Pterins as Diagnostic Markers of Mechanical and Impact-Induced Trauma: A Systematic Review

We performed a systematic review of the literature to evaluate pterins as biomarkers of mechanical and impact-induced trauma. MEDLINE and Scopus were searched in March 2019. We included in vivo human studies that measured a pterin in response to mechanical or impact-induced trauma with no underlying prior disease or complication. We included 40 studies with a total of 3829 subjects. Seventy-seven percent of studies measured a significant increase in a pterin, primarily neopterin or total neopterin (neopterin + 7,8-dihydroneopterin). Fifty-one percent of studies measured an increase within 24 h of trauma, while 46% measured increases beyond 48 h. Pterins also showed promise as predictors of post-trauma complications such as sepsis, multi-organ failure and mortality. Exercise-induced trauma and traumatic brain injury caused an immediate increase in neopterin or total neopterin, while patients of multiple trauma had elevated pterin levels that remained above baseline for several days. Pterin concentration changes in response to surgery were variable with patients undergoing cardiac surgery having immediate and sustained pterin increases, while gastrectomy, liver resection or hysterectomy showed no change. This review provides systematic evidence that pterins, in particular neopterin and total neopterin, increase in response to multiple forms of mechanical or impact-induced trauma.


Introduction
Pterins are biologically active bi-cyclic compounds. Pterins have been utilised as biomarkers because of their involvement in inflammation [1], oxidative stress [2] and aromatic amino acid hydroxylation [3] since they were discovered in 1889 [4]. Neopterin and tetrahydrobiopterin (BH 4 ) are examples of pterins used to assess changes in immune system activation and oxidative status, as well as deficiencies in monoamine neurotransmitter synthesis and nitric oxide availability, respectively. Neopterin is the oxidative product of 7,8-dihydroneopterin, a metabolic derivative of guanosine triphosphate (GTP) produced by monocytes and macrophages during γ-interferon (γ-IFN)-stimulated immune activation [5]. Neopterin has been used a useful measure of exercise-induced stress [6], cancer [7], neuromuscular disease [8], human immunodeficiency virus (HIV) [9], sepsis [10] and vascular disease [11]. BH 4 is also a product of GTP metabolism and used to diagnose numerous neurological diseases, including phenylketonuria, due to its involvement as a co-factor in many hydroxylase enzyme reactions [12].
Accurate diagnosis and management of mechanical or impact-induced trauma is critical for patient monitoring and treatment. Major mechanical incident can cause immediate structural damage that results in the loss of muscle plasmalemma integrity, organ perforation or bone breaks and a secondary can remain elevated following an MMA training session up to 24 h post-contest. Interestingly, only a continuous increase in neopterin and total neopterin showed evidence of sustained oxidative stress/inflammation over an extended training camp in MMA (6 weeks), while a whole season of rugby union did not affect concentrations, indicating the intensity and repetition of impact during MMA training may be of a greater intensity than rugby union. Collectively, the data indicates that pterins, in particular neopterin and total neopterin, are sensitive markers of exercise/sport-induced trauma and reflect changes in oxidative stress and inflammation.  Overall Finding: A total of 3829 subjects from 40 studies have been compiled ( Figure 2). Seventy-seven percent of the studies reported an increase in a pterin in response to mechanical or impact-induced trauma (Figure 2A). In the studies that reported a pterin response timeline to trauma, 51% increased within 24 hours ( Figure 2B), while 46% remained elevated for greater than 48 hours ( Figure 2C).   Exercise Trauma: Nine total articles met the inclusion criteria for exercise-induced trauma (Table 1). Articles were published between 2015-2017, indicating the use of pterins in exercise-induced trauma is relatively novel. All nine articles measured total neopterin (neopterin + 7,8-dihydroneopterin), six measured neopterin and total neopterin and one measured biopterin, total biopterin (biopterin + 7,8-dihydrobiopterin + BH 4 ) and xanthopterin. Each study included elite amateur or professional athletes and measured a change in urinary pterins associated with rugby union or mixed martial arts (MMA), two sports that both endure repeated high-force impacts resulting in muscle damage and inflammation [20]. The evidence indicates a single game of rugby union or an MMA contest or training session causes an immediate increase in all pterins measured. It is noted that an increase in pterins may be specific to the impacts of the sport because neopterin concentrations do not always change in response to exercise devoid of impacts [21]. Changes in neopterin are transient following rugby union, returning to baseline within 24 h of a game while it can remain elevated following an MMA training session up to 24 h post-contest. Interestingly, only a continuous