Serum S100 protein: A potential marker for cerebral events during cardiopulmonary bypass

Serum $100 Protein: A Potential Marker for Cerebral Events During Cardiopulmonary Bypass Stephen Westaby, FRCS, Per Johnsson, MD, PhD, Andrew J. Parry, FRCS, Sten Blomqvist, MD, PhD, Jan-Otto Solem, MD, PhD, Christer Ailing, MD, PhD, Ravi PiUai, FRCS, David P. Tag,gaff, MD (Hons), Catherine Grebenik, MD, and Erik St~hl, MD, PhD Oxford Heart Centre, Oxford, England, and Departments of Cardiothoracic Surgery, Anaesthesia, Psychiatry, and Neurochemistry, University Hospital of Lund, Lund, Sweden

Background. There is no simple method to determine the incidence or severity of brain injury after a cardiac operation. A serum marker equivalent to cardiac enzymes is required. $100 protein leaks from the cerebrospinal fluid to blood after cerebral injury. We sought to determine the pattern of release after extracorporeal circulation (ECC). Methods. Thirty-four patients without neurologic problems underwent coronary bypass using ECC. Four had carotid stenoses. Nine others underwent coronary bypass without ECC. Serum $100 levels were measured before, during, and after the operation. Results. $100 was not detected before sternotomy.

Postoperative levels of $100 were related to duration of perfusion (r = 0.89, p < 0.001). Patients who did not have ECC had undetectable or fractionally raised levels except in I w h o suffered a stroke. No patient in whom ECC was used suffered an event, but those with carotid stenoses had greater $100 levels. Conclusions. $100 protein leaks into blood during ECC and may reflect both cerebral injury and increased permeability of the blood brain barrier. $100 is a promising marker for cerebral injury in cardiac surgery if elevated levels can be linked with clinical outcome.

erebral injury manifest as overt neurologic deficit or subtle neuropsychological dysfunction continues to blight the outcome after corrective cardiac operations. Although the anatomic and functional extent of embolic stroke can be determined through physical examination and imaging techniques, 70% of the brain is intellectually silent, and there are inherent dil~icuRies with the detection of "subclinical" problems such as cognitive dysfunction [1, 2]. Although experimental studies suggest that the blood-brain barrier is unaltered by cardiopulmonary bypass, apparently normal patients after cardiopulmonary bypass exhibit cerebral edema when investigated by magnetic resonance imaging [3, 4]. Cerebral edema results from cytotoxic or vasogenic disorders, both of which compromise the blood-brain barrier. An early marker for neuronal damage (equivalent to creatine kinase or troponin-T for myocardial infarction) would be valuable to gauge the timing and extent of cerebral injury and assist with prognostication. Several biochemical markers have been considered, but for many the need to sample cerebrospinal fluid prohibits their use in clinical practice [5-7]. $100 is an acidic, calcium binding protein (molecular weight, 21 kD) found in high concentration in glial and Schwann cells [8, 9]. The appearance of $100 in serum

indicates both neuronal damage and increased permeability of the blood-brain barrier [10]. $100 is eliminated by the kidney, with a biological half-life of about 2 hours [11]. To date, assays in cerebrospinal fluid at 24 hours after cardiopulmonary bypass have shown $100 to be in the normal range in patients without a neurologic complication [12]. $100 levels in patients with a cerebral tumor reflect the extent of cerebral involvement and may be a valuable prognostic indicator [13]. We therefore considered that perioperative measurement of serum $100 within the half-life of the molecule might be used to determine the occurrence and extent of cerebral events in patients undergoing cardiac operations. We sought to characterize the pattern of release with increasing duration of cardiopulmonary bypass.

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Accepted for publication Aug 14, 1995. Address reprint requests to Mr Westaby, Oxford Heart Centre, Oxford RadcliffeHospital, Oxford,England, OX3 9DU. © 1996 by The Society of Thoracic Surgeons Published by Elsevier Science Inc

(Ann Thorac Surg 1996;61:88-92)

Patients a n d M e t h o d s

Patients Ethical Committee approval to perform this study was obtained in both Oxford and Lund (but not to sample cerebrospinal fluid). In Lund we prospectively studied 34 nonconsecutive patients (21 male, 13 female) aged 46 to 78 years who underwent coronary artery bypass operations of varying complexity to encompass a broad spectrum of cardiopulmonary bypass times (ranging from 15 to 180 minutes). All had normal renal function. None of the patients had a previous neurologic event, but 3 had Doppler echocardiography-documented bilateral internal carotid stenoses (greater than 50%) and 1 a right 0003-4975/96/$15.00 SSDI 0003-4975(95)00904-3

