Associate Professor, Dr.Med. V.V. Dorofeykov¹
Professor, Dr.Hab. S.M. Ashkinazi¹ 
Professor, Dr.Med. V.A. Bukharin¹
PhD T.I. Oparina²
Associate Professor, Dr.Biol. A.N. Vetosh¹
Professor, Dr.Med. S.Y. Kalishevich^1
1Lesgaft National State University of Physical Education, Sport and Health, St. Petersburg
²Obstetrics, Gynaecology and Reproduction Research Institute n.a. D.O. Ott, St. Petersburg

Keywords: brain damage, biomarkers, creatine kenase, neuron-specific enolase, protein S100.

Background. Since brain concussion is a metabolic rather than organic damage, modern visualizing diagnostic tools like computed tomography (CT) and magnetic resonance (MR) tomography are virtually worthless for diagnosing purposes, though still allow to exclude more serious damages (e.g. intracranial haemorrhage) which can be caused even by a light fall. Therefore, negative decisions of the clinical visualizing methods cannot rule out the probability of a brain damage nor may be used as a basis for a permission to come back to sports. It is a matter of common knowledge that in some cases such permissions based on CT data or assurance of the athlete that he/she feels well – were premature since athletes in such cases are always at high risk of repeated brain concussion from an insignificant injury, that normally requires much more time for rehabilitation.

Objective of the study was to offer a new approach based on the reputable visualizing diagnostic methods being supported by a set of new laboratory biochemical methods.

Study results and discussion. Prerequisites for the new approach were created by the studies of cardiac troponins since 1990 which resulted in total, revolutionary changes – first in the acute myocardial infarction diagnostics followed by the impaired cardiac function diagnostics and curing, and at present the methods are being used in combination with the modern multimarker strategy on a very broad basis, particularly in economically developed countries with advanced cardiology and survival medicine [1, 2]. In modern sport medicine, this new approach is of high promise for early diagnostics of heart damages [3, 4] with the brain micro- and macro-damage diagnostics being considered not less important subject for the method. Such injuries are quite common in boxing and other striking sports and in team sports where collisions and falls of athletes are multiple (like ice hockey, football, rugby, handball etc.). It may be pertinent to highlight such sport disciplines as diving, alpine skiing, sledding, trampoline tumbling, artistic gymnastics, mountain biking, motorcycle racing, freestyle, bobsledding etc. It is commonly acknowledged that neurocognitive deficit and other complications of brain concussion usually appear at early stages after injury, stay for a long time and largely determine the social (including professional) performance and psychosocial adaptation disorders in athletes, military servicemen and other people. 

For the last few years, many damage markers (molecules) have been comprehensively studied including myoglobin, intracellular enzymes (including liver and heart transaminases) and С-reactive protein [5]. These markers and the relevant indices have been added to the diagnostic toolkits applied by physicians, traumatologists and sport physicians. However, the organ-specificity of these laboratory tests has been considered insufficient, whilst the laboratory biochemical tools of brain damage diagnostics are still new and unusual. It should be noted that some molecules specific for brain conditions and well known to biochemists and physiologists have long not been inaccessible for laboratory studies. There are a few reasons for this situation. Mentioned first may be the hematoencephalic (blood-brain) barrier that does not let through large-size protein molecules from liquor (cerebrospinal fluid) to blood; plus liquor is a body liquid largely inaccessible for routine tests. Second, these molecules are normally found in nano-quantities that cannot be detected using regular laboratory methods in disposal of physicians. And the last but not least reason is the need in the clinical diagnostics process being fast and efficient. The tests is to be made for a few minutes or at most within a few hours after the injury to be helpful. However, the immune-enzyme assay systems presently applied at clinical diagnostics laboratories all over the world include long manual procedures to find the biomarker concentrations and normally use 96-reagents assay kits. Therefore, every single test, when performed on a commercial basis, is very costly for an injured person: in Russia, for instance, it may cost many thousand Roubles. However, for the last few years the situation has been rapidly changing for the better, particularly since the laboratories were equipped with the last-generation automatic immune-enzyme assay systems that may make the high-technology tests for 15-30 minutes after the blood sample is delivered to the laboratory.

We offer hereby three biomarkers to be used separately or all together as the most promising brain micro-damage diagnostic indicators in the assays to efficiently rate the degree of damage and make prognoses in the patient’s rehabilitation process. Regular tests to find variations of the indices may be helpful in rating the medical treatment process efficiency and rehabilitation progress for further competitive career of the athlete. In addition, the method may be beneficial in diagnostics of home injuries, car accident injuries, other accidental injuries, military servicemen’s injuries and in the survival medicine.  

