- Email: [email protected]
Biochemical analysis of the cerebrospinal fluid: evidence for catastrophic energy failure and oxidative damage preceding brain death in severe head injury: a case report
Clinical Biochemistry 38 (2005) 97 – 100
Biochemical analysis of the cerebrospinal fluid: evidence for catastrophic energy failure and oxidative damage preceding brain death in severe head injury: a case report Luciano Cristoforia, Barbara Tavazzib, Roberta Gambina, Roberto Vagnozzic, Stefano Signorettid, Angela M. Amorinie, Giovanna Fazzinae, Giuseppe Lazzarinoe,* a Department of Neurosurgery, University Hospital of Verona, Italy Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy c Department of Neurosciences, Chair of Neurosurgery, University of Rome bTor Vergata,Q Italy d Department of Neurosciences, Division of Neurosurgery, bSan CamilloQ Hospital, Rome, Italy e Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Italy b
Received 24 June 2004; received in revised form 2 September 2004; accepted 20 September 2004
Abstract Objectives: To compare biochemical and clinical parameters in a case of fatal severe traumatic brain injury (TBI) with secondary insult. Design and methods: A TBI patient was catheterized for intracranial pressure (ICP) monitoring and cerebrospinal fluid (CSF) analysis of ascorbate, malondialdehyde, oxypurines, and nucleosides. Results: Oxidative brain damage preceded ATP catabolite increment in the CSF even with ICP below 20 mm Hg. Sustained oxidative stress caused irreversible energy state derangement followed by a refractory ICP rise. Massive oxypurine and nucleoside release was recorded 36 h before brain death. Conclusions: Molecular events, detected by biochemical CSF analysis and preceding modification of clinical parameters in severe TBI with secondary insult, are discussed. D 2004 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Cerebrospinal fluid; Energy metabolism; High-performance liquid chromatography; Oxidative stress; Traumatic brain injury
Introduction Experimental traumatic brain injury (TBI) causes release of excitatory amino acids [1], changes to cell ionic permeability [2], early occurrence of ROS-mediated oxidative stress, and is followed by profound impairment of cerebral energy metabolism [3,4]. The amount of oxidative damage, energy imbalance, and the eventual metabolic recovery are closely related to trauma severity [5]. In
* Corresponding author. Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Fax: +39 095337036. E-mail address: [email protected] (G. Lazzarino).
humans, these occurrences have not yet been clearly established, requiring research into what molecular events correlate with clinical evolution and with TBI patient outcome. By assaying cerebrospinal fluid (CSF) samples, we demonstrated that severe TBI patients are subjected to oxidative stress and energy metabolism alterations already during the time interval between TBI and hospital admission [6]. Similar results were obtained recently in infants and children suffering from TBI [7,8]. In this case report of a severe TBI patient with secondary insult, we present the time-course changes of biochemical parameters representative of ATP catabolism (oxypurines and nucleosides) and of ROS-mediated tissue damage (ascorbate and malondialdehyde) in CSF samples. On the basis of the biochemical and clinical patient evolution, we
0009-9120/$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2004.09.013
98
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
hypothesize a conceivable sequence of molecular events (dramatic oxidative stress followed by catastrophic energy failure) indicating irreversible loss of neurological functions or brain death.