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carotid stenosis. All operations were performed with a p u m p flow of 2.4 L • m - 2 . min -1, moderate hypothermia (32° to 34°C), and a perfusion pressure maintained pharmacologically between 50 and 80 m m Hg, We used a Cobe CML membrane oxygenator and a roller p u m p with nonpulsatile flow (Cobe Cardiovascular, Arvada, CO). Alpha-stat carbon dioxide tension management was employed. Myocardial protection was with cold anterograde St. Thomas" cardioplegic solution. In addition, 9 other patients underwent coronary bypass to the left anterior descending or right coronary artery without cardiopulmonary bypass (in Oxford). In this case the bypass grafts were applied during continuous ventilation with a heart supporting the circulation and the grafted arteries temporarily occhadecL There was no hemodilution, but the patients were heparinized (10,000 IU). For all patients undergoing cardiopulmonary bypass blood samples for analysis of $100 were collected before anesthesia, after heparinization (but before cardiopulmonary bypass), after extracorporeal circulation, and at 24 hours postoperatively. In the 9 patients operated on without cardiopulmonary bypass the samples were taken before anesthesia, at the time of limited heparinization, at skin closure, and postoperatively. The blood samples were centrifuged to separate the serum, which was frozen to -20°C and stored for analysis.

The Assay All $100 levels were measured by the same monoclonal two-site immunoradiometric assay (Sangtec 100; Sangtec Medical AB, Bromma, Sweden). The method is defined by the three monoclonal antibodies SMST 12, SMSK 25, and SMSK 28. The monoclonal antibodies detect the $100 beta beta and alpha beta dimer, which are isoforms specific for astroglial cells [14]. This protein is not released from striated muscle, heart, or kidney. The serum samples were diluted with phosphate buffer and incubated with a plastic bead coated with monoclonal anti$100 antibodies. During incubation $100 is bound to the antibody-coated bead. After 1 hour of incubation the beads were washed to remove any unbound material and incubated with iodine-125-1abeled anti-S100 antibody. This antibody binds to the $100 captured by the bead antibody. After a 2-hour incubation and subsequent washing, the amount of radioactive label bound to immobilized $100 was measured by gamma counter. Samples were analyzed in duplicate with the intention of rejecting any with more than 10% variation. This did not apply to any of the trial patients. A value of 0.2/zg/L in serum was the lower level of sensitivity of the assay. Levels in excess of 0.5/zglL were considered pathologic

[15].

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WESTABY ET AL CARDIOPULMONARY BYPASS AND $100 PROTEIN

Table 1. $100 Values and Related Data for 30 Patients Without Carotid Stenosis Undergoing Coronary Bypass Operation With ECC (A) and 8 Patients Undergoing Uneventful Coronary Bypass Operation Without ECC (B)a

Perfusion Time (min)

Age (y)

Mean Mean + SD Range + SD

Group A B

Range

S100 Level 30 Min After Perfusion (A) or at Skin Closure (B) (/zg/L) Mean - SD

Range

62 -+ 8.1 46-80 79 _+42 14-181 0.35 -+ 0.28b 0.2-1.16 60 _+7.4 48-71 ...... 0.2 -+ 0.2 ND-0.6

a Not i n c l u d e d is the p a t i e n t o p e r a t e d on without ECC w h o suffered a perioperative c e r e b r o v a s c u l a r accident with a n $100 level at the time of skin closure of 0.83/zg/L. b p value for difference b e t w e e n b y p a s s a n d n o n b y p a s s patients < 0.001. ECC = extracorporeal circulation; s t a n d a r d deviation.

N D = not detected;

SD =

Results $100 was not detected in serum before sternotomy in any patient. $100 was detected at the time of heparinization in only 1 patient. This man was operated on without cardiopulmonary bypass and suffered a perioperative stroke with expressive dysphasia, which resolved completely over a 2-week period. Analysis showed the $100 value to reach 0.83 /zg/L before bypass grafting. This probably resulted from hemodynamic instability and a neurologic event during induction of anesthesia. There were no symptoms or focal neurologic events in other trial patients. Table I shows $100 values at the end of extracorporeal circulation or at the time of chest closure for patients without extracorporeal circulation. For the cardiopulmonary bypass patients perfusion time ranged from 14 minutes (single graft) to 181 minutes (six grafts including endarterectomies). Figure 1 shows the relationship between $100 levels 30 minutes after perfusion and duration of cardiopulmonary bypass. For the 30 patients who did not have a carotid stenosis, there is a

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Statistical Analysis Computerized statistical analysis was performed using standard statistical method programs from BBN Software Products Corp (Cambridge, MA). $100 levels are expressed as mean _+ standard deviation. Means were compared using the t test.

