**3.1 Biomarkers of brain damage**

Biochemical tests are useful diagnostic tools in the examinations of functional brain disorders. Elevated serum concentrations of the markers of brain damage indicate that there has been a neuronal and/or glial injury. The biomarkers are released as a consequence of either transient ischemia or ultimate cell degradation, and their serum concentration depends on the localization of pathological changes, the degree of tissue damage and the time that has passed since the onset of changes.

The ideal marker of brain damage should be: [1] highly specific, [2] highly sensitive, [3] released in cases of an irreversible damage to cerebral neurons only, [4] possible to detect in the blood and/or cerebrospinal fluid within a short period of time after the injury, and [5] released in well-known time sequences after the injury; furthermore, it ought to be [6] age- or sex-independent, [7] easily detectable in the blood since frequent drawing of cerebrospinal fluid samples is impractical, and [8] its concentration should be easily measureable in laboratory tests (Ingebrigtsen T). Many compounds were investigated for this purpose of founding such a marker. In the '70s, it was lactic dehydrogenase (LDH) and aspartate aminotransferase (AspAT); in the '80s, creatine kinase BB isoenzyme (CK-BB); and later, the S100B protein and neuron-specific enolase (NSE). Nowadays, glial fibrillary acidic protein (GFAP) seems most promising (Ingebrigtsen & Romner, 2003). The latter three (the S100B protein, NSE and GFAP) were the best known biomarkers of brain damage (Ingebrigtsen & Romner, 2003; L. E. Pelinka, 2004). Of these the S100B protein is thought to correspond the best to the optimal indicator of a neuronal injury (Ingebrigtsen & Romner, 2003).

Postoperative Cognitive Dysfunction (POCD)

released S100B protein (Raabe, et al., 2003).

**3.3 S100B protein in orthopedic patients** 

et al., 2003).

There were a few papers on the S100B protein in orthopedics only.

and Markers of Brain Damage After Big Joints Arthroplasty 7

because of its short (below 30 minutes) half-life (Ingebrigtsen & Romner, 2003; Raabe, et al., 2003) as well as sex and age non-dependent serum concentrations (Ingebrigtsen & Romner, 2003). Neither alcohol overdose nor hemolysis altered its concentration (Raabe, et al., 2003). The S100B protein was stable in solution, there was no need to centrifuge and freeze its samples (Raabe, et al., 2003). Contemporary biochemical tests measure either S100B or S100A1B and S100BB serum levels. Because S100BB is considered to be the most brain-specific unit, its determination could eliminate the influence of the extracerebrally

Kinoshita et al. (Kinoshita et al., 2003) examined 14 patients; half of them underwent total knee arthroplasty (TKA) with bone cement, the other half underwent intramedullary nail stabilization of the tibia. All the procedures were performed with tourniquet and ischemia. In the TKA group, in blood samples withdrawn 15 minutes after tourniquet release, there was a statistically significant elevation of the S100B serum level in comparison to the group where tibial fracture was stabilized with an intramedullary nail. The authors suggested that the increase was due to a transient injury of brain tissues caused by bone cement (Kinoshita,

An increased serum concentration of the S100B protein was observed after injuries which did not include brain damage. The highest levels were noted in patients with long bones fractures (Anderson, Hansson, Nilsson, Dijlai-Merzoug, & Settergren, 2001). Studies on people with isolated bone fractures without brain injury revealed that patients with hip, radius or tibia fractures had significantly higher concentrations of the S100B protein, but those with phalanges, hand or foot fractures did not (Undén et al., 2005). In animal studies there were increased S100B serum levels after bilateral femur fractures in rats (L. Pelinka et al., 2003). These results indicated that bone marrow could be a potential source of the S100B protein. During orthopedic cement was used for fixing the elements of the prosthesis to the bone basis. The use of cement can lead to hemodynamic instability, a decrease in cardiac output, heart contractility, systemic vascular resistance and blood pressure, i.e. to the so-called bone cement implantation syndrome (Donaldson, Thomson, Harper, & Kenny, 2009). Hemodynamic changes have an impact upon cerebral perfusion, as in the relation between the S100B concentration and the degree of shock that has been discussed above (L. E. Pelinka, 2004). Although the presence of bone cement inside the medullar cavity itself did not produce hypotension, hemodynamic instability is often observed when hammering the prosthesis stem into the bone (Sharrock, Beckman, Inda, & Savarese) - the pressure inside the marrow cavity increases. The higher the pressure, the better the penetration of the cement into the bone and the greater the strength of osteosynthesis. But due to the increased pressure, the translocation of the cellular material originating at the site of the surgery into the systemic circulation was facilitated, and the said material could reach the lungs via the bloodstream. The diameter of the lung capillaries is about 8 µm. In 1956, Niden and Aviado showed that there was a possibility of transferring glass spheres up to 420 µm in diameter through the pulmonary vessels (Nunn, 1981). The second way of going round the pulmonary filter was via the foramen ovale. In 1/3 of the population it is closed only functionally. Thus, it is a way for transferring the embolic material into the brain. The presence of cellular material from the site of the surgery in the circulation can be shown with an ultrasound examination as a "snow flurry." In the work of Hayakawa et al.