It is protein S100 that acts as one of the mediators in the glia-neuron and glia-glial interactions in the brain, protein being located in astro- and oligodendrocytes that are probably responsible for its secretion [6]. Effects of the extracellular protein S100 are known to be dose-specific: in nano-molar concentrations, protein has an autocrine effect on the astrocytes stimulating their proliferation in vitro; whilst the S100(ββ) dimer modulates long-term synaptic plasticity and has a trophic effect on the developing and regenerating neurons. Normally, protein S100B is always found in small quantities in cerebrospinal fluid and blood serum; and when brain tissue is damaged, its content grows dozens of times and makes it possible to use the agent for the damage diagnostics and process prognosis in many cases, including a traumatic brain damage, stroke, hematoencephalic barrier damage and brain involvement in systemic inflammation process as diagnosed by active expression of protein S100B.

One more biomarker of different neurons accessible for laboratory assays at present is enolase (NSE), an intracellular enzyme found in the cells of neuro-ectodermal origin. Presently NSE is the commonly known and well-studied neuron-specific protein adequately indicative of the purely membrane functions of the hematoencephalic barrier. NSE is being applied for acute condition diagnostics associated with cerebral ischemia and brain hypoxia, and for studies of pathogenic neurological conditions associated with blood-brain barrier dysfunction [7].

When cells are damaged, the intracellular enzymes come to the blood flow through action of a universal mechanism. Hence, a brain isoform of creatine phosphokenase (CK) may be used as the third biomarker of brain damage. Modern studies report that there are three forms of creatine kenase in blood, namely the (cerebral) ВВ-isoform, (muscular) ММ-isoform and the (heart) МВ-isoform. Each molecular of the enzyme is composed of two subunits having similar enzymatic activity. Healthy men are tested with the ВВ-isoform activity/ content varying under 1% of the total activity and, therefore, it is normally neglected in the myocardial and skeletal muscle damage diagnostics. Activity of the МВ-isoform in the patients and healthy people showing no signs of acute coronary syndrome make up under 2-4% of the total activity; and the MM-isoform activity is dominating and expressed in units per litre in the standard laboratory tests, with the normal value for adults varying within 175-195 u/l. In case of heart damage, the МВ-isoform activity in the blood grows within 3-6 hours, and this is the ground for the МВ-isoenzyme test being applied as the main diagnostic test by clinical cardiologic departments. However, myocardial infarctions and strokes result in serious cellular breakdown with a significant part of proteins including CK being damaged with the relevant fall in the enzymatic activity. Therefore, the new-generation laboratory tests implemented at the leading clinical diagnostics laboratories of the world are designed to identify the mass of molecular rather than activity of the CK isoforms, i.e. are driven by the immunochemical principle. The above assays to rate the CK activity and mass and its MB-isoform are presently applied as routine procedures by the modern clinical diagnostic laboratories and normally take under 20 minutes since the patient’s blood comes to the test. However, the cerebral ВВ-isoform of CK has been tested using specific versions of electrophoretic analysis which have their serious drawbacks including the following: high difficulty of the test procedure; need for highly skilled service personnel; long time required for the test; high cost of the assay kit; and the need for special precursors of narcotic substances being used as components of the buffer solutions in the electrophoretic test. These difficulties have resulted in the electrophoretic segregation of isoforms virtually disappearing from the test sets applied at the clinical diagnostics laboratories.

We have developed a new fast diagnosing method for the cerebral isoform of CK readily applicable in operations of the clinical diagnostics laboratories, with the priority of application for invention registered by the RosPatent with registration #2015124963 of 2015. The new assay is designed to test the total creatine phosphokenase activity (CK-TOTact); the heart creatine phosphokenase isoform activity (СК-МВact) and the heart creatine phosphokenase isoform content by mass (СК-МВmass); followed by the cerebral creatine phosphokenase isoform activity (СК-BВact) being calculated using the following formula:

СК-ВВact = K₁ × (СК-МВact/СК-МВmass) + K₂ × (СК-МВact/СК-TOTact) + K₃ × (СК-МВmass),

Where:

СК-ВВact is the cerebral creatine phosphokenase isoform activity, u/l; and

СК-TOTact is the total creatine phosphokenase activity including cerebral, heart and muscular creatine phosphokenase isoform activity as determined by the kinetic method, u/l.

