Microbial production of hydroxy and oxo fatty acids by several microorganisms as a model of adipocere formation

Some varieties of aerobic or anaerobic microorganisms from the human stool and adipocere were separated and identified. These separated microorganisms together with other authentic ones produced 10-hydroxystearic acid from oleic acid. Some bacteria could convert oleic acid to 10-oxostearic acid as well as 10-hydroxystearic acid. These findings indicate that the microbial enzyme(s) catalyzes the hydration of oleic acid and probably the oxidation of this hydrated product. Aerobic bacteria as well as anaerobic microorganisms were found to be involved in the formation of adipocere.     

同时裂解甲基化气相色谱法快速鉴别人体脂肪及动植物油脂

采用同时裂解甲基化气相色谱法（SPM－GC），首次分析人体脂肪，并且观察人体脂肪、猪、鸡、牛、羊脂和豆油中7种脂肪酸的组份含量变化。根据7种主要脂肪酸（肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸和亚麻酸）甲酯的百分含量可有效地进行鉴别。结果表明：通过SPM－GC法分析油脂，可将传统的油脂酯化法时间由2个多小时缩短到1分钟左右。数据C．V．％＜4％，最小检测量为1．Oμg。最佳比例四甲基氢氧化按（TMAH）甲醇液（TMAH：甲醇＝1：10，V／V），可消除油脂中多不饱和脂肪酸的异构化和降解。     

Evidence of adipocere in a burial pit from the foot and mouth epidemic of 1967 using gas chromatography-mass spectrometry

Gas-chromatography-mass spectrometry was used to characterise the fatty acids from soils and associated tissues excavated from a 1967 Foot and Mouth burial pit. Subcutaneous fats were mainly comprised of 55-75% palmitic acid, 17-22% stearic acid and 3-16% oleic acid as well as 5-7% myristic acid. The distribution of fatty acids confirmed that the tissues were decayed to adipocere. The loss of oleic acid to <3% in two of the decayed fats suggested advanced stages of adipocere. However, adipocere formation was limited in a third tissue sample recovered from greater depth. Inductively coupled plasma atomic emission spectrometry of the pore waters revealed a decrease in Ca concentration and concurrent increase in Na concentrations this suggested that insoluble calcium salt had formed through displacement of sodium. The use of fatty acid profiles from soils and soil interstitial pore waters provide complementary evidence of adipocere formation in foot and mouth burial pits.     

裂解器金属衬管对油脂定量分析诸因素的影响

目的在裂解甲基化油脂分析中,考察并讨论了裂解器金属衬管对油脂定量分析的影响因素。方法采用气相色谱仪,FID检测器,FFAP毛细柱,热丝型裂解器及居里点裂解器,对大豆色拉油的五种主要组分:软脂酸,硬脂酸,油酸,亚油酸,亚麻酸进行了定量分析。结果(1)当在金属衬管内裂解分析时,裂解产物往往随极性的增大而吸附性增强;(2)在有酸,碱媒介裂解分析时,衬管的金属材质会对活性组分起到降解和催化转化作用;(3)这些因素的存在,会严重影响测定结果的准确性。结论采用金属衬管时,亚麻酸(n=5)RSD(%)高达14.07,而采用石英衬管时,RSD(%)则降至1.92。     

Free Fatty Acids Composition in Adipocere of the Kwäday Dän Ts’ìnchí Ancient Remains Found in a Glacier*

Abstract: Adipocere is a postmortem decomposition product consisting of mostly a mixture of free fatty acids (FFAs) that are formed because of the hydrolysis of triglycerides in adipose tissues. This article describes a simple and robust method for the extraction, identification, and quantification of FFA commonly found in adipocere using gas chromatography–mass spectrometry (GC/MS). This method was applied to analyze tissues from Kwäday Dän Ts’ìnchí, ancient remains discovered in a retreating glacier in the Tatshenshini‐Alsek Park, British Columbia, Canada in August 1999. The lyophilized tissues were grinded and extracted with hexane. The trimethylsilyl fatty acid derivatives were analyzed by GC/MS, and the relative abundances of myristic acid, palmitic acid, oleic acid, and stearic acid were determined. Milligram per gram levels of saturated fatty acids were found in the tissues of the ancient remains, while the levels of unsaturated fatty acids, such as palmitoleic acid, were found to be negligible. The results provided further evidence of the existence of adipocere found during forensic examination of the Kwäday Dän Ts’ìnchí ancient remains.     