### **3.2 S100B protein**

In 1965, Moore isolated from the bovine brain a fraction containing brain specific proteins (Kleindienst & Ross Bullock, 2006). Since then, our knowledge about the above mentioned compounds has increased. Currently, the S100B proteins family consist of 24 members, with similar structures and like functions (Marenholz, Heizmann, & Fritz, 2004).

They play different roles in the human body and are present in many types of cells and tissues (Eckert et al., 2003). Some members of the S100 protein family are specific for certain localizations (Heizmann, 2004). High S100B protein concentrations were present inside the brain, mainly in astroglial and Schwann cells (Ali, Harmer, & Vaughan, 2000; L. E. Pelinka, 2004) as well as in adipocytes, chondrocytes and melanocytes (L. E. Pelinka, 2004; Raabe et al., 2003). The S100B protein can be either actively released into the extracellular space or passively excreted after cell death (L. E. Pelinka, 2004). An animal study revealed high S100B concentrations in cerebral regions injured by chronic ischemia (Ohtani et al., 2007).

In 2003, there was published a review of 18 clinical studies (involving 1085 patients) of the S100B protein as a marker of brain damage (Kleindienst & Ross Bullock, 2006). In 2004 and 2005, another 6 papers appeared (involving more than 600 adults) on the correlations between an elevated concentration of the S100B protein and poor outcome after brain injury (Kleindienst & Ross Bullock, 2006). The highest S100B protein serum level was observed just after an injury (Ingebrigtsen & Romner, 2003), then normalized within 24 hours, even in patients with poor outcome (Kleindienst & Ross Bullock, 2006). The study of Raabe and Seifert showed an increased concentration of the S100B protein on the 6th day after head trauma, probably as a result of a secondary injury (Kleindienst & Ross Bullock, 2006). The elevated posttraumatic S100B protein concentration was also demonstrated in an animal model (Kleindienst & Ross Bullock, 2006).

The increased concentration of the S100B protein can be a result of an increased permeability of the blood-brain barrier, regardless of cerebral damage (Kleindienst & Ross Bullock, 2006). The results of animal studies suggested that S100B protein levels correlated with the degree of shock: in moderate shock they were higher than in a severe one (L. E. Pelinka, 2004). The S100B protein concentration was increased just after bilateral long bones fractures as well as after local ischemia and the reperfusion of the liver, gut and kidneys (L. E. Pelinka, 2004). Elevated levels of S100B protein were shown in basketball and hockey players after competitions as well as in runners, boxers (Stalnacke, Tegner, & Sojka, 2003), swimmers and soccer players; although in the latter there was a correlation between an increased protein concentration and the frequency of head injury (Stålnacke, Ohlsson, Tegner, & Sojka, 2006). There was a possibility that some amounts of the S100B protein were released from red cells, melanocytes and steatocytes. The protein's origin – whether it was extra- or intracerebral - remains unclear. If it was of cerebral origin, the question is whether it was released due to an astroglial injury or activation, or as an effect of the blood-brain barrier impairment (Stalnacke, et al., 2003). Stress and physical effort can lead to an increased permeability of the blood-brain barrier (Stålnacke, et al., 2006). In some cases, a raised plasma level of the S100B protein was an effect of long bones fractures, multiple traumas and surgical procedures. It also occurred in melanoma patients (Salama, Malone, Mihaimeed, & Jones, 2008) and in sepsis-associated encephalopathy (Piazza, Russo, Cotena, Esposito, & Tufano, 2007).

