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Ann Thorac Surg 2000;69:750-754
© 2000 The Society of Thoracic Surgeons

Original Articles

Neuron-specific enolase increases in plasma during and immediately after extracorporeal circulation

Per Johnsson, MD, PhDa,c, Sten Blomquist, MD, PhDb,c, Carsten Lührs, MDa,c, Gunnar Malmkvist, MD, PhDb,c, Christer Alling, MD, PhDa,c, Jan-Otto Solem, MD, PhDa,c, Erik Ståhl, MD, PhDa,c

a Department of Cardiothoracic Surgery, University Hospital, Lund, Sweden
b Department of Anesthesiology, University Hospital, Lund, Sweden
c Department of Medical Chemistry, University Hospital, Lund, Sweden

Address reprint requests to Dr Blomquist, Department of Anesthesiology and Intensive Care, University Hospital, S-22185 Lund, Sweden


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Comment
 References
 
Background. Minor cerebral complications are common after cardiac surgery. Several biochemical markers for brain injury are under research; one of these is neuron-specific enolase (NSE). The purpose of this study was to investigate the release of this enzyme into the blood during and immediately after extracorporeal circulation and to evaluate the effect of hemolysis on this release.

Methods. Sixteen patients scheduled for elective heart surgery were included in the study. Blood samples for analysis of NSE and free hemoglobin in plasma were drawn before, during, and up to 48 hours after the end of extracorporeal circulation. The release of NSE from erythrocytes and its correlation to the release of free hemoglobin was studied by serial dilution and hemolysis in vitro.

Results. The peri- and postoperative course was uneventful in all patients. Extracorporeal circulation initiated a release of NSE that reached a maximum 6 hours after the end of perfusion. Thereafter, the levels declined with an estimated t1/2 of 30 hours. The concentration of free hemoglobin increased during the perfusion, with maximum levels at the end of perfusion, after which they fell rapidly to normal values. The in vitro study showed a strong linearity between the release of NSE and free hemoglobin after induced hemolysis.

Conclusions. The increased levels of enolase at the end of cardiopulmonary bypass can, to a major part, be explained by the release from hemolysed erythrocytes. The value of NSE as a marker for brain injury in these situations is therefore doubtful.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Comment
 References
 
Cerebral injury continues to be a major source of complication after extracorporeal circulation. The incidence of stroke after cardiac surgery is 1% to 5%, however, the incidence of minor cerebral complications have been reported to be 60% to 70% [1–4]. Patients with such complications show subtle dysfunction concerning memory, concentration ability, as well as disturbance in personality. These cognitive dysfunctions can only be detected by prospective neuropsychological testing, which is both cumbersome to the patients and expensive. Methods to identify these patients at an early stage are lacking, thus justifying the search for specific biochemical markers for brain injury.

Enolase is a cytoplasmatic glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate. The enzyme exists as a dimer and has three immunologically distinct subunits, {alpha}, ß, and {gamma}. Five isoenzymes can be found: {alpha}{alpha}, ßß, {gamma}{gamma}, {alpha}ß, and {alpha}{gamma}. The brain contains both {alpha} and {gamma} subunits but not ß. The dimeric {alpha}{alpha} form is specific for glial cells, whereas the {gamma}-enolase has been shown to be located in neurons and neuroectodermal tissue. The term "neuron-specific enolase" (NSE) refers to both the {gamma}{gamma} and {alpha}{gamma} forms. The molecular weight for the dimeric form of the enzyme is 77 kDa [5–7].

In vitro studies have shown that NSE is released from cultured neurons when exposed to cytotoxic agents and that the levels of NSE can serve to quantify the amount of neuronal cell death [8]. In animals, increased levels of NSE in cerebrospinal fluid (CSF) have been reported in models of traumatic and ischemic brain damage [9, 10]. Studies in humans have shown increased levels of NSE in CSF to be associated with a variety of central nervous injuries and diseases, eg, stroke, traumatic head injury, multiple sclerosis, Alzheimer’s disease, and epileptic seizures [11–17]. We have previously reported results that show an association between neurological outcome after open heart surgery and serum levels of NSE assessed on the second postoperative day [18]. However, NSE can also be found in platelets and erythrocytes [6, 7], both of which are significantly affected by extracorporeal circulation (ECC) and cardiac surgery. In this study, we report the appearance and elimination of NSE from serum during and after ECC. To evaluate the effect of hemolysis on the release of NSE, we also studied the appearance of free hemoglobin (fHb) during and after ECC, as well as the effect of hemolysis on the release of NSE in vitro.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Comment
 References
 
