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WO2007091033A1 - Trousse portative et procédé permettant d'estimer l'heure du décès d'un cadavre par détermination du taux d'hypoxanthine dans le cadavre relativement à la température et au temps écoulé - Google Patents

Trousse portative et procédé permettant d'estimer l'heure du décès d'un cadavre par détermination du taux d'hypoxanthine dans le cadavre relativement à la température et au temps écoulé Download PDF

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Publication number
WO2007091033A1
WO2007091033A1 PCT/GB2007/000390 GB2007000390W WO2007091033A1 WO 2007091033 A1 WO2007091033 A1 WO 2007091033A1 GB 2007000390 W GB2007000390 W GB 2007000390W WO 2007091033 A1 WO2007091033 A1 WO 2007091033A1
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Prior art keywords
hypoxanthine
kit
amount
time
sample
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Ceased
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PCT/GB2007/000390
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English (en)
Inventor
Torleiv Ole Rognum
Ole Didrik Saugstad
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GORDON KIRSTEEN HELEN
Universitetet i Oslo
Original Assignee
GORDON KIRSTEEN HELEN
Universitetet i Oslo
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Publication of WO2007091033A1 publication Critical patent/WO2007091033A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7769—Measurement method of reaction-produced change in sensor
    • G01N2021/7786—Fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2201/00—Features of devices classified in G01N21/00
    • G01N2201/02—Mechanical
    • G01N2201/022—Casings
    • G01N2201/0221—Portable; cableless; compact; hand-held
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/12—Meat; Fish

Definitions

  • the present invention provides a kit and a device for estimating the time of death of a corpse that may be taken to, and used, at the scene of an accident, natural death, suicide or crime.
  • the invention also concerns use of the portable kit and portable device for estimating the time of death of a corpse as well as methods for estimating the time of death of a corpse.
  • time of death is of great importance in forensic medicine, particularly in cases of murder or manslaughter where the estimated time often significantly impacts upon the course of the resulting criminal investigation.
  • the estimated time of death is almost always used as a means to identify, as well as to eliminate, suspects hence it is important that time of death is reliably determined since an erroneous time of death window can lead investigations in the wrong direction. It is crucial therefore that an accurate estimate of the time of death time is provided as soon as possible after the discovery of the corpse to aid the investigation.
  • forensic scientists tend to rely on rudimentary techniques to estimate time of death, particularly at the scene of a crime.
  • the most commonly used technique is based on the temperature drop models which describe the cooling of the body after death. This method is, however, unreliable with the results being affected by variables such as the amount of body fat on the corpse, the environment in which the corpse is present, the type and amount of clothing worn and so on.
  • the temperature drop models can therefore only provide an approximation of time of death.
  • a further biochemical marker which may be used in a method of determining time of death is disclosed by Rognum et al. in Forensic Science International, 51 (1991) 139-146. This method is based on the observation that the level of hypoxanthine (Hx) in the vitreous humor of a corpse increases linearly with time after death. When death occurs, Hx accumulates because cells can no longer catalyse the conversion of AMP into ATP, thus AMP is degraded to form Hx.
  • the procedure disclosed involves analysis of the samples obtained using HPLC. More specifically the samples are diluted 3-fold with the HPLC mobile phase and then filtered prior to being separated by reverse phase chromatography. Peak height ratios are then used to quantify the amount of Hx present.
  • this method suffers from the disadvantages of being time consuming and requiring sophisticated equipment which limits its use to a laboratory environment.
  • the inventors have surprisingly developed a kit and a device which can be taken to the scene of crime and used to determine the time of death
  • the present invention provides a portable kit for estimating the time of death of a corpse comprising:
  • the invention provides a portable device for estimating the time of death of a corpse comprising:
  • the invention provides a method for estimating the time of death of a corpse comprising:
  • steps (b) and (c) are carried out using a kit or device as hereinbefore defined.
  • the invention provides use of a portable kit or portable device as hereinbefore described for estimating the time of death of a corpse.
  • the term "corpse" is intended to encompass any formerly living animal, preferably a mammal. It is envisaged, however, that the invention will be most useful for humans.
  • the corpse is preferably a human corpse.
  • kits and devices according to the present invention may be, for example, transported in a carry case or holdall.