Methods This study was approved by the Azienda Ospedaliera Ethical Committee in Verona, Italy. An otherwise healthy 61-year-old man was found unresponsive with a left parietooccipital scalp wound presumably after falling down his staircase. The accident was not due to alcohol abuse. He was intubated on site and admitted to the hospital, approximately 6 h post-trauma. After resuscitation, the patient remained unconscious, showing bilateral extension posturing to painful stimuli as well as a nonreactive left pupil with Glasgow Coma Scale (GCS) = 4. A computerized tomography (CT) scan documented a large acute left frontotemporal subdural hematoma, associated with a temporal brain contusion. Basal cisterns and the third ventricle were compressed with a 10-mm shift of midline structures. These radiological features prompted the immediate surgical removal of the hematoma, which was removed with temporal laceration debridement. After operation, a right ventricular catheter was positioned for intracranial pressure (ICP) monitoring and the patient was admitted to the Neurosurgical Intensive Care Unit. Mechanical ventilation and mild sedation allowed ICP maintenance below 20 mm Hg and cerebral perfusion pressure (CPP) at 70 mm Hg. Ventricular drainage, mannitol, vasopressors, and mild episodic hyperventilation were also used to control ICP. During the next 2 days, the patient clinical conditions were unchanged, and at 48 h postsurgery, a CT scan confirmed that hematoma was removed. However, 72 h postinjury, anisocoria worsened, the patient became poliuric with less than satisfactory ICP control, and a CT scan showed the presence of swelling in the left hemisphere. At 96 h after trauma, ICP begun to monotonously rise with a refractory course. Five days postinjury, the pupils were midriatic and fixed, brainstem reflexes were absent, and the EEG showed only low amplitude activity. The next day, the EEG was silent and cerebral death was documented. Sampling and analysis of CSF CSF samples were collected immediately after catheter insertion in the operating room (zero time = 8 h postinjury) and after 12, 24, 48, 72, 96, and 101 h. To remove blood contamination, the CSF was centrifuged at 1860 g for 10 min at 48C; the clear supernatant was then deproteinized (800 Al sample) by adding 2.30 ml of HPLC-grade acetonitrile. After centrifugation at 20,690 g for 10 min at 48C, deproteinized CSF was extracted with 3 ml of HPLC-grade chloroform, centrifuged again
at 20,690 g for 5 min at 48C, and the upper aqueous phase was saved. After two additional chloroform extractions, each aqueous phase was filtered through a 0.45 Am Millipore HV filter and analyzed in triplicate by HPLC using a Kromasil 250 4.6 mm, 5-Am particle size column (Eka Chemicals AB, Bohus, Sweden), along with a SpectraSystem P2000 pump, a SpectraSystem UV6000LP diode array detector (ThermoFinnigan Italia, Rodano, Milano, Italy) with a 5-cm light-path flow cell that was set up with a wavelength between 200 and 300 nm. Malondialdehyde (MDA), ascorbic acid, oxypurines (hypoxanthine, xanthine, uric acid), and nucleosides (inosine, adenosine) were determined from a 200-Al sample by an ion-pairing HPLC method described in detail elsewhere [6,9,10].
Results Fig. 1 reveals ICP variations (Panel A) and CSF metabolite changes (Panel B), which occurred in the time interval between catheter insertion and 24 h before cerebral death. Zero time values of all biochemical parameters were remarkably different from corresponding values previously recorded in the CSF of noncerebral patients [2]. In particular, ascorbic acid concentration in this patient was 42.28 Amol/l CSF at time zero compared to 151.66 Amol/l CSF in noncerebral patients [2]. It is worthwhile recalling that plasma ascorbate at this time point was in the normal range (39.15 Amol/l plasma). Twenty-four hours after catheter insertion, MDA tripled and the ascorbate level halved with respect to corresponding zero time values; 72 h after the first CSF sampling, MDA in the CSF was 7-fold higher (2.62 Amol/l CSF) than the zero time value (0.35 Amol/l CSF) and ascorbate was further depleted to 12.51 Amol/l CSF. In the last CSF sample, cerebral oxidative stress exacerbation provoked a steady MDA increase to a maximum of 3.39 Amol/l CSF and continuous dramatic ascorbate decrease to a minimum of 6.48 Amol/l CSF. ATP catabolite values modestly increased 12 and 24 h after catheter insertion while they tripled 48 h after catheter insertion. With respect to corresponding zero time values, a 4- to 6-fold oxypurine and nucleoside increase was registered 72 h after catheter insertion, and 36 h before brain death, a further massive release was observed. CSF concentrations of these compounds increased by 5.8-fold (uric acid) to 14-fold (hypoxanthine) in comparison with corresponding zero time values. It is worth noting that in all HPLC runs of CSF samples, compounds deriving from nucleic acid breakdown (adenine, guanine, guanosine, cytosine, cytidine, etc.) were always undetectable (data not shown). Only after these profound metabolic changes had occurred (88 h after catheter insertion) did ICP rise to the point where it was pharmacologically uncontrollable.
