highly sensitive serum marker of transient myocardial ischemia induced by Coronary vasospasm. Coron Artery Dis 2007;18(2):83-7.
  12. Lippi G, Montagnana M, Guidi GC. Albumin cobalt binding and ischemia modified albumin generation: an endogenous response to ischemia. Int J Cardiol 2006;108(3):410-1.
  13. Keating L, Benger JR, Beetham R, Bateman S, Veysey S, Kendall J, et al. The PRIMA study: presentation ischaemia-modified albumin in the emergency department. Emerg Med J 2006;23:764-8.
  14. Boie ET. Initial evaluation of chest pain. Emerg Med Clin North Am 2005;23(4):937-57.
  15. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report. J Emerg Med 2000;19:311-5.
  16. Gunduz A, Turedi S, Mentese A, Karahan SC, Hos G, Tatli O, et al. Ischemia modified albumin in the diagnosis of Acute mesenteric ischemia: a preliminary study. Am J Emerg Med 2008;26(2):202-5.
  17. Mitsudo S, Brandt LJ. Pathology of intestinal ischemia. Surg Clin North Am 1992;72:43-63.
  18. Apple FS, Quist HE, Otto AP, Mathews WE, Murakami MM. Release characteristics of cardiac biomarkers and ischemia-modified albumin as measured by the albumin cobalt-binding test after a marathon race. Clin Chem 2002;48(7):1097-100.
  19. Apple FS, Wu AH, Mair J, Ravkilde J, Panteghini M, Tate J, et al. Committee on Standardization of Markers of Cardiac damage of the IFCC. Future biomarkers for detection of ischemia and risk stratification in acute coronary syndrome. Clin Chem 2005;51(5): 810-24.
  20. Refaai MA, Wright RW, Parvin CA, Gronowski AM, Scott MG, Eby CS. Ischemia-modified albumin increases after skeletal muscle ischemia during arthroscopic knee surgery. Clin Chim Acta 2006; 366(1-2):264-8.