  • the equipment required to carry out HPLC in a laboratory is not intended to fall within the definition of portable.
  • kits are typically meant a collection or array of certain equipment and chemicals (e.g. enzymes, reporter molecules, buffers etc).
  • device is typically meant a piece of equipment.
  • the devices herein described may form, or may form part of, the kits of the present invention. Preferred features of the kits and methods of using the kits of the invention, thus also form preferred features of devices and methods of using the devices of the invention.
  • assessing it is meant that a quantitative value for the amount of Hx is determined.
  • the value may be a concentration of Hx or may alternatively be an index, ratio, percentage or similar number reflective of the concentration of Hx.
  • the value is the absolute concentration of Hx in the sample.
  • comparing it is typically meant that the amount of Hx in the sample obtained from the corpse is evaluated against one or more reference samples which are identical to the sample obtained from the corpse with the exception that the reference samples contain a known amount of Hx.
  • the reference samples typically comprise the same body fluid as the samples obtained from the corpse. Additionally the reference samples preferably undergo the same treatment steps as the sample obtained from the corpse.
  • any known portable means for the assessment of Hx may be used in the kit of the invention. Whilst several such means are known and may be used in the method of the invention, there has not previously been any suggestion that these may be used to provide a portable kit or device for estimating time of death.
  • said means for assessing the amount of Hx comprises a portable detector.
  • Hx may, for example, be assessed spectrophotometrically (e.g. by absorption spectroscopy), by an electrochemical cell, by a biosensor (e.g. an electrochemical biosensor) or by luminescence spectroscopy.
  • spectrophotometrically e.g. by absorption spectroscopy
  • an electrochemical cell e.g. an electrochemical biosensor
  • a biosensor e.g. an electrochemical biosensor
  • luminescence spectroscopy e.g. an electrochemical biosensor
  • Hx is assessed by a biosensor (e.g. an electrochemical - biosensor) or by luminescence spectroscopy as these methods can detect Hx at low concentrations (e.g. as low as 1-10 ⁇ M).
  • a biosensor e.g. an electrochemical - biosensor
  • luminescence spectroscopy as these methods can detect Hx at low concentrations (e.g. as low as 1-10 ⁇ M).
  • Any known biosensor for the detection of Hx may be used (e.g. the biosensors described in Biosensors and Bioelectronics, 2002, 17(3), 147-157 or Analytica Chimica Acta, 1996, 319, 325-333).
  • the recently developed biosensors employing carbon nanotubes may be employed (e.g. the biosensor described in Analytical Sciences, 2003, 19(9), 1309- 1312).
  • Hx is assessed spectrophotometrically or by luminescence spectroscopy.
  • a preferred spectrophotometric method is absorption spectroscopy, more preferably absorption spectroscopy at 540-580 run (e.g. 571 nm). Any conventional portable detector may be used to determine absorption, e.g. a charge-coupled device or photodiode.
  • Hx is assessed by luminescence spectroscopy, e.g. chemiluminescence or fluorescence.
  • Chemilurninescence may, for example, be carried out using a lurninol-hydrogen peroxide-horseradish peroxidase system.
  • an enzyme is used to convert Hx into hydrogen peroxide which, in the presence of horseradish peroxidase, reacts with luminol to generate electromagnetic radiation which can be measured.
  • kits of the present invention therefore comprise a portable fluorometer.
  • Hx may be assessed directly or indirectly. When Hx is to be measured directly, it is preferably assessed by a portable biosensor as hereinbefore described. Particularly preferred biosensors specifically assess Hx (i.e. they distinguish between Hx and xanthine).
  • Hx is assessed indirectly.
  • directly is meant herein that Hx is converted in a known manner to another compound that is detected.
  • Many of the known methods for the assessment of Hx are based on its conversion to uric acid and/or hydrogen peroxide and detection of either or both of these compounds.
  • the portable kit of the present invention preferably comprises an enzyme which can decompose Hx into at least one of these compounds.
  • a preferred enzyme for use in the kit is xanthine oxidase (EC 1.2.3.2).
  • any conventional xanthine oxidase may be used in the kit of the invention.
  • the xanthine oxidase may be produced by culturing a microorganism belonging to the genus Enterobacter as described in EP-A-0016845. More preferably the. xanthine oxidase is derived from milk (e.g. buttermilk).