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WESTABY ET AL C A R D I O P U L M O N A R Y BYPASS A N D $100 PROTEIN

Arm Thorac Surg 1996;61:88-92

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highly significant correlation between duration of perfusion and level of $100 measured in the serum (r = 0.89; p < 0.001). Five patients with bypass times greater than 100 minutes had $100 levels greater than 0.5/~g/L (pathologic range), the highest being 1.16/~glL. At 24 hours $100 levels were undetectable in all but this last patient, where the level fell from 1.16 ~g/L postoperative to 0.46 ~g/L the following day. When patients with carotid stenoses were included in the graph, all had $100 levels outside the 95% confidence limit (Fig 2), but all returned to undetectable levels at 24 hours. For the nonbypass coronary patients, $100 was not detected in 3 patients and was elevated only fractionally in 3 others. The highest level detected in an apparently normal patient in this group was 0.6 ~g/L. Comment Our findings suggest that the serum assay for $100 may be used as an index of cerebral changes during cardiopulmonary bypass and a vehicle with which to compare the influence of perfusion equipment and management strategies on the brain. The highly significant correlation between serum $100 level and duration of cardiopulmonary bypass in this set of patients without neurologic signs or symptoms suggests that subclinical cerebral injury or increased permeability of the blood-brain bartier occurs progressively in a dose-related fashion. The etiology of cerebral injury during cardiac operations is multifactorial. The release of $100 protein documented in this study is compatible with flow-related microembolization of the brain as suggested by previous authors [16]. Higher levels of $100 noted in patients with carotid stenosis may result from maldistribution of cerebral blood flow, microembolization from atheromatous arterial walls, or periods of global ischemia [17]. However, overall cerebral blood flow is not necessarily impaired by a carotid stenosis [18]. Flow-dependent delivery of microemboli to the brain may be exacerbated by the so-called luxury perfusion

syndrome. This occurs through loss of cerebral autoregulation or inappropriate acid-base management hypothermia [19, 20]. We employed the so-called alpha-stat method of carbon dioxide tension management to avoid this problem. Profound changes in blood pressure, either hypotension or hypertension, may also contribute to the incidence of stroke, particularly in patients more than 60 years old and those with carotid occlusion. Independent of cardiopulmonary bypass is the risk of atheromatous embolism from a diseased ascending aorta subject to cross-clamping or side-clamping during a coronary operation. Aortic side-clamping is as likely to affect nonbypass patients who undergo vein grafts and would have been implicated in the nonbypass patient with stroke had it not been for the fact that prebypass $100 levels suggested that the problem occurred before the chest was opened. The remaining nonbypass patients did not show an important increase in $100 level. Currently, documentation of neurologic injury in cardiac operations depends on relatively crude physical examination and radiologic imaging techniques [21]. Postoperative cognitive dysfunction or confusion is rarely assessed objectively, and few surgeons request a spinal tap in patients with an acute cerebral event. The battery of neuropsychological and neurophysiological tests for cognitive dysfunction employed on a research basis require specialist personnel to perform them, are prolonged and tedious for the patient, and are inappropriate for infants and children [22-24]. They have never been translated into clinical practice. In contrast, measurement of $100 by a simple blood test may provide evidence of neurologic damage and with further experience provide a prognostic indicator for patients with overt injury. Confirmation of cerebral injury depends on clincopathological correlation, and we have already documented substantially elevated $100 levels (>0.5 ~g/L) in patients who suffered perioperative stroke after cardiopulmonary bypass. In patients with extensive cerebral infarction and stroke, $100 levels increase progressively from the postbypass value and remain greatly elevated at 24 and 48 hours. In 1 paraplegic aortic dissection patient with anterior spInal artery syndrome, we peformed therapeutic cerebrospinal fluid drainage and recorded similar greatly elevated levels of $100 in both serum and cerebrospinal fluid. In pa@ents who recovered without overt neurologic injury after prolonged perfusion, high levels of $100 were recorded soon after bypass but fell by 24 hours postoperatively. A speculative interpretation is that this reflects d i ~ s e microembolic cerebral injury together with increased permeability of the blood-brain barrier but not irreversible cerebral damage through neuronal ischemia and death. The blood-brain barrier is based on the endothelial lining of the vasculature and is not permeable to proteIn in normal circumstances [3]. We suspect that increased permeability of the blood-brain barrier after prolonged cardiopulmonary bypass resolves in the absence of a focal lesion. Consequently leakage of S100 protein from cerebrospInal fluid ceases and serum $100 is cleared by the kidney. This suggestion is at odds with