Proton nuclear magnetic resonance spectroscopy (NMR) methods for determining the purity of reference drug standards and illicit forensic drug seizures

A rapid, sensitive, accurate, precise, reproducible, and versatile method for determining the purity of reference drug standards and the routine analysis of illicit drugs and adulterants using proton (1H) Nuclear Magnetic Resonance (NMR) Spectroscopy is presented. The methodology uses a weighed sample dissolved in a deuterated solvent or solvent mixture containing a high purity internal standard. The NMR experiment employs 8 scans using a 45 second delay and 90 degrees pulse. In the determination of purity of reference standards, the number of quantitative determinations available is equal to the number of peak groups that are baseline resolved. The relative standard deviation (RSD) of these signals is usually < 1% for pure standards, and the results agree well with other purity determining methods. This method can also aid in the determination of correct molecular weight for standards containing an unknown number of waters of hydration or an unknown number of acids per drug in salts. Because the molar response for the hydrogen nucleus is 1 for all compounds, and since no separation media are used, only one linearity study is required to test a probe. In the presented study, the linearity of the NMR probe was determined using methamphetamine HCl dissolved in deuterium oxide (D2O) with maleic acid as the internal standard (5 mg) for a range of concentrations from 0.033 to 69.18 mg/ml with a resulting correlation coefficient of >0.9999 for all 6 methamphetamine peak groups. The spectra of complex illicit heroin, methamphetamine, MDMA, and cocaine samples are presented, as well as an extensive list of compounds, their solubilities and the solvent(s) and internal standard used.     

Monitoring of Levamisole Concentration in Serum of Traffic Participants after Cocaine Consumption

This study highlights the problem of levamisole‐adulterated cocaine in context of active traffic participation. For the purposes of levamisole concentration monitoring in human serum, an analytical method based on LC‐MS/MS and solid‐phase extraction was applied. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm column and a mobile phase consisting of A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and with 0.1% acetic acid (pH = 3.2), were used. The validation experiments demonstrated that the method applied was appropriate for levamisole quantification in human serum. For 23% of levamisole‐positive samples, the concentrations exceeded 20 ng/mL. Therefore, the interaction of this drug with cocaine has to be considered as important for active traffic participation. As a consequence, monitoring of levamisole concentration in human serum is recommended, as long as it is used as cocaine adulterant.     

Separation and identification of 9-chloro-10-methoxy (9-methoxy-10-chloro)hexadecanoic and octadecanoic acids in adipocere

Two homologs of 9-chloro-10-methoxy(9-methoxy-10-chloro) fatty acids were found in adipocere from the human neonate, and identified as 9-chloro-10-methoxy(9-methoxy-10-chloro)hexadecanoic acid and 9-chloro-10-methoxy(9-methoxy-10-chloro)-octadecanoic acid. The chemical structures of these compounds were confirmed by thinlayer chromatography, gas-liquid chromatography, gas chromatography-mass spectrometry, nuclear magnetic resonance spectrometry and elemental analysis. The adipocere contained approximately 7.2% 9-chloro-10-methoxy(9-methoxy-10-chloro) fatty acids in the total fatty acids.     

Large droplet lipids in blood in the right ventricle

In several cases of fatal blunt-trauma injuries or after exposure to severe burning, large-drop fatty substances, discernible to the naked eye, were found in the venous blood of the right ventricle. Thin-layer chromatographic separation of the extracted lipids demonstrated an increase in the triglyceride fraction. Analysis of the fatty acids by means of combined gas chromatography/mass spectrometry showed a shift towards oleic acid. The distribution pattern of the molecular weights was determined by means of DCI-mass spectrometry in order to characterize the triglycerides in detail. The total chemical findings indicated that the visible lipids present in the right ventricle had predominantly derived from the adipose tissues. In cases of post mortem burning (without vital traumata) no typical pulmonary fat embolism had developed despite massive lipemia in the venous blood.     

Changes in the lipid composition of latent fingerprint residue with time after deposition on a surface

The work described in this report was focused on generating increased knowledge of fingerprint chemistry, particularly the composition of a latent fingerprint at the time it is deposited, and the chemical changes in lipid components that occur over time. Fingerprints from five male donors (aged 25-34 years) were collected and aged under controlled conditions. The prints were then sampled at set intervals, solvent extracted with dichloromethane, co-derivatized with MSTFA and analyzed by gas chromatography-mass spectrometry (GC-MS). It was shown that there was loss of squalene from prints stored in the light or in the dark. Loss was more rapid in the light, with squalene in prints from some donors not detected after 9 days storage. For these same donors, squalene was still detected after 33 days storage in the dark, but at much lower levels than in fresh prints. For saturated fatty acids (tetradecanoic, palmitic and stearic acid) there was a trend towards an increase in levels of these substances during storage (up to about 20 days) followed by a decrease back to original levels or below. This was the case for samples stored in the light or in the dark. For palmitoleic acid, a similar trend was seen. For oleic acid, this trend was seen for samples stored in the dark. For samples stored in the light the general trend was a decrease in level over the storage period (up to 33 days).     