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
99
Fig. 1. Time course changes of ICP (panel A) and CSF concentrations of biochemical markers of oxidative stress (MDA and ascorbate) and ATP catabolism (hypoxanthine, xanthine, uric acid, inosine and adenosine) (panel B) in a patient with severe head injury associated with secondary insult. At each time point, different compounds were assayed in triplicate by HPLC on 200 Al of acetonitrile-deproteinized CSF samples.
The last ICP recording before cerebral death was 55 mm Hg.
Discussion Data referring to the present clinical case suggest a temporal sequence of molecular events preceding changes of the clinical status (ICP) and leading to cerebral death. On the basis of the patient clinical report and CSF HPLC analysis, it might be hypothesized the following bmolecular time courseQ: (i) the patient suffered from
severe TBI, a primary insult that triggered ROS overproduction, coupled with secondary insult (i.e., hypoxia), which further aggravated cerebral oxidative stress. This was documented in the CSF by both the abnormally low level of ascorbate and the high MDA concentration at time zero; the sustained oxidative stress caused a progression of ascorbate depletion and MDA increase. At 24 h postinjury, when ascorbate halved and MDA tripled with respect to corresponding zero time values, ATP catabolites modestly increased and ICP was still below 20 mm Hg; (ii) further ROS generation probably caused extensive cell damage, jeopardizing mitochondrial integrity and function, which
100
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
limited the ATP supply and provoked an imbalance between energy production and consumption. This supposition was supported by the late oxypurine and nucleoside increase of the ATP catabolism major products recorded in the fourth CSF sampling, when ICP was still pharmacologically controlled, and which occurred with no increase of other compounds deriving from nucleic acid breakdown; (iii) severity of the cerebral energy state modification was even more evident in the next CSF samples (72 h after catheter insertion) and was probably one of the major factors leading to cytotoxic edema with modification of the clinical status, in particular causing an uncontrollable ICP rise between 88 and 92 h after catheter insertion; (iv) 36 h before brain death was ascertained, that is, 101 h after ventricular catheter insertion, catastrophic energy failure took place, with massive release of oxypurines and nucleosides in the CSF. At this time point, 222.92 Amol/ l CSF of oxypurines, 39.78 Amol/l CSF of nucleosides, minimal ascorbate (6.48 Amol/l CSF), and maximal MDA (3.39 Amol/l CSF) levels were recorded, and a concomitant refractory ICP (at 55 mm Hg) was observed. Hence, the biochemical and clinical evolutions of this case report allow us to hypothesize that if ROS-mediated cell damage did not compromise mitochondrial function, energy metabolism was not irreversibly altered and ICP responded to classical pharmacological treatments, remaining below 20 mm Hg. Our results, besides indicating a relationship between oxidative stress and cerebral ATP homeostasis based on the different time at which they occurred (ROS-mediated damages preceding increase of ATP catabolites), suggest that metabolic changes appear several hours prior to modification of clinical signs. This finding might open a new perspective for the biochemical monitoring of CSF as a potential predictor of clinical evolution, at least in TBI patients. CSF analysis might represent a crucial tool for clinicians to drive the pharmacological treatment of severely head-injured patients. Since the present findings are not conclusive because they are limited to a single case report, a multicentric study, to substantiate these results and to correlate biochemical markers of oxidative stress and energy metabolism with the outcome of TBI patients, is currently in progress.
Acknowledgments This work was made possible by Research Funds of Rome bTor VergataQ University (R. Vagnozzi) and Catania University (G. Lazzarino).