Sixteen consecutive patients scheduled for elective open heart surgery were studied. Patients with a history of cerebral disease were excluded from the study. Informed consent was obtained and the study protocol was approved by the Ethics committee of the University. Indications for surgery were coronary disease in 14 patients and valvular disease in 2 patients. The male/female ratio was 12:4, and the median age was 63 years (range 30 to 78 years). The anesthetic procedure was similar in all patients, and consisted of 5 to 15 µg/kg of fentanyl (Leptanal; Janssen Pharmaceutica, Beerse, Belgium) in combination with midazolam (Dormicum; Roche, Basel, Switzerland), 3 to 5 mg at induction. Intubation was performed during succinylcholine relaxation and anesthesia was maintained by additional doses of midazolam and fentanyl. No volatile anesthetic was given before ECC; however, during and after bypass inhalation of isoflurane (Forene; Abbot, Chicago, IL) 0.7% to 1.0% was administered.

Surgery was performed in moderate hypothermia (30°C to 32°C) using a COBE compact membrane lung (CML) membrane oxygenator and a roller pump generating a nonpulsatile flow. An arterial filter was included in the circuit. Mean arterial pressure was maintained above 50 mm Hg during bypass; if necessary, intermittent doses of norepinephrine were administered. Distal anastomosis was performed with the aorta cross-clamped, while the ensuing proximal anastomosis was performed on a beating heart with a side-biting clamp on the aorta. Cold anterograde St. Thomas’ cardioplegic solution and topical ice slush were used for myocardial protection.

Arterial blood samples for analysis of NSE and fHb were collected during anesthesia immediately after administration of heparin, 20 minutes after the start of ECC, immediately after the end of ECC (t = 0 in the figures), and then 3, 6, 12, 24, and 48 hours after the end of ECC.

In vitro study
Blood from 6 healthy donors was used. After centrifugation and separation, the erythrocytes were serially diluted and hemolysed by distilled water to obtain hemolysis of 0.2%, 0.5%, 1%, and 2.5%. The concentrations of free Hb and NSE were measured at each stage.

Biochemical assay
Hemoglobin in plasma was determined by measuring the absorption of plasma at 577 nm. The concentration of hemoglobin was then calculated after correction for nonspecific absorbance at 660 nm.

NSE in serum was analyzed using a monoclonal two-site single incubation immunoradiometric assay (Prolifigen NSE; Sangtec Medical, Bromma, Sweden). The assay uses monoclonal antibodies that bind to the {gamma}-subunit of the enzyme. Both the {gamma}-{gamma} and {alpha}-{gamma} forms of the enzyme are thus detected. The sample is incubated with a plastic bead coated with the antibody to NSE labeled with 125I. The antibodies bind to different epitopies of the NSE molecule, whereby the labeled antibody is indirectly bound to the bead. After washing unreacted radioactive antibody off the bead, the radioactivity bound to the bead was measured using a gamma counter. The amount of NSE in the samples was then calculated using standards with known concentrations of NSE. The upper limit of normal values with the method of analysis used in this study has been reported to be 9.2 µg/L.

Statistics
Linear and multiple regression tests were performed using the Sigma-Stat software (SPSS Inc, Chicago, IL). Mann-Whitney rank sum test was used for groupwise comparison in Table 1. A p level of less than 0.05 was considered significant.


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  		Table 1. Perfusion Time and NSE Values at the End of ECC in Each Individual Patient

 

   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Comment
 References
 
The peri- and postoperative course was uneventful in all patients included in the study. The median time of ECC was 86 minutes (range 35 to 144 minutes). The serum levels of NSE are shown in Figure 1. The mean time between administration of heparin, when the first blood sample was drawn, and the end of ECC (t = 0) was 120 minutes (range 78 to 134 minutes). The mean perfusion time was 86 minutes (range 36 to 144 minutes) (Table 1). NSE concentrations were within normal levels until the end of ECC, when a sharp increase from 9.2 ± 1.2 to 17.0 ± 2.0 µg/L was seen (Fig 1, Table 1). A further increase occurred and the maximum NSE concentration (19.4 ± 1.8 µg/L) was noted 6 hours after the end of ECC. Thereafter, the concentrations declined to 12.1 ± 1.3 µg/L at 48 hours after the end of ECC. Estimation of t1/2 for the elimination of NSE from plasma resulted in a value of 30 hours.