Estimation of age from a tooth by means of racemization of an amino acid, especially aspartic acid--comparison of enamel and dentin.

In a study of age estimation from teeth by means of measuring racemization of aspartic acid (Asp), a representative amino acid, the accuracy of data from enamel and dentin in the same tooth was compared. The correlation of D/L ratio of aspartic acid with actual age gave the following parameters: r = 0.928, sigma = +/- 5.2, k = 4.47 x 10(-4) yr-1 in enamel and r = 0.995, sigma = +/- 1.4, k = 5.75 x 10(-4) yr-1. The difference in ages between one estimated by the D/L ratio and the actual one was within +/- 3 years in dentin, while in enamel an error of from 2 to 11 years was observed. Reaction rate constants of the racemization in a dry postmortem state (15 degrees C) were calculated as k = 9.70 x 10(-8) yr-1 in enamel, and k = 1.33 x 10(-7) yr-1 in dentin. Compared to rates determined from teeth recently extracted from living subjects, the rate was higher in dentin than in enamel. These data reconfirmed that dentin is superior to enamel in making exact age estimations from teeth.     

Development of Rapid and Economical Colorimetric Screening Method for p‐Phenylenediamine in Variety of Biological Matrices and its Application to Eleven Fatal Cases of p‐Phenylenediamine Poisoning

A rapid colorimetric method for detection of p‐phenylenediamine (PPD) in various biological samples is developed. The o‐cresol test for acetaminophen detection has been modified to detect PPD in blood, urine, gastric contents, and liver. After precipitating protein with trichloroacetic acid solution (2 mL, 10% w/v), biological specimens were required to convert PPD metabolites to PPD by acid hydrolysis. Finally, o‐cresol solution (1 mL, 1% w/v), hydrogen peroxide (200 μL, 3%v/v), and concentrated ammonium hydroxide (0.5 mL) were added in the biological samples. The presence of PPD was indicated by formation of violet color which was turned to bluish green color within 10–15 min. The limit of detection was found to be 2 mg/L in blood, urine, and gastric contents and 2 mg/Kg in liver. This method is also free from any potential interference by p‐aminophenol, acetaminophen, and other amine drugs under test conditions. This method was successfully employed to thirteen fatal cases of PPD poisoning.     

Mineralogical and elemental data for soil discriminating and geolocation tracing

One of the key tasks of soil analysis in forensic sciences is to provide information about its diversities and geolocation. In fact, soil analysis is relevant for forensic geologists. In this study, a total of 80 soil samples were collected from eight Chinese cities (10 samples per city). Different minerals and their relative percentages were analyzed by the X-ray diffraction (XRD) method. In addition, the relative amounts of montmorillonite, kaolinite, amphibole, feldspar, calcite, and dolomite provided information about the origin of a soil, either if it came from a northern or southern city of China. The oxide weight percentages of 10 elements of Al₂O₃, SiO_2, Fe₂O₃, K₂O, Na₂O, MgO, CaO, P₂O₅, MnO, and TiO₂ were also obtained by using X-ray fluorescence (XRF) from the 80 soil samples. Moreover, principal component analysis (PCA) and hierarchical clustering analysis (HCA) methods were performed for dimensionality reduction, elemental marker identification and soils classification to the city they came from purposes. The eighty soils analyzed in this study could be tracked correctly to their city of origin. The K-Nearest Neighbors (KNN) model was done to evaluate the prediction ability based on the soil elemental composition, and it was confirmed by cross validation methods. The results demonstrated that mineralogical and elemental composition can provide powerful information for soil discrimination and source tracing.     

