TECHNICAL NOTE Eloy Girela, 1 Ph.D.; Enrique Villanueva, 2 Ph.D.; Pilar Irigoyen, 2 Ph.D.; Victorino Girela, 2 M.D.; Claudio Hernµndez-Cueto, 2 Ph.D.; and JosØ M. Peinado, 3 Ph.D. Free Amino Acid Concentrations in Vitreous Humor and Cerebrospinal Fluid in Relation to the Cause of Death and Postmortem Interval ABSTRACT: We studied free amino acids in vitreous humor and cerebrospinal fluid from 58 cadavers in the course of routine medicolegal autopsies in the city of Granada. The main objective was to establish whether free amino acids contents in these fluids were related with the cause of death, postmortem interval, and severity of the classic signs of asphyxia. The amino acids (aspartic acid, glutamic acid, serine, glutamine, gly- cine ⁄ threonine ⁄ histidine, citruline, arginine, alanine, taurine, GABA, tirosine, valine, methionine, isoleucine, phenylalanine ⁄ tryptophan, leucine, and lysine) were quantified by high performance liquid chromatography. There were no statistically significant differences in amino acids concentrations in vitreous humor when the different causes of death were considered. Our results did not show any statistically significant relationship when asphyx- ial score was plotted against the vitreous content of each amino acid. A statistically significant increase with postmortem interval was observed in vit- reous taurine (r = 0.3191, p = 0.01461), glutamate (r = 0.4323, p = 0.0007) and particularly in aspartate (r = 0.4508, p = 0.0003). KEYWORDS: forensic science, vitreous humor, cerebrospinal fluid, amino acids, postmortem interval, postmortem chemistry, cause of death Numerous methods have been used to estimate the time after death, particularly for short postmortem intervals (PMI). These methods involve most of the biochemical compounds in blood, vit- reous humor, cerebrospinal fluid (CSF), and to a lesser extent in pericardial fluid. Nonetheless, many of these methods show a broad margin of error even under controlled conditions, and most of them have considered only the first day after death (1). One exception to these criticisms is potassium concentration in the vitreous humor, which has been investigated in detail by differ- ent groups (2–5). Despite its large margin of error within the first 24 h after death, it is generally accepted as the best biochemical test available for longer PMI. In 1963, Sturner (2) was the first to report a formula to calculate PMI from vitreous potassium concen- tration; this approach was reformulated later using modern statisti- cal methods (3–5), and improved by the use of potassium as the independent variable, instead of the dependent variable (5,6). In recent dates, new methods developed in radiology, like H magnetic resonance spectroscopy (H-MRS), have been applied for the identification of metabolites emerging during decomposition of brain tissue, both in sheep brain models by Scheurer et al. (7) and in porcine brain model by Banaschak et al. (8). Their results show more precision in estimation of longer PMI. Few publications deal with the possible role of amino acids in postmortem chemistry. Purcher and Burd (9) and Schourup (10) described a sharp rise in free amino acid nitrogen level both in blood and in CSF, probably as a result of enzymatic hydrolysis of proteins. Schleyer (11) investigated samples obtained up to 80 h postmortem, and found results in accordance with those published by Schourup. These findings suggested that the concentrations of amino acids could be used to determine PMI during the first 20 h after death: values of amino acid nitrogen not over 14 mg% proba- bly correspond to PMI of <10 h. Vass et al. (12) studied aminoac- ids, neurotransmitters, and other decompositional products in various organs in relation to PMI, revealing distinct patterns useful for determining PMI up to a range of c. 3 weeks. The most signifi- cant biomarkers for the establishment of PMI in all tissues studied were GABA, proline, methionine, and oxalic acid. Erdei and Vass (13) and Durham et al. (14) reported the pres- ence of free amino acids in the vitreous humor. Later, Patrick and Logan (15) established, in sudden infant death syndrome (SIDS), that the concentrations of all vitreous amino acids rose as PMI increased. However, samples needed to be obtained within the first 24 h after death to give reliable results. These results showed no indication of a specific aminoacidopathy as a factor in SIDS. With the exception of the publication by Patrick and Logan (15) and Vass et al. (12) most analysis of amino acid have been per- formed with old methods, such as amino acid nitrogen analysis with the Folin naphthoquinone method (11) or paper chromatogra- phy (13). In the present study, we tried to correlate free amino acid concentrations in CSF and vitreous humor with the PMI, and also to investigate a possible relationship with the cause of death, by the use of modern high performance liquid chromatography (HPLC). Materials and Methods Material Samples were taken from 58 cadavers (18 females and 40 males aged 13–97 years) in the course of routine medicolegal autopsies at 1 Department of Legal Medicine, Faculty of Medicine, University of Cór- doba, 14004 Córdoba, Spain. 2 Department of Legal Medicine, Faculty of Medicine, University of Gra- nada, 18071 Granada, Spain. 3 Institute of Neuroscience, Department of Biochemistry, Faculty of Medi- cine, University of Granada, 18071 Granada, Spain. Received 17 Feb. 2007; and in revised form 11 Oct. 2007; accepted 14 Oct. 2007. J Forensic Sci, May 2008, Vol. 53, No. 3 doi: 10.1111/j.1556-4029.2008.00726.x Available online at: www.blackwell-synergy.com 730 Ó 2008 American Academy of Forensic Sciences