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  		Fig 1. The concentrations in serum of NSE (µg/L) (filled circles, left y-axis) and fHb (g/L) (open circles, right y-axis) during and after ECC. End of ECC is at t = 0. (Data are mean ± SEM, n = 16).

 
The concentration of NSE at the end of perfusion as a function of perfusion time is shown in Figure 2. A positive correlation was observed with r = 0.68 and p less than 0.01.



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  		Fig 2. The correlation between concentration of NSE (µg/L) at the end of ECC and perfusion time (min). (Linear regression, n = 16. r = 0.68, p < 0.01).

 
The concentration of fHb was 0.015 ± 0.003 g/L after administration of heparin, then increased to 0.122 ± 0.031 g/L after 20 minutes of ECC. Maximum concentration (0.404 ± 0.06 g/L) was reached at the end of ECC. Twelve hours after the end of ECC, the concentration of fHb had decreased to 0.012 ± 0.003 g/L, and at the end of the study, 0.017 ± 0.005 g/L (Fig 1). The elimination t1/2 of fHb was estimated to be 4 hours after the end of ECC.

There was no correlation between the concentration of fHb at the end of the perfusion and the duration of perfusion. The concentration of NSE at the end of perfusion as a function of fHb at the same time is shown in Figure 3. A weak positive correlation was found, with r = 0.55 and p less than 0.05. A multiple linear regression test with the concentration of NSE at the end of perfusion as dependent variable and the concomitant concentration of fHb and perfusion time as independent variables resulted in a correlation coefficient of r = 0.806. Both variables contributed significantly to the model, with p less than 0.05 and less than 0.01, respectively.



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  		Fig 3. The correlation between concentrations of NSE (µg/L) and fHb (g/L) at the end of ECC. (Linear regression, n = 16, r = 0.55, p < 0.05).

 
The relationships between hemolysis, fHb, and NSE from the in vitro study are shown in Figure 4. The concentration of NSE in whole blood with no hemolysis was 5.28 ± 0.23 µg/L. The concentration increased with the degree of hemolysis and was 7.07 ± 0.29, 10.66 ± 0.89, 15.49 ± 1.06, and 33.30 ± 2.60 µg/L at 0.2%, 0.5%, 1%, and 2.5% hemolysis, respectively. The concentrations of fHb at the corresponding time points were 0.23 ± 0.1, 0.58 ± 0.01, 1.15 ± 0.03, and 2.88 ± 0.07 g/L, respectively. Linear regression test resulted in the equation NSE (µg/L) = 4.89 + 9.78 x fHb (g/L), with r = 0.99 and p less than 0.0001. Using this equation, estimation of the amount of NSE that could be attributed to hemolysis was performed by using the measured fHb at the end of ECC and adding the concentration of NSE before ECC for each individual. The difference between this estimated NSE and the measured NSE was also calculated; this difference would then give the amount of NSE derived from other sources than hemolysis. The results for each individual patient are shown in Table 1. There was no statistically significant difference between estimated and measured concentration of NSE at the end of perfusion.



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  		Fig 4. The correlation between concentrations of NSE (µg/L), fHb (g/L), and degree of hemolysis (%) in vitro. (r = 0.99, p < 0.0001).

