Preliminary Study Of Fatal Hypothermia Via ATR-FTIR Spectroscopy

Objective:Attenuated total reflection Fourier transform infrared spectroscopy combined with chemometric methods were used to analyze the biochemical changes of Vitreous humor in New Zealand white rabbits after fatal hypothermia,which provided a new way for identification fatal hypothermia in forensic.Methods:54 adult male New Zealand white rabbits,weighing 1.9².3 kg,provided by Shanghai Animal Experimental Company,were given free access to food and drinking water at 24°C,50⁶0%ambient humidity for one week.Adult male New Zealand white rabbits were randomly divided into low temperature freezing group and control group by random number method.The control group included sudden death,hemorrhagic shock,and mechanical asphyxia.Using a 1 ml syringe and slowly move the needle about 2 mm outside the limbus.When the needle is at the center of the pupil,slowly extract all the vitreous humor（about 0.5¹ ml）from the eyes.Numbered into an EP tube,centrifuged at 12,000 r pm,and the supernatant was extracted and frozen at-80℃.The vitreous humor samples were analyzed by ATR-FTIR 6700 spectrometer.SPSS 22 statistical software was used to analyze the characteristic spectral differences of vitreous humor between fatal cryopreservation and control group;The ATR-FTIR 6700 spectrometer was used to detect the biochemical information of the vitreous samples of each death group,and the spectral data set was analyzed by Matlab R2016a software.In order to ensure the quality of the subsequent analysis of the spectral data set of the vitreous humor sample,the spectral data set pretreatment was performed by the following method.First,the second-order derivative spectral transform of the vitreous humor spectral data set is used to eliminate interference from other backgrounds,distinguish overlapping peaks,and improve resolution and sensitivity.Secondly,standard normal transformation is used to eliminate interference factors such as optical path difference.Finally,the fingerprint region of the vitreous humor spectrum was taken for further analysis.The pre-processed spectral data set was combined with the chemometric method for principal component analysis（PCA）and partial least squares discriminant analysis.A partial least squares discriminant analysis（PLS-DA）model of fatal hypothermia vitreous humor was established by genetic algorithm（GA）to predict the frozen individual.Results:1.The mean second-order derivative spectra of the vitreous humor of the New Zealand white rabbits in the fatal hypothermia group and the control group were different at 989 cm^-1 and1080 cm^-1,and the difference between the two groups was significant（P<0.05）.And the level of nucleic acid and glucose in the fatal hypothermia group was higher.2.Principal component analysis combined with load factor analysis showed differences between the fatal hypothermia group and the control group.The load factors positively correlated with the fatal hypothermia group were mainly concentrated at 1107 cm^-1,1078 cm^-1,1030 cm^-1 and 989 cm^-1 nearby,and the load factors negatively correlated with the fatal hypothermia group were mainly distributed around 1122 cm^-1,1092 cm^-1 and 1045 cm^-1,indicating that the vitreous humor in the fatal hypothermia group contained more nucleic acid,glucose and Less lactic acid,phosphate,glycogen relative to other causes of death in the control group.3.Two types of partial least squares analysis models are established,which show that the partial least squares discriminant model established by genetic algorithm has better performance.With only 88 variables,the accuracy and sensitivity of internal cross-validation are 100%and 95%,respectively.The partial least squares discriminant model guided by genetic algorithm is used to predict the additional samples,and the accuracy rate reaches 94%.Although the predicted sample is a complex post-mortem storage environment,the predicted results obtained are not affected by different ambient temperatures.Conclusion:Infrared spectroscopy combined with chemometric methods can directly and effectively reflect the biochemical differences of vitreous humor in New Zealand rabbits between fatal hypothermia and death in control group,and it provides new methods for fatal hypothermia forensic identification.