References [1] Stover JF, Schoning B, Beyer TF, Woiciechowsky C, Unterberg AW. Temporal profile of cerebrospinal fluid glutamate, interleukin-6, and tumor necrosis factor-alpha in relation to brain edema and contusion following controlled cortical impact injury in rats. Neurosci Lett 2000;288:25 – 8. [2] Obrenovitch TP, Urenjak J. Is high extracellular glutamate the key to excitotoxicity in traumatic brain injury? J Neurotrauma 1997;14: 677 – 98. [3] Smith SL, Andrus PK, Zhang JR, Hall ED. Direct measurement of hydroxyl radicals, lipid peroxidation, and blood–brain barrier disruption following unilateral cortical impact head injury in the rat. J Neurotrauma 1994;11:393 – 404. [4] Vagnozzi R, Marmarou A, Tavazzi B, et al. Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury. J Neurotrauma 1999;16:903 – 13. [5] Signoretti S, Marmarou A, Tavazzi B, Lazzarino G, Beaumont A, Vagnozzi R. N-acetyl-aspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury. J Neurotrauma 2001;18:977 – 94. [6] Cristofori L, Tavazzi B, Gambin R, et al. Early onset of lipid peroxidation after human traumatic brain injury: a fatal limitation for the free radical scavenger pharmacological therapy? J Investig Med 2001;49:450 – 8. [7] Bayir H, Kagan VE, Tyurina YY, et al. Assessment of antioxidant reserves and oxidative stress in cerebrospinal fluid after severe traumatic brain injury in infants and children. Pediatr Res 2002;51: 571 – 8. [8] Varma S, Janesko KL, Wisniewski SR, et al. F2-isoprostane and neuron-specific enolase in cerebrospinal fluid after severe traumatic brain injury in infants and children. J Neurotrauma 2003;20:781 – 6. [9] Lazzarino G, Di Pierro D, Tavazzi B, Cerroni L, Giardina G. Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography. Anal Biochem 1991;197: 191 – 6. [10] Di Pierro D, Tavazzi B, Perno CF, et al. An ion-pairing highperformance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts. Anal Biochem 1995;231:407 – 12.
Biochemical analysis of the cerebrospinal fluid: evidence for catastrophic energy failure and oxidative damage preceding brain death in severe head injury: a case report Luciano Cristoforia, Barbara Tavazzib, Roberta Gambina, Roberto Vagnozzic, Stefano Signorettid, Angela M. Amorinie, Giovanna Fazzinae, Giuseppe Lazzarinoe,* a Department of Neurosurgery, University Hospital of Verona, Italy Institute of Biochemistry and Clinical Biochemistry, Catholic University of Rome, Italy c Department of Neurosciences, Chair of Neurosurgery, University of Rome bTor Vergata,Q Italy d Department of Neurosciences, Division of Neurosurgery, bSan CamilloQ Hospital, Rome, Italy e Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Italy b
Received 24 June 2004; received in revised form 2 September 2004; accepted 20 September 2004
Abstract Objectives: To compare biochemical and clinical parameters in a case of fatal severe traumatic brain injury (TBI) with secondary insult. Design and methods: A TBI patient was catheterized for intracranial pressure (ICP) monitoring and cerebrospinal fluid (CSF) analysis of ascorbate, malondialdehyde, oxypurines, and nucleosides. Results: Oxidative brain damage preceded ATP catabolite increment in the CSF even with ICP below 20 mm Hg. Sustained oxidative stress caused irreversible energy state derangement followed by a refractory ICP rise. Massive oxypurine and nucleoside release was recorded 36 h before brain death. Conclusions: Molecular events, detected by biochemical CSF analysis and preceding modification of clinical parameters in severe TBI with secondary insult, are discussed. D 2004 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Cerebrospinal fluid; Energy metabolism; High-performance liquid chromatography; Oxidative stress; Traumatic brain injury
Introduction Experimental traumatic brain injury (TBI) causes release of excitatory amino acids [1], changes to cell ionic permeability [2], early occurrence of ROS-mediated oxidative stress, and is followed by profound impairment of cerebral energy metabolism [3,4]. The amount of oxidative damage, energy imbalance, and the eventual metabolic recovery are closely related to trauma severity [5]. In
* Corresponding author. Department of Chemical Sciences, Laboratory of Biochemistry, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Fax: +39 095337036. E-mail address: [email protected] (G. Lazzarino).