increase in neopterin and total neopterin showed evidence of sustained oxidative stress/inflammation over an extended training camp in MMA (6 weeks), while a whole season of rugby union did not affect concentrations, indicating the intensity and repetition of impact during MMA training may be of a greater intensity than rugby union. Collectively, the data indicates that pterins, in particular neopterin and total neopterin, are sensitive markers of exercise/sport-induced trauma and reflect changes in oxidative stress and inflammation. Surgery: Fifteen articles met the inclusion criteria for surgery-induced trauma (1988-2019; Table 2). Pterins were measured primarily following cardiac surgery with or without cardiopulmonary bypass, but were also measured following herniotomy, cholecystectomy, rectal or colon surgery, ypsilon graft, liver resection, hernia, vulvectomy or hysterectomy, gastrectomy or knee replacement. All 15 articles measured either neopterin or total neopterin, while only one measured BH 4 , indicating a preference for a marker of immune system activation. Furthermore, all but one study measured these pterins in either plasma or serum. All but two articles also had specific exclusion criteria to evaluate the change in biomarker concentrations to the surgical procedure, including pterins (detailed in Table 2). One study investigated the change in neopterin in neonates and infants/children, while all other studies recruited adults. Procedures that involved a form of cardiac surgery always resulted in a significant increase in neopterin within 6-hours post-surgery that remained elevated for several days. Neopterin was also higher in those patients that developed post-surgical complications such as sepsis, delirium, cardiac dysfunction or acute kidney injury following surgery. A similar neopterin response was also observed in patients undergoing hernia surgery or knee replacement surgery; however, there were limited changes in neopterin in response to all other surgeries while some patients showed a decrease in concentration post-surgery, likely because of pre-surgical complications associated with immune system activation or interactions with general anaesthesia down-regulating the immune system. Taken together, the evidence indicates neopterin may only increase in response to cardiac surgery and other limited surgical procedures and identify patients at greater risk of post-surgical complications.  Traumatic Brain Injury: Two articles met the inclusion criteria for pterin changes in response to TBI ( [44,45]; Table 3). Both articles, published in 2001 and 2002, were by the same group and measured neopterin concentrations in patients in response to an "isolated" or "severe" TBI. Patients presented with a Glasgow Coma Score at admission equal to or less than eight and associated abnormalities of the brain via computer-aided tomography. Neopterin was measured in serum and CSF. In one study, serum neopterin increased in response to the TBI and remained elevated 14 days post-surgery. Neopterin in CSF also increased and remained elevated up to 21 days post-surgery. The second study only presented CSF neopterin concentrations in relation to another biomarker, but some patients had levels > 30 ng/mL. No further studies have measured any pterin in response to TBI since 2002. The limited data available from these two studies does indicate TBI increases plasma neopterin concentrations. Multiple Injuries (Polytrauma): Sixteen total articles published between 1989 and 2017 met the inclusion criteria for multiple injury-induced trauma on changes in pterins (Table 4). All studies involved severe trauma or hip-fracture and patients were greater than 16 years of age. In severe trauma studies, patients were included based on their injury severity score (≥ 16 to a maximum of 66 in a single case-study) or APACHE (acute physiology, age, chronic health evaluation) score (severity of disease; 18.9). Exclusion criteria, where stated, included patients with an underlying disease, immunosuppressive therapy, immunodeficiences, corticosteroid treatment or in some cases, patients did not survive during the data collection period. All 16 studies measured neopterin with only one measuring total neopterin. No studies measured other pterins. Neopterin and total neopterin were measured in urine, serum, plasma and CSF. In all studies, neopterin and total neopterin significantly increased. Neopterin concentrations remained elevated 2-14 days post-trauma, which corresponded with the final measurement of a study design. Neopterin concentrations, irrespective of the bio-fluid, correlated with the severity of trauma [(ISS (injury severity score) and APACHE score] and were higher in non-surviving patients, patients suffering from organ failure as a result of trauma (one study did not see a relationship), patients with delirium and/or cognitive impairment. Neopterin also continually increases in the days following trauma and is a good predictor of mortality up to one-year post-bone fracture in the elderly. Overall, multiple trauma or bone fracture causes an increase in neopterin concentrations that are associated with clinically relevant outcomes, including death.