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investigations that suggest that the b l o o d - b r a i n b a r r i e r is not altered b y c a r d i o p u l m o n a r y b y p a s s [3]. In patients with cerebral infarction confirmed b y c o m p u t e d t o m o graphic scan, elevated levels of s e r u m $100 are s u s t a i n e d a n d even increase with time, suggesting a progressive release of $100 into the circulation t h r o u g h a less reversible d i s r u p t i o n of the b l o o d - b r a i n barrier. Because the biological half-life for $100 is short, the s e r u m concentration m u s t be m a i n t a i n e d b y persistent release. This is in contrast to the glycolytic e n z y m e neuron-specific enolase, which has a m u c h longer hip logical half-life [25--27]. Large areas of the b r a i n are silent, s o that elevation of a m a r k e r does not g u a r a n t e e clinically detectable injury. Paradoxically, we suspect that only a m o d e s t increase in $100 was seen in the n o n b y p a s s patient with a stroke because the b l o o d - b r a i n barrier r e m a i n e d intact in the absence of perfusion a n d diffuse microembolism. W e w o u l d n o t expect to d o c u m e n t cerebral injury clinically without elevation of $100 protein level in the cerebrospinal fluid or s e r u m at s o m e time. Increasingly, p r o l o n g e d c a r d i o p u l m o n a r y b y p a s s is recognized to cause transient a n d s o m e t i m e s p e r m a n e n t cerebral dysfunction [28, 29]. O u r findings are c o m p a t i b l e with this observation, a n d w e speculate that p r o l o n g e d perfusion with increasing d a m a g e to the b l o o d - b r a i n b a r r i e r m a y p r e d i s p o s e the b r a i n to irreversible injury. $100 has a l r e a d y p r o v e d effective in quantifying the extent of injury after stroke, w h e r e the levels correlated with the size of cerebral infarction a n d the short-term patient d e p e n d e n c y [9]. Persson a n d co-workers [30] d e t e r m i n e d the time course of $100 release into the cerebrospinal fluid for stroke patients. N o r m a l levels were found at 8 hours after the stroke b u t i n c r e a s e d a n d r e m a i n e d elevated 18 hours to 4 days after the acute event. The severity of stroke correlated with $100 levels. Reports of $100 m e a s u r e m e n t in s e r u m are infrequent, b u t Fagnart a n d associates [13] r e p o r t e d i n c r e a s e d s e r u m levels of $100 in the majority of patients with ischemic stroke, i n t r a c e r e b r a l h e m o r r h a g e , a n d s u b a r a c h n o i d h e m o r r h a g e . Ingebrightsen a n d co-workers [31] have recently shown a relationship b e t w e e n early levels of $100 in s e r u m a n d the prognosis of m i n o r h e a d injury. In conclusion, our findings s h o w a correlation b e t w e e n $100 level a n d duration of perfusion. W i t h characterization of the p a t t e r n of release in those with uneventful recovery, it m a y be possible to e m p l o y d i s p r o p o r t i o n a t e elevation of $100 as a p r e d i c t o r of adverse neurologic outcome. As a m a r k e r for cerebral events, it is possible that $100 will have the potential to differentiate b e t w e e n the benefits a n d adverse effects of different t r e a t m e n t strategies.

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Notice From the American Board of Thoracic Surgery The American Board of Thoracic Surgery began its recertification process in 1984. Diplomates interested in participating in this examination should maintain a documented list of the operations they performed during the year prior to application for recertification. This practice review should consist of I year's consecutive major operative experiences~ (If more than 100 cases occur in I year, only 100 need to be listed.) They should also keep a record of their attendance at approved postgraduate medical education activities for the 2 years prior to application. A minimum of 100 hours of approved CME activity is required. In place of a cognitive examination, candidates for recertification will be required to complete both the general thoracic and cardiac portions of the SESATS VI syllabus (Self-Education/Self-Assessment in Thoracic Surgery). It is not necessary for candidates to purchase

SESATS VI booklets prior to applying for recertification. SESATS VI booklets will be forwarded to candidates after their applications have been accepted. Diplomates whose 10-year certificates will expire in 1998 may begin the recertification process in 1996. This new certificate will be dated 10 years from the time of expiration of the original certificate. Recertification is also open to any diplomate with an unlimited certificate and wilt in no way affect the validity of the original certificate. The deadline for submission of applications is May 1, 1996. A recertification brochure outlining the rules and requirements for recertification in thoracic surgery is available u p o n request from the American Board of Thoracic Surgery, One Rotary Center, Suite 803, Evanston, IL 60201.