  • the xanthine oxidase may be provided in any conventional form. It may, for example, be immobilised on a solid phase or more preferably present in a buffer.
  • Xanthine oxidase suitable for use in the kits of the present invention is commercially available from, for example, Fluka, Roche, Cayman Chemicals and Sigma-Aldrich.
  • the kit of the present invention further comprises a means for assessing the amount of uric acid and/or hydrogen peroxide.
  • Any conventional portable means may be used. The key to portability often lies in the device used for the detection and assessment of uric acid and/or hydrogen peroxide.
  • the means for assessing the amount of uric acid and/or hydrogen peroxide preferably comprises a portable detector and a reporter molecule.
  • a reporter molecule is a compound which reacts, optionally in the presence of a catalyst (e.g. enzyme), with uric acid and/or hydrogen peroxide to produce a moiety that can be detected by the detector.
  • the portable detector is a portable biosensor, a portable spectrophotometer, a portable luminescence (e.g. chemiluminescence) detector or a portable fluorometer (preferably a portable fluorometer).
  • a portable fluorometer preferably a portable fluorometer
  • the means for assessing the amount of uric acid and/or hydrogen peroxide may comprise a portable electrochemical cell or a biosensor.
  • kits according to the present invention provide means to assess the amount of uric acid and/or hydrogen peroxide, preferably hydrogen peroxide, fluorometrically. Unlike portable electrochemical cells, hand held fluorometers are readily available and are easy to set up and use. Fluorometric measurements are also less prone to interference than, for example, those determined chemiluminescently and hence are much more reliable.
  • kits for use in the present invention therefore comprise (i) a portable fluorometer, (ii) a compound which reacts, optionally in the presence of an enzyme, with said hydrogen peroxide and/or uric acid (preferably hydrogen peroxide) to produce a fluorescent compound; and (iii) optionally an enzyme which catalyses a reaction between said compound and hydrogen peroxide.
  • the compound present in the kits of the invention maybe any compound which produces a fluorescent, compound upon reaction with hydrogen peroxide and/or uric acid (preferably hydrogen peroxide).
  • fluorescent compounds that may be produced by the compound present in the kit of the invention include haematoporphyrin, merocyanine, phycoerythrine, pyronin, resorufm, rhodamine, fluorescein, texas red, thiazin red and derivatives thereof.
  • the fluorescent compound produced is resorufm, rhodamine, fluorescein or derivatives thereof. More preferably the fluorescent compound produced is resorufm or a resorufm derivative (e.g. resorufin).
  • Resorufm and derivatives thereof are resistant to oxidation and have excitation/emission maxima (e.g. ⁇ 570/585 nm) which are not significantly affected by interference from autofluorescence.
  • excitation/emission maxima e.g. ⁇ 570/585 nm
  • use of these fluorescent compounds in the kits of the present invention increases their sensitivity and reliability.
  • Preferred compounds for use in the kits of the invention are therefore capable of producing the afore-mentioned fluorescent compounds (e.g. resorufm, rhodamine, fluorescein or derivatives thereof), upon reaction, optionally in the presence of an enzyme, with hydrogen peroxide and/or uric acid.
  • Suitable compounds for use in the kits of the invention therefore include the dihydro analgoues of fluorescent compounds.
  • Examples of such compounds include 2 ⁇ T - dichlorodihydrofluorescein (DCFH); peroxyfluor-1 (as described in JACS, 2004, 126, 15392-15393), dihydrorhodamine 123 and 10-acetyl-3,7- dihydroxyphenoxazine.
  • DCFH dichlorodihydrofluorescein
  • peroxyfluor-1 as described in JACS, 2004, 126, 15392-15393
  • dihydrorhodamine 123 2-acetyl-3,7- dihydroxyphenoxazine.
  • a particularly preferred compound for use in the kits of the present invention is 10-acetyl-3,7-dihydroxyphenoxazine (sold by Invitrogen Detection Technologies under the tradename Amplex Red).
  • the compound sold by Invitrogen Detection Technologies under the tradename Amplex UltraRed may be used in the kits of the present invention.