Discussion
The purpose of this systematic review was to critically evaluate studies that measured pterins in response to mechanical or impact-induced trauma. Only studies measuring a pterin in humans in response to mechanical or impact-induced trauma were considered and it is the first survey to date of pterins as diagnostic and prognostic biomarkers of trauma. Overall, this review provides evidence that pterins increase in response to trauma. However, different effects and changes in pterin concentration were identified when the type of trauma was considered. Collectively, pterins, particularly neopterin and total neopterin, increase after trauma, remain elevated and may be a predictor of several clinically important patient outcomes.
Review of the literature identified 40 articles that measured pterins in response to trauma, all of which measured either neopterin, total neopterin or both. 7,8-Dihydroneopterin is an antioxidant synthesized from γ-IFN-activated monocytes/macrophages during immune system activation and can be oxidized to neopterin. Neopterin's highly fluorescent properties and easy detection methods [6,61,62] make it a popular candidate to measure an inflammatory response, which may explain why every article considered in this review measured it in reaction to various forms of trauma. However, the use of neopterin and not the simultaneous measurement of 7,8-dihydroneopteirn as a marker of immune system activation has been a topic of discussion. It is suggested that the measurement of neopterin alone might only be an indicator of oxidative stress and thus, does not reflect the true level of inflammation [2,63]. Therefore, the increase in neopterin associated with mechanical or impact-induced trauma might be a reflection of elevated oxidative stress rather than cumulative inflammation. However, the simultaneous increase in neopterin and total neopterin following knee replacement surgery [43], an MMA contest [27,29,30] or rugby game [22] suggests neopterin might reflect immune system activation in response to trauma as well as oxidative stress.
While neopterin was measured in every study, biopterin, BH 4 and total biopterin were also measured in response to trauma. BH 4 is enzymatically synthesized from GTP and metabolized to dihydrobiopterin while acting as a co-factor for the amino acid hydroxylases and nitric oxide synthases. Dihydrobiopterin is salvaged to BH 4 or oxidized to biopterin. Thus, the measurement of biopterins can be indicative of oxidative stress, monoamine neurotransmitter synthesis and nitric oxide availability (nitric oxide-induced vasodilation and other functions [64]). Given the biopterins ability to indicate several acute and chronic biological conditions, and trauma is associated with oxidative stress and blood flow [65][66][67], it was surprising the biopterins have not been measured in abundance. The lack of biopterins as biomarkers may be due to the multitude of processes that produce biopterin, or use BH 4 as a co-factor. The only article that clearly states a change in biopterins noted significant increases following four rugby games that was positively associated with the number of impacts a player experienced during the game [28]. Therefore, we suggest, on limited data, that biopterins may provide a general but sensitive biomarker of trauma and should be considered in future studies. Furthermore, xanthopterin was the only other pterin measured, once again in the same study on several rugby games. Xanthopterin, a catabolite of biopterin, was also increased following a rugby game and associated with the number of impacts experienced by a player. Collectively, this evidence and other research showing isoxanthopterin is elevated in muscle degenerative diseases [68], indicates other pterins may be useful indicators of trauma.