humans, these occurrences have not yet been clearly established, requiring research into what molecular events correlate with clinical evolution and with TBI patient outcome. By assaying cerebrospinal fluid (CSF) samples, we demonstrated that severe TBI patients are subjected to oxidative stress and energy metabolism alterations already during the time interval between TBI and hospital admission [6]. Similar results were obtained recently in infants and children suffering from TBI [7,8]. In this case report of a severe TBI patient with secondary insult, we present the time-course changes of biochemical parameters representative of ATP catabolism (oxypurines and nucleosides) and of ROS-mediated tissue damage (ascorbate and malondialdehyde) in CSF samples. On the basis of the biochemical and clinical patient evolution, we
0009-9120/$ - see front matter D 2004 The Canadian Society of Clinical Chemists. All rights reserved. doi:10.1016/j.clinbiochem.2004.09.013
98
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
hypothesize a conceivable sequence of molecular events (dramatic oxidative stress followed by catastrophic energy failure) indicating irreversible loss of neurological functions or brain death.
Methods This study was approved by the Azienda Ospedaliera Ethical Committee in Verona, Italy. An otherwise healthy 61-year-old man was found unresponsive with a left parietooccipital scalp wound presumably after falling down his staircase. The accident was not due to alcohol abuse. He was intubated on site and admitted to the hospital, approximately 6 h post-trauma. After resuscitation, the patient remained unconscious, showing bilateral extension posturing to painful stimuli as well as a nonreactive left pupil with Glasgow Coma Scale (GCS) = 4. A computerized tomography (CT) scan documented a large acute left frontotemporal subdural hematoma, associated with a temporal brain contusion. Basal cisterns and the third ventricle were compressed with a 10-mm shift of midline structures. These radiological features prompted the immediate surgical removal of the hematoma, which was removed with temporal laceration debridement. After operation, a right ventricular catheter was positioned for intracranial pressure (ICP) monitoring and the patient was admitted to the Neurosurgical Intensive Care Unit. Mechanical ventilation and mild sedation allowed ICP maintenance below 20 mm Hg and cerebral perfusion pressure (CPP) at 70 mm Hg. Ventricular drainage, mannitol, vasopressors, and mild episodic hyperventilation were also used to control ICP. During the next 2 days, the patient clinical conditions were unchanged, and at 48 h postsurgery, a CT scan confirmed that hematoma was removed. However, 72 h postinjury, anisocoria worsened, the patient became poliuric with less than satisfactory ICP control, and a CT scan showed the presence of swelling in the left hemisphere. At 96 h after trauma, ICP begun to monotonously rise with a refractory course. Five days postinjury, the pupils were midriatic and fixed, brainstem reflexes were absent, and the EEG showed only low amplitude activity. The next day, the EEG was silent and cerebral death was documented. Sampling and analysis of CSF CSF samples were collected immediately after catheter insertion in the operating room (zero time = 8 h postinjury) and after 12, 24, 48, 72, 96, and 101 h. To remove blood contamination, the CSF was centrifuged at 1860 g for 10 min at 48C; the clear supernatant was then deproteinized (800 Al sample) by adding 2.30 ml of HPLC-grade acetonitrile. After centrifugation at 20,690 g for 10 min at 48C, deproteinized CSF was extracted with 3 ml of HPLC-grade chloroform, centrifuged again
at 20,690 g for 5 min at 48C, and the upper aqueous phase was saved. After two additional chloroform extractions, each aqueous phase was filtered through a 0.45 Am Millipore HV filter and analyzed in triplicate by HPLC using a Kromasil 250 4.6 mm, 5-Am particle size column (Eka Chemicals AB, Bohus, Sweden), along with a SpectraSystem P2000 pump, a SpectraSystem UV6000LP diode array detector (ThermoFinnigan Italia, Rodano, Milano, Italy) with a 5-cm light-path flow cell that was set up with a wavelength between 200 and 300 nm. Malondialdehyde (MDA), ascorbic acid, oxypurines (hypoxanthine, xanthine, uric acid), and nucleosides (inosine, adenosine) were determined from a 200-Al sample by an ion-pairing HPLC method described in detail elsewhere [6,9,10].