Phenibut screening and quantification with liquid chromatography–tandem mass spectrometry and its application to driving cases

An analytical strategy for identification by an LC–MS/MS multitarget screening method and a suitable LC–MS/MS based quantification were developed for the psychotropic drug phenibut. The samples analyzed were collected during traffic control and were associated with driving under the influence of drugs. A positive sample for phenibut was identified in a single case of driving under the influence. The quantification revealed a drug concentration of 1.9 μg/mL. An interaction with blood alcohol (BAC = 0.10%) was discussed as the explanation of the way of driving and deficit manifestations observed (swaying, nystagmus, quivering of the eyelid, and reddened eyes). According to the available information, the quantified phenibut concentration could be explained by an intake of four tablets (self-reported) during the day containing 250 mg of the drug. Chromatography was performed with a Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column, and a buffer system consisted of 10 mM ammonium acetate and 0.1% acetic acid (v/v) included in mobile phases marked as A (H₂O/methanol = 95/5, v/v) and B (H₂O/methanol = 3/97, v/v). An effective limit of detection (LOD = 0.002 μg/mL) could be achieved for the multitarget screening method. The quantification of phenibut was performed on a second LC–MS/MS system with LOD/LOQ values of 0.22/0.40 μg/mL. Since phenibut quantification data are rare, the presented information can be used with caution for evaluation of positive cases in the future.     

柱切换HPLC法分析尿中吗啡和O^6—单乙酰吗啡的研究

目的建立用柱切换HPLC技术分析尿中吗啡和06-单乙酰吗啡的方法.方法尿样用硼砂缓冲液(pH9.2)稀释后进入预处理柱(YWG-C18,33mm×5.0mm,10μm),用H2O洗去杂质,再用CH3OHH2O(6040)将被分析组分洗脱进入分析柱(Lichrospher(R)100CN,125mm×4.0mm,5μm),分析流动相为CH3OH磷酸盐缓冲液(pH6.86)=2278.紫外检测器波长为286nm.结果尿中吗啡和06--单乙酰吗啡的线性范围分别为50～1 600n/ml和100～1 600n/ml.吗啡和O6--单乙酰吗啡的精密度均小于4%.吗啡和O6-单乙酰吗啡的检测限均为40n/ml.结论用CSHPLC测定尿中吗啡和O6-单乙酰吗啡,方法准确、灵敏、快速、简便.     

大鼠肥大细胞的法医学实验研究──过氧化物酶活性变化、脱颗粒超微结构改变及分布

采用植物血凝素（PHA）致敏S、D大鼠，2周后处死，灌洗腹腔，用阿拉伯胶分离液分离肥大细胞（MC），将PHA与MC在37C水治中孵育，攻击MC使之受损伤，于伤后5、10、15、20、30和60分钟，分别吸取孵有液作聚丙烯酰胺凝胶圆盘电泳，分离MC过氧化物酶；用抗坏血酸-联苯胺染色显带，并计算酶的相对活性。另取20分钟时孵育液，常规固定，包埋，切片及染色后用透射电镜观察MC脱颗粒叶的形态。用常规石蜡切片，甲苯胺蓝染色，对SD大鼠的35种组织中MC的分布进行光镜观察。结果发现；（1）大鼠腹腔MC显示-条过氧化物酶电泳区带；（2）MC经PHA攻击受损伤10分钟内，过氧化物酶相对活性升高不明显；15、ZO、3O分钟时实验组酶活性升高，与对照组的差异明显；到60分钟时两组酶活性无显著差异；（3）MC与PHA孵育20分钟时，脱颗粒率为76％，MC浆内可见有膜性和无膜性两种类型的空泡．对照组大鼠MC脱颗粒仅为16％；（4）大鼠皮肤、肺、呼吸道、消化道、骨髓及胸腺MC含量较多；泌尿生殖道次之；心、肝、脾、肾等组织较少；脑、脊髓、脑垂体、肾上腺及睾丸等组织中未见MC，从而为法医损伤时间的推测提供方法与依据。     

Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers

In previous investigations hair analysis for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) proved to be suitable for the detection of excessive alcohol consumption. The aim of this study was to compare EtG and FAEE concentrations in hair of alcoholics, social drinkers and teetotallers. Hair samples from 10 alcoholics in withdrawal treatment, 11 fatalities with documented excessive alcohol consumption, four moderate social drinkers who consumed up to 20 g ethanol per day, and three strict teetotallers were analysed. After external degreasing with n-heptane, extraction with a dimethyl sulfoxide/n-heptane mixture and headspace solid-phase microextraction of the extracts, four fatty acid ethyl esters (FAEEs) (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) were analysed by gas chromatography-mass spectrometry (GC-MS) with deuterated internal standards. EtG was determined by GC-MS/NCI after ultrasonication of the samples with H2O, cleanup by SPE with aminopropyl columns and PFP derivatisation. The following concentrations were measured for the four groups: teetotallers EtG < 0.002 ng/mg, FAEE 0.05-0.37 ng/mg, moderate social drinkers EtG < 0.002 ng/mg, FAEE 0.26-0.50 ng/mg, alcoholic patients EtG 0.030-0.415 ng/mg, FAEE 0.65-20.50 ng/mg and the fatalities with alcohol history EtG 0.072-3.380 ng/mg, FAEE 1.30-30.60 ng/mg. The results confirm that by using a cut-off value of the sum of FAEE > 1 ng/mg and/or a positive EtG result in hair, excessive alcohol consumption can be identified using hair analysis. However, no significant correlation between the EtG and FAEE concentrations in the positive cases could be shown. Segmental analysis of some of the specimens did not reveal the same distribution for EtG compared to FAEE in hair, and no chronological accordance compared to the self-reported alcohol consumption could be observed for both parameters. These different results of both methods are discussed in terms of differences between EtG and FAEE in mechanism of formation and incorporation into hair and elimination from hair.     