Pterins are synthesized from all cell types and tissues. Therefore, measurement of pterins in response to mechanical or impact-induced trauma can be taken in multiple bio-fluids with similar efficacy. While pterin measurements were limited to urine and CSF in exercise and brain injury studies, respectively, pterins were measured almost selectively in plasma or serum in multi-trauma or surgical studies. Blood and CSF media do provide a reliable and direct measurement of pterins in circulation. However, urinary pterins also increase in response to various trauma and it does provide a medium that can be readily accessed for multiple measurements through catheterization or non-invasively using a collection container. Urinary pterins are also able to be measured by HPLC, RIA or ELISA and require less sample clean up prior to measurement; however, urinary pterin concentrations require normalization by creatinine, specific gravity or osmolality to account for changes in hydration. Collectively, pterins increase in all bio-fluids following mechanical or impact-induced trauma, but for longitudinal analyses that require follow-up measurements, urine may be a suitable media.
Exercise Trauma: Studies on exercise-induced trauma, typically those associated with repeated high-force impacts like rugby or MMA, have utilized biomarkers like creatine kinase [69], myoglobin [70] or C-reactive protein [71] to measure changes in muscle damage and inflammation. The results of this review provide evidence that repeated high-force impacts in rugby union or MMA always result in a transient increase in several pterins and that this increase is mostly associated with the number of total impacts experienced by a competitor. Furthermore, the studies that collected samples at multiple time-points following exercise noted that neopterin or total neopterin was sustained for up to 24 hours, indicating they can be used to monitor post-traumatic changes in immune system activation following impact-induced trauma [30]. Two studies further defined the potential of total neopterin to measure the longitudinal response to repetitive insult, which might mimic a multiple-trauma patient undergoing repeated surgical intervention and long hospital stays. During a 20-week rugby season, where players complete several training sessions (fitness and weight-lifting) and a competitive game on a weekly basis, total neopterin levels did not change [25]. However, during a six-week MMA training camp where athletes complete weight-lifting and several contact sessions/week, neopterin and total neopterin steadily increased throughout the duration [29]. When assessing exercise-induced trauma using pterins, the evidence suggests pterins should be immediately quantified to understand the acute stress, while weekly monitoring may provide important information about adaptation to repeated insults or development of over-training. Overall, the evidence indicates neopterin and total neopterin are sensitive biomarkers of impact-induced trauma in sport and exercise.
Surgery: Surgical intervention is highly invasive and initiates a cascade of inflammatory processes [72]. The results of this review indicate neopterin and/or total neopterin increase with selective surgeries. Therefore, neopterin and/or total neopterin is a useful diagnostic and prognostic marker of surgical intervention. Interestingly, the pterins were only responsive to selective surgeries, which suggests they may only be useful under specific circumstances. For example, any thoracic surgery, with or without cardiopulmonary bypass, always resulted in an increase [35], which likely reflects the level of trauma inflicted on the patient to access the heart. It would be interesting to compare neopterin levels following cardiac key hole surgery. Surgeries such as hysterectomy [39] or gastrectomy [42] did not affect neopterin concentrations, suggesting there is a much lower level of trauma involved in these particular procedures. Differentiating between the level of stress associated with each surgical procedure is difficult but the rise in neopterin is not thoracic-specific, with knee replacement surgery [43] also resulting in an increase. There is also evidence that neopterin remains elevated for several days post-surgery [37] and is higher in patients that develop sepsis [31] and become delirious [38]. Therefore, in a clinical setting, the pre to post change in pterins could provide an assessment of acute surgical stress while long-term daily monitoring of pterins might be advantageous for selective patients that develop post-surgical complications. Because neopterin and total neopterin are considered reliable and sensitive markers of oxidative stress and inflammation, clinicians should consider daily measurements until concentrations either return to "normal" values or remain steady below pre-surgical levels for several days. These data suggest neopterin can provide indicative data on the acute and chronic response to selective surgical interventions, especially where post-operative infection complications are suspected.