The possibility that the S100B protein could be released from extracerebral localizations as well confines its utility as a marker of brain damage, which, nonetheless, still ranges from 70 to 80% (Raabe, et al., 2003). The S100B protein was a very useful biochemical tool

In 1965, Moore isolated from the bovine brain a fraction containing brain specific proteins (Kleindienst & Ross Bullock, 2006). Since then, our knowledge about the above mentioned compounds has increased. Currently, the S100B proteins family consist of 24 members, with

They play different roles in the human body and are present in many types of cells and tissues (Eckert et al., 2003). Some members of the S100 protein family are specific for certain localizations (Heizmann, 2004). High S100B protein concentrations were present inside the brain, mainly in astroglial and Schwann cells (Ali, Harmer, & Vaughan, 2000; L. E. Pelinka, 2004) as well as in adipocytes, chondrocytes and melanocytes (L. E. Pelinka, 2004; Raabe et al., 2003). The S100B protein can be either actively released into the extracellular space or passively excreted after cell death (L. E. Pelinka, 2004). An animal study revealed high S100B concentrations in cerebral regions injured by chronic ischemia (Ohtani et al., 2007). In 2003, there was published a review of 18 clinical studies (involving 1085 patients) of the S100B protein as a marker of brain damage (Kleindienst & Ross Bullock, 2006). In 2004 and 2005, another 6 papers appeared (involving more than 600 adults) on the correlations between an elevated concentration of the S100B protein and poor outcome after brain injury (Kleindienst & Ross Bullock, 2006). The highest S100B protein serum level was observed just after an injury (Ingebrigtsen & Romner, 2003), then normalized within 24 hours, even in patients with poor outcome (Kleindienst & Ross Bullock, 2006). The study of Raabe and Seifert showed an increased concentration of the S100B protein on the 6th day after head trauma, probably as a result of a secondary injury (Kleindienst & Ross Bullock, 2006). The elevated posttraumatic S100B protein concentration was also demonstrated in an animal

The increased concentration of the S100B protein can be a result of an increased permeability of the blood-brain barrier, regardless of cerebral damage (Kleindienst & Ross Bullock, 2006). The results of animal studies suggested that S100B protein levels correlated with the degree of shock: in moderate shock they were higher than in a severe one (L. E. Pelinka, 2004). The S100B protein concentration was increased just after bilateral long bones fractures as well as after local ischemia and the reperfusion of the liver, gut and kidneys (L. E. Pelinka, 2004). Elevated levels of S100B protein were shown in basketball and hockey players after competitions as well as in runners, boxers (Stalnacke, Tegner, & Sojka, 2003), swimmers and soccer players; although in the latter there was a correlation between an increased protein concentration and the frequency of head injury (Stålnacke, Ohlsson, Tegner, & Sojka, 2006). There was a possibility that some amounts of the S100B protein were released from red cells, melanocytes and steatocytes. The protein's origin – whether it was extra- or intracerebral - remains unclear. If it was of cerebral origin, the question is whether it was released due to an astroglial injury or activation, or as an effect of the blood-brain barrier impairment (Stalnacke, et al., 2003). Stress and physical effort can lead to an increased permeability of the blood-brain barrier (Stålnacke, et al., 2006). In some cases, a raised plasma level of the S100B protein was an effect of long bones fractures, multiple traumas and surgical procedures. It also occurred in melanoma patients (Salama, Malone, Mihaimeed, & Jones, 2008) and in sepsis-associated encephalopathy (Piazza,

The possibility that the S100B protein could be released from extracerebral localizations as well confines its utility as a marker of brain damage, which, nonetheless, still ranges from 70 to 80% (Raabe, et al., 2003). The S100B protein was a very useful biochemical tool

similar structures and like functions (Marenholz, Heizmann, & Fritz, 2004).

**3.2 S100B protein** 

model (Kleindienst & Ross Bullock, 2006).

Russo, Cotena, Esposito, & Tufano, 2007).

because of its short (below 30 minutes) half-life (Ingebrigtsen & Romner, 2003; Raabe, et al., 2003) as well as sex and age non-dependent serum concentrations (Ingebrigtsen & Romner, 2003). Neither alcohol overdose nor hemolysis altered its concentration (Raabe, et al., 2003). The S100B protein was stable in solution, there was no need to centrifuge and freeze its samples (Raabe, et al., 2003). Contemporary biochemical tests measure either S100B or S100A1B and S100BB serum levels. Because S100BB is considered to be the most brain-specific unit, its determination could eliminate the influence of the extracerebrally released S100B protein (Raabe, et al., 2003).