Results Fig. 1 reveals ICP variations (Panel A) and CSF metabolite changes (Panel B), which occurred in the time interval between catheter insertion and 24 h before cerebral death. Zero time values of all biochemical parameters were remarkably different from corresponding values previously recorded in the CSF of noncerebral patients [2]. In particular, ascorbic acid concentration in this patient was 42.28 Amol/l CSF at time zero compared to 151.66 Amol/l CSF in noncerebral patients [2]. It is worthwhile recalling that plasma ascorbate at this time point was in the normal range (39.15 Amol/l plasma). Twenty-four hours after catheter insertion, MDA tripled and the ascorbate level halved with respect to corresponding zero time values; 72 h after the first CSF sampling, MDA in the CSF was 7-fold higher (2.62 Amol/l CSF) than the zero time value (0.35 Amol/l CSF) and ascorbate was further depleted to 12.51 Amol/l CSF. In the last CSF sample, cerebral oxidative stress exacerbation provoked a steady MDA increase to a maximum of 3.39 Amol/l CSF and continuous dramatic ascorbate decrease to a minimum of 6.48 Amol/l CSF. ATP catabolite values modestly increased 12 and 24 h after catheter insertion while they tripled 48 h after catheter insertion. With respect to corresponding zero time values, a 4- to 6-fold oxypurine and nucleoside increase was registered 72 h after catheter insertion, and 36 h before brain death, a further massive release was observed. CSF concentrations of these compounds increased by 5.8-fold (uric acid) to 14-fold (hypoxanthine) in comparison with corresponding zero time values. It is worth noting that in all HPLC runs of CSF samples, compounds deriving from nucleic acid breakdown (adenine, guanine, guanosine, cytosine, cytidine, etc.) were always undetectable (data not shown). Only after these profound metabolic changes had occurred (88 h after catheter insertion) did ICP rise to the point where it was pharmacologically uncontrollable.
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
99
Fig. 1. Time course changes of ICP (panel A) and CSF concentrations of biochemical markers of oxidative stress (MDA and ascorbate) and ATP catabolism (hypoxanthine, xanthine, uric acid, inosine and adenosine) (panel B) in a patient with severe head injury associated with secondary insult. At each time point, different compounds were assayed in triplicate by HPLC on 200 Al of acetonitrile-deproteinized CSF samples.
The last ICP recording before cerebral death was 55 mm Hg.
Discussion Data referring to the present clinical case suggest a temporal sequence of molecular events preceding changes of the clinical status (ICP) and leading to cerebral death. On the basis of the patient clinical report and CSF HPLC analysis, it might be hypothesized the following bmolecular time courseQ: (i) the patient suffered from
severe TBI, a primary insult that triggered ROS overproduction, coupled with secondary insult (i.e., hypoxia), which further aggravated cerebral oxidative stress. This was documented in the CSF by both the abnormally low level of ascorbate and the high MDA concentration at time zero; the sustained oxidative stress caused a progression of ascorbate depletion and MDA increase. At 24 h postinjury, when ascorbate halved and MDA tripled with respect to corresponding zero time values, ATP catabolites modestly increased and ICP was still below 20 mm Hg; (ii) further ROS generation probably caused extensive cell damage, jeopardizing mitochondrial integrity and function, which
100
L. Cristofori et al. / Clinical Biochemistry 38 (2005) 97–100