The invention was based on the assumption that the total creatine phosphokenase activity is composed of the activities of all the three isoenzymes (muscular, heart and cerebral) – with or without brain damage. Following the CK isoenzymes being segregated, they are visualized using a specific chromogenic substrate as recommended by the instruction manuals for the test instruments. The test data obtained by the new method have been found highly compatible with the traditional test data, whilst the test time of the new laboratory procedure is more than 5 times faster. 

Conclusion. The innovative laboratory tests developed by the authors to quantify biomarkers of brain damage are designed as a standard, highly efficient and promising approach to diagnostics of both micro- and macro-damages of brain in elite athletes; home injuries of brain; brain concussions in military/ rescue service personnel; and medical treatment efficiency assessments and rehabilitation prognoses based on the test data variations. We believe that it is the multi-marker test approach that will play a special role in the next decade in health condition prognoses. The first steps have been made. Much further efforts need to be taken, and the work appears to be highly promising.

The study was performed under the State Order by the Federal State Higher Education Establishment Supported by Federal State Budgetary Educational Institution of Higher Education P.F. Lesgaft National State University, St. Petersburg, for the research report “Modern athletic training system design for Olympic sports, case study of freestyle wrestling”, pursuant to the Ministry of Sports of Russia Order #318 of April 07, 2015.

References

1. Dorofeykov V.V. Sovremennye laboratornye tekhnologii i risk serdechno-sosudistykh oslozhneniy (Modern laboratory technologies and risk of cardiovascular complications) / V.V. Dorofeikov // Translational Medicine / Ed. by E.V. Shlyakhto. – St. Petersburg, 2015. – P. 290–321.
2. Dorofeykov V.V. Novye laboratornye tekhnologii v otsenke povrezhdeniya i peregruzki serdtsa u sportsmenov (New laboratory technologies to assess heart damage and overload in athletes) / V.V. Dorofeykov, E.N. Kuryanovich // Aktual'nye problemy fizicheskoy i spetsial'noy podgotovki silovykh struktur. – 2015. – # 3. – P. 38–45.
3. Laboratornaya diagnostika mikropovrezhdeniy miokarda vo vremya koronarnoy ballonnoy angioplastiki so stentirovaniem (Laboratory diagnostics of cardiac micro-injuries during coronary balloon angioplasty and stenting) / V.V. Dorofeykov [et al.] // Klinicheskaya laboratornaya diagnostika. – 2011. – # 2. – P. 15–18.
4. Laboratorny monitoring sostoyaniya organizma u sportsmenov (Laboratory monitoring of athlete's body condition) / Sergey Aleksandrovich Tsvetkov [et al.]; Lesgaft National State University of Physical Education, Sport and Health, St. Petersburg (NSU n.a. P.F. Lesgaft, St. Petersburg); State Research Institute of Socio-economic Problems and Health and Fitness Technologies (GNII SEP i SOT), St. Petersburg // Uch zapiski un-ta im. P.F. Lesgafta. – 2013. – # 6 (100). – P. 159–163.
5. Sovremennye algoritmy otsenki prognoza u bolnykh HSN (Modern prognosis estimation algorithms in patients with CHF) / E.V. Shlyakhto [et al.] // Serdechnaya nedostatochnost. – 2009. – V. 10, № 1. – P. 4–7.
6. The Prognostic Value of Serum Neuron-Specific Enolase in Traumatic Brain Injury: Systematic Review and Meta-Analysis / F. Cheng [et al.] // Ai J, ed. PLoS ONE. – 2014. – № 9 (9). – e106680.
7. Trajectory Analysis of Serum Biomarker Concentrations Facilitates Outcome Prediction after Pediatric Traumatic and Hypoxemic Brain Injury / R.P. Berger [et al.] // Developmental Neuroscience. – 2011. – № 32 (5-6). – Р. 396–405.

Corresponding author: vdorofeykov@yandex.ru

Abstract

Brain damage diagnostics and brain rehabilitation progress rating studies are ranked among the top priorities in modern sport medicine serving elite and youth sports. The authors offer a new approach to these problems based on their own study data on the brain, muscular and heart isoforms of creatine kinase using a set of computerized and standardized innovative biochemical and laboratory research methods, with due consideration for the available study data on the brain damage assessment biomarkers including neuron-specific enolase and protein S100. The new multimarker laboratory strategy to measure the above biomarkers and trace variations in the athlete’s health will help assess the injury, success of the applied therapy and the rehabilitation progress on a timely and objective basis.