Traumatic Brain Injury: Traumatic brain injury is associated with inflammation [73] and oxidative stress [74]. While limited studies have measured any pterin in response to TBI, two studies which have noted a significant increase in neopterin also reported a sustained elevation for 14-21 days post-surgery [44,45]. Whether the increase in neopterin is a measure of oxidative stress or inflammation is unknown but it provides evidence that neopterin may be a sensitive marker of TBI. However, because surgery followed the TBI, the surgical intervention could have an effect on neopterin concentrations. It is also interesting that no further studies have explored the use of neopterin or any other pterin in response to TBI given the sensitivity of the two published studies. While numerous studies are exploring biomarkers of TBI [75], perhaps the inclusion of neopterin and/or total neopterin may provide some added diagnostic and prognostic benefit. Therefore, in cases of TBI that arise from sport, accidents or work-related injuries, neopterin and/or total neopterin should be measured at patient admittance and daily until values return to "normal".
Multiple Trauma (Polytrauma): Multiple trauma always results in an acute and chronic increase in neopterin and total neopterin, the latter only being measured in one study [59]. The sensitivity of neopterin in response to multiple trauma seems greatest compared to the three other categories of this review. Neopterin concentrations, whether in the urine, plasma, blood or CSF, significantly increase, but because 15 of the 16 studies only measured neopterin and not total neopterin, the full scope of neopterin's diagnostic and prognostic capability is unclear. Nonetheless, it is evident that multiple trauma [54] or a bone fracture [60] results in a sustained increase in neopterin. Most importantly, it is the association of neopterin with clinically relevant outcomes like organ failure [50], survival [49] and the severity of the trauma [53] that indicate neopterin is a strong biomarker candidate for identifying the degree of multiple trauma and the likelihood of developing post-trauma complications. Therefore, in cases where patients experience multi-trauma, the evidence suggests neopterin and/or total neopterin should be immediately assessed to quantify the extent of injury and then repeatedly measured every 12-24 hours to monitor development of other complications. Because neopterin was always greater in non-surviving patients, it may provide sensitive and reliable information to clinicians regarding an appropriate course of treatment.
Limitations: There are limitations to this systematic review: (1) the number of studies measuring a pterin following traumatic brain injury is severely limited. It is difficult to assess the effectiveness of pterins as biomarkers of TBI, specifically neopterin, in their clinical capacity to monitor the severity and progression of injury. (2) The purpose of this review was to evaluate the response of all pterins to mechanical or impact-induced trauma, yet the current literature is severely limited to neopterin studies. Because other pterins like isoxanthopterin, sepiapterin, pterin and xanthopterin have been measured in diseases like cancer [76] and Duchenne muscular dystrophy [68], and these diseases are associated with inflammation, oxidative stress and ischemia, these pterins may also provide some benefit in situations of trauma. Furthermore, the development of novel nanotechnology/biomedical engineering platforms for biomarker discovery in conditions associated with inflammation and oxidative stress [77][78][79] could further enhance the validity and utility of specialist diagnostic biomarkers such as pterins. (3) It was evident that the media used to measure a selected pterin both within and between each of the four categories was different. For example, all exercise-based studies used urine, whereas all surgical studies used plasma or serum. To understand the capacity of pterins to act as diagnostic and prognostic markers, all media should be considered for measurement of pterins.

Conclusions
This systematic review evaluated 40 articles that measured pterins in response to mechanical or impact-induced trauma. While neopterin and/or total neopterin were the most quantified pterin, the majority of studies in each of the four categories showed a transient and/or chronic increase. The evidence suggests neopterin and/or total neopterin provide a quality assessment of oxidative stress and inflammation in response to mechanical or impact-induced trauma. Further research on the response of other pterins to mechanical or impact-induced trauma is required before drawing conclusions.

Conflicts of Interest:
The authors declare no conflict of interest.