  • Any enzyme which catalyses the reaction to produce a fluorescent compound may be used in the kit of the invention. Preferably such an enzyme is present.
  • An example of an enzyme which catalyses a reaction between hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenoxazine is a peroxidase (e.g. horseradish peroxidase).
  • kits according to the present invention further comprise a calibration curve (e.g. a graph of [Hx] versus fluorescence) for determining the concentration of Hx. More preferably the kits may comprise Hx for the preparation of reference samples and a calibration curve.
  • the Hx may be provided in a range of concentrations or more preferably is provided in a single concentration which may be diluted to provide a number of reference samples.
  • a suitable diluent is also preferably provided.
  • Typical reference samples have Hx concentrations of up to 1000 ⁇ mol/1, more preferably up to 100 ⁇ mol/1.
  • kits according to the present invention additionally comprise one or more buffers which are suitable for diluting the various agents used in the assessment method; ' The skilled man will be readily determine suitable buffers.
  • a particularly preferred kit according to the present invention comprises as a means of assessing the amount of Hx at least the following agents: xanthine oxidase, a peroxidase (e.g. horseradish peroxidase)* a compound (e.g. 10-acetyl-3,7- dihydroxyphenoxazine) which, in the presence of peroxidase, reacts with hydrogen peroxide to produce a fluorescent compound and optionally Hx.
  • the various agents which may comprise the kit of the invention may be presented in any conventional form. Each of the different agents may be present in separate containers.
  • the enzyme which can convert Hx to hydrogen peroxide e.g.
  • xanthine oxidase may be present in the same container as the peroxidase enzyme and/or the compound which produces the fluorescent compound. IfHx is present, this must obviously be present in a separate container to the enzyme which catalyses its conversion to hydrogen peroxide.
  • the means for assessing the amount of hypoxanthine in a sample obtained from said corpse is provided by the Amplex® Red Xanthine Assay kit commercially available from Invitrogen Detection Technologies.
  • the fluorometer present in the kit of the invention may be any commercially available device.
  • the device sold under the tradename Picofiuor by Turner Biosystems may be used.
  • Preferred kits, or fiuorometers for use in the kit include a means for heating samples (e.g. a. water bath).
  • Fluorescence is generally measured in the range 350-600 run.
  • excitation is carried out in the wavelength range 540-580 nm.
  • emission occurs in the range 570-600 nm.
  • excitation is carried out at ⁇ 570 nm and emission occurs at 585 nm.
  • the kit of the invention also comprises a means for comparing the amount of hypoxanthine present in said sample with known values for amounts of hypoxanthine with time since death.
  • a means for comparing the amount of hypoxanthine present in said sample with known values for amounts of hypoxanthine with time since death.
  • This may take any conventional form, e.g. a table, graph or computer program.
  • the kit of the invention comprises several such means which each demonstrate the relationship between Hx concentration and time since death at different temperatures. Still more preferably the kit comprises data showing the correlation between Hx concentration and time since death substantially as shown in Figure 2.
  • kits of the present invention comprise a computer program which calculates time of death based on Hx concentration.
  • the computer program preferably utilises reference data correlating Hx concentration with time since death, preferably at a range of environmental temperatures. Use of a computer program is advantageous as a time of death measurement maybe obtained very quickly.
  • the program may run, for example, on a lap top computer or PDA.
  • the program is preferably based on equation [I]:
  • ⁇ T is the time (in hours) between the time when the sample is taken and the time of death
  • F is Hx (in ⁇ mol/1 ) in the sample obtained from the corpse as assessed by the means hereinbefore described (e.g. by fiuorometry);
  • K 1 is a correction factor for the content of xanthine present in the sample and is preferably 5-20 % of F, more preferably 10-15% of F, e.g. about 12.3 % of F;
  • K 2 is the correction factor for the content of Hx present in living human beings and is preferably 0.05 to 50 ⁇ mol/1, more preferably 0.1 to 10 ⁇ mol/1, e.g. about 0.3 to 0.7 ⁇ mol/1; and a is the slope or rate of increase of Hx, preferably related to the environmental temperature, and is preferably taken from reference data, e.g. from Figure 2 or 3.
  • the body sample used for assessing the amount of Hx may be any Hx-containing sample, e.g. a body fluid or tissue sample.