GHB free acid: I. Solution formation studies and spectroscopic characterization by 1HNMR and FT-IR

In forensic evidence, gamma-hydroxybutyric acid (GHB) has frequently been encountered in one of its salt forms (gamma-hydroxybutyrate), but has also been encountered in its free acid form (GHB). Owing to the physical properties, encounters of the free acid have been largely restricted to forensic exhibits comprising aqueous solutions, such as acidic beverages that have been "spiked" or formulated with GHB salts or gamma-butyrolactone (GBL). The analysis of GHB free acid presents particular difficulties including the potential for altering the original proportions of GHB free acid, GHB carboxylate, and GBL in the course of analysis, and discrimination between GHB free acid and carboxylate forms. In this work, the formation of GHB free acid in aqueous solutions (water and/or D2O) was studied as a function of solution pH. Proton nuclear magnetic resonance (1HNMR) and Fourier-transform infrared spectrometry (FT-IR) measurements were obtained on freshly prepared mixtures of NaGHB and HCl stock solutions representing a series of points along the GHB titration curve. Both 1HNMR and FT-IR were shown to track the changing proportions of GHB free acid and carboxylate forms as a function of pH, while simultaneously monitoring for the formation of the lactone (GBL). The results were consistent with acid-base conversion behavior for a carboxylic acid. 1HNMR was shown to provide an ideal means for analysis of aqueous-based GHB/GBL forensic exhibits based on simple dilution of the neat liquid exhibit, without altering the original proportions of GHB free acid, carboxylate, and GBL in the samples.     

Determination of a mechanism of the coming of death by morphological methods in strangulation mechanical asphyxia

Described in the paper are morphological data characterizing the tempo and type of thanatogenesis in strangulation asphyxia. The cerebral thanatogenesis was shown to prevail in mechanical asphyxia. The results of determination of a degree of hydration of the brain by drying its samples are described. Medullary substance was demonstrated to be hypohydrated in such death.     

The relationship between bedding and face-down death in infancy: mathematical analysis of a respiratory simulation system using an infant mannequin to assess gas diffusibility in bedding

Rebreathing is a model for the relationship between a prone sleeping position and sudden infant death syndrome. This study used a mechanical simulation model to establish the relationship between types of bedding and rebreathing potential for an infant placed prone (face down) at different postnatal ages. The infant mannequin was connected to a respirator set to deliver physiologically appropriate combinations of tidal volume (V(T)) and respiratory rates (RR) across a range of postnatal ages (0-18 months). Before measurements were made, CO(2) flow was regulated to 5+/-0.1% of end-tidal PCO(2) (EtCO(2)). After the model was placed in a prone position, any increase in the fractional concentration of inspired CO(2) (FiCO(2)) was measured. FiCO(2) increased immediately and rapidly, and reached a maximum value within a few minutes. The maximum FiCO(2) ranged from under 2% to over 10%, depending on the bedding. FiCO(2) was also affected by V(T) and RR. This model is not applicable to actual infants because of the large tissue stores of CO(2) in infants; however, it is useful for evaluation of gas diffusibility of bedding and will simplify the investigation of sleeping environments when a baby is found dead with its face covered by soft bedding. In general, the higher the FiCO(2), the greater the rebreathing potential. Theoretically, considering the paucity of body stores of O(2), changes in FiO(2) would be affected not by changes in FiCO(2), but by CO(2) production and gas movement around the infant's face. The rapid decrease of FiO(2) is approximated at the inverse of the FiCO(2) timecourse, suggesting the significance of not only CO(2) accumulation but also O(2) deprivation in the potential space around the baby's face.