 

   Comment
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
References
 
The results of this study show that there is a release of NSE to plasma during ECC. To a part, this release can be attributed to hemolysis caused by the destruction of erythrocytes and platelets during ECC. Two earlier studies have reported attempts to correlate the release of NSE to the degree of hemolysis in the same sample. According to the first [6], even limited hemolysis can increase NSE levels by 5 to 10 µg/L, but no relationship between the degree of hemolysis and the release of NSE was given. The second study [7] showed a correlation between the degree of hemolysis and release of NSE, expressed as NSE (µg/L) = 0.03 + 5.73 x %hemolysis. Because the concentration of fHb in our study reached its maximum at the end of ECC, estimation of the contribution of hemolysis to the NSE levels is of special interest at this point. The results from the calculation of the amount of NSE derived from hemolysis in each patient show that there was no significant difference between these and the measured values at the same time. This observation leads us to conclude that the appearance of increased levels of NSE in plasma after ECC does not necessarily indicate an affection on the central nervous system in these patients with uneventful outcomes, but can to a great extent be explained by the concomitant hemolysis. This conclusion is in contradiction with that of Gao and associates [19], who in in vitro studies found that the introduction of a correction factor in the spectroscopic measurement of hemolysis eliminated the influence of hemolysis on NSE measurements. Nevertheless, we believe that the results of our study clearly suggest that NSE levels after ECC must be interpreted with caution.

As can be seen from Figure 1, the levels of NSE continue to rise after the end of ECC even though the levels of fHb started to decline at the same time. When considering the relationship between hemolysis and NSE in blood after ECC, differences in elimination need to be noted. The free hemoglobin produced by hemolysis is rapidly taken up by the reticuloendothelial system and transformed in the liver. When fHb reaches critical levels, hemoglobinuria appears, usually at 0.5% hemolysis. NSE is a fairly large protein and is probably metabolized in the liver, although the rate of elimination is unknown. The fact that fHb decreased rather instantly after the end of ECC in our study does not necessarily mean that hemolysis has ceased. Therefore, due to the differences in elimination rates, the sustained increase in NSE could in part be explained by the release caused by ongoing hemolysis. Our results raise the question as to whether NSE could be used as a marker for cerebral injury after cardiac surgery. The advantage of NSE as a marker for neuronal injury is the well-proven correlation between the release of NSE and neuronal death in in vitro studies mentioned before. Likewise, increased levels of NSE in CSF and blood after experimental brain injury in animals and cerebral injury, not associated with hemolysis, in humans suggest the usefulness of NSE in these respects. The main disadvantage would be the association between NSE and hemolysis, especially early in the course after ECC, coupled with the relatively slow elimination of NSE from the circulation. However, we believe that analysis of NSE can be used as a marker for cerebral injury after ECC provided that serial tests are analyzed and ample time is allowed between the tests in order to avoid possible contamination from hemolysis caused by ECC. In our study, all patients had an uneventful outcome with no clinical signs of cerebral injury. The clinical value of NSE as a marker for cerebral injury after ECC needs to be validated in patients with a known postoperative brain lesion, preferably where the location and volume of the lesion is known.


   Acknowledgments
 
This study was in part supported by grants from Sangtec AB, Bromma, Sweden.


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Comment
 References
 

  1. Shaw P.J., Bates D., Cartlidge N.E.F., et al. Early intellectual dysfunction following coronary bypass surgery. Q J Med 1986;58:59-68.[Abstract/Free Full Text]
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  5. Schmechel D., Marangos P.J., Brightman M. Neuron-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 1978;276:834-836.[Medline]
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  9. Hrdemark H.-G., Ericsson N., Kotwica Z., et al. S-100 protein and neuron-specific enolase in CSF after experimental traumatic or focal ischemic brain damage. J Neurosurg 1989;71:727-731.[Medline]
  10. Usui A., Kato K., Murase M., et al. Neural tissue-related proteins (NSE, G0{alpha}, 28-kDa calbindin-D, S-100b and CK-BB) in serum and cerebrospinal fluid after cardiac arrest. J Neurol Sci 1994;123:134-139.[Medline]
  11. Persson L., Hrdemark H.-G., Gustafsson J., et al. S-100 protein and neuron-specific enolase in cerebrospinal fluid and serum. Stroke 1987;18:911-918.[Abstract/Free Full Text]
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  13. Kärkelä J., Bock E., Kaukinen S. CSF and serum brain-specific creatinine kinase isoenzyme (CK-BB), neuron-specific enolase (NSE) and neural cell adhesion molecule (NCAM) as prognostic markers for hypoxic brain injury after cardiac arrest in man. J Neurol Sci 1993;116:100-109.[Medline]
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Accepted for publication August 2, 1999.




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