limited the ATP supply and provoked an imbalance between energy production and consumption. This supposition was supported by the late oxypurine and nucleoside increase of the ATP catabolism major products recorded in the fourth CSF sampling, when ICP was still pharmacologically controlled, and which occurred with no increase of other compounds deriving from nucleic acid breakdown; (iii) severity of the cerebral energy state modification was even more evident in the next CSF samples (72 h after catheter insertion) and was probably one of the major factors leading to cytotoxic edema with modification of the clinical status, in particular causing an uncontrollable ICP rise between 88 and 92 h after catheter insertion; (iv) 36 h before brain death was ascertained, that is, 101 h after ventricular catheter insertion, catastrophic energy failure took place, with massive release of oxypurines and nucleosides in the CSF. At this time point, 222.92 Amol/ l CSF of oxypurines, 39.78 Amol/l CSF of nucleosides, minimal ascorbate (6.48 Amol/l CSF), and maximal MDA (3.39 Amol/l CSF) levels were recorded, and a concomitant refractory ICP (at 55 mm Hg) was observed. Hence, the biochemical and clinical evolutions of this case report allow us to hypothesize that if ROS-mediated cell damage did not compromise mitochondrial function, energy metabolism was not irreversibly altered and ICP responded to classical pharmacological treatments, remaining below 20 mm Hg. Our results, besides indicating a relationship between oxidative stress and cerebral ATP homeostasis based on the different time at which they occurred (ROS-mediated damages preceding increase of ATP catabolites), suggest that metabolic changes appear several hours prior to modification of clinical signs. This finding might open a new perspective for the biochemical monitoring of CSF as a potential predictor of clinical evolution, at least in TBI patients. CSF analysis might represent a crucial tool for clinicians to drive the pharmacological treatment of severely head-injured patients. Since the present findings are not conclusive because they are limited to a single case report, a multicentric study, to substantiate these results and to correlate biochemical markers of oxidative stress and energy metabolism with the outcome of TBI patients, is currently in progress.
Acknowledgments This work was made possible by Research Funds of Rome bTor VergataQ University (R. Vagnozzi) and Catania University (G. Lazzarino).
References [1] Stover JF, Schoning B, Beyer TF, Woiciechowsky C, Unterberg AW. Temporal profile of cerebrospinal fluid glutamate, interleukin-6, and tumor necrosis factor-alpha in relation to brain edema and contusion following controlled cortical impact injury in rats. Neurosci Lett 2000;288:25 – 8. [2] Obrenovitch TP, Urenjak J. Is high extracellular glutamate the key to excitotoxicity in traumatic brain injury? J Neurotrauma 1997;14: 677 – 98. [3] Smith SL, Andrus PK, Zhang JR, Hall ED. Direct measurement of hydroxyl radicals, lipid peroxidation, and blood–brain barrier disruption following unilateral cortical impact head injury in the rat. J Neurotrauma 1994;11:393 – 404. [4] Vagnozzi R, Marmarou A, Tavazzi B, et al. Changes of cerebral energy metabolism and lipid peroxidation in rats leading to mitochondrial dysfunction after diffuse brain injury. J Neurotrauma 1999;16:903 – 13. [5] Signoretti S, Marmarou A, Tavazzi B, Lazzarino G, Beaumont A, Vagnozzi R. N-acetyl-aspartate reduction as a measure of injury severity and mitochondrial dysfunction following diffuse traumatic brain injury. J Neurotrauma 2001;18:977 – 94. [6] Cristofori L, Tavazzi B, Gambin R, et al. Early onset of lipid peroxidation after human traumatic brain injury: a fatal limitation for the free radical scavenger pharmacological therapy? J Investig Med 2001;49:450 – 8. [7] Bayir H, Kagan VE, Tyurina YY, et al. Assessment of antioxidant reserves and oxidative stress in cerebrospinal fluid after severe traumatic brain injury in infants and children. Pediatr Res 2002;51: 571 – 8. [8] Varma S, Janesko KL, Wisniewski SR, et al. F2-isoprostane and neuron-specific enolase in cerebrospinal fluid after severe traumatic brain injury in infants and children. J Neurotrauma 2003;20:781 – 6. [9] Lazzarino G, Di Pierro D, Tavazzi B, Cerroni L, Giardina G. Simultaneous separation of malondialdehyde, ascorbic acid, and adenine nucleotide derivatives from biological samples by ion-pairing high-performance liquid chromatography. Anal Biochem 1991;197: 191 – 6. [10] Di Pierro D, Tavazzi B, Perno CF, et al. An ion-pairing highperformance liquid chromatographic method for the direct simultaneous determination of nucleotides, deoxynucleotides, nicotinic coenzymes, oxypurines, nucleosides, and bases in perchloric acid cell extracts. Anal Biochem 1995;231:407 – 12.