  • the sample is a body fluid, e.g. vitreous humor.
  • the sample is substantially all (e.g. all) of the vitreous humor that can be removed from an eye. Typically about 1 to 2 ml of fluid may be obtained. Removal of the vitreous humor may be done by any conventional method known in the art, e.g. by insertion of a vacuum filled syringe into the eye.
  • kits according to the invention additionally comprise a means (e.g. a syringe) for obtaining vitreous humor.
  • the sample may be treated prior to assessing the amount of Hx present therein.
  • the sample may, for example, be treated to remove any cells or tissue present therein.
  • the sample may be treated to transfer the Hx to a separate medium, e.g. a solid substrate.
  • An advantage of the kit of the invention is that the sample may be used without any treatment prior to assessing the amount of Hx. Preferably the sample is used without any treatment.
  • the sample may be concentrated or diluted. Dilution may be carried out using a buffer or another aqueous medium. Most preferably the sample (e.g. vitreous humor) is used directly and/or after dilution (e.g. after dilution). Dilution may be carried out using any buffer which does not interfere with the subsequent reactions.
  • a representative example of a suitable buffer is Tris-HCl.
  • the buffer concentration is preferably in the range 0.01 to IM. Still more preferably a sample obtained from the corpse is used without any dilution.
  • the concentration of Hx in the sample obtained from the corpse will depend on the time since death. Typically, however, the concentration of Hx will be in the range 1-1000 ⁇ mol/1.
  • the sample obtained from the corpse will be divided into several (e.g. 3-10) different aliquots (e.g. each of 20-70 ⁇ L) and diluted to varying degrees.
  • a typical dilution series might involve mixing the sample obtained with buffer in volume/volume ratios of 100:0, 80:20, 60:40, 50:50, 40:60, 20:80, 10:90, 1:100, 1:200 and 1:400.
  • the sample(s) obtained from the corpse is reacted with an enzyme which is capable of converting it into uric acid and/or hydrogen peroxide (e.g. xanthine oxidase).
  • an enzyme which is capable of converting it into uric acid and/or hydrogen peroxide (e.g. xanthine oxidase).
  • the resulting mixture is then contacted with a fluorescent compound which in the presence of an enzyme (e.g. peroxidase) reacts with hydrogen peroxide.
  • an enzyme e.g. peroxidase
  • the reactions are carried out simultaneously (i.e. a pre-prepared mixture of, for example, xanthine oxidase, peroxidase and a • fluorescent compound as defined above are added to the sample obtained from the corpse).
  • the reactions may be carried out in any conventional format, e.g.
  • the pH of the reaction mixture is preferably in the range 6.5 to 8.0, still more preferably 7.0 to 8.0.
  • the reaction is preferably allowed to proceed for 1 to 90 minutes, preferably 10 to 60 minutes, more preferably 20 to 40 minutes, e.g. about 30 minutes. Still more preferably the reaction proceeds for 1 to 10 minutes, more preferably 2 to 5 minutes.
  • the mixtures are preferably protected from light.
  • the fluorescence of the sample may be measured. Typically fluorescence is measured using excitation in the range of 540- 580 run and emission detection at 570-600 nm. For example, when 10-acetyl-3,7- dihydroxyphenoxazine is used as the fluorescent compound excitation is typically in range -571 nm and emission detection at about 585 nm.
  • the fluorescence levels of reference samples with known concentrations of Hx are determined at the same time. These can be used to plot a calibration curve from which the Hx content of the sample obtained from the corpse can be obtained. This Hx correlation can in turn be correlated with a certain time since death, preferably using Figure 2. Still more preferably a computer program as hereinbefore described converts the Hx content of the sample into a time since death.
  • the temperature of the environment in which the corpse is present is also measured.
  • the most appropriate Hx concentration with time since death curve can then be used to obtain the most reliable estimate of time since death.
  • the time of death is additionally estimated by a second method, e.g. by the determination of vitreous potassium concentration as is well known in the art or by the determination of vitreous Hx concentration by HPLC as described in Forensic Science International, 51 (1991) 139-146.
  • the kit/device of the invention advantageously provides an immediate estimate of time of death (i.e. an on the spot assessment) and the second method provides validation of the estimate.
  • the means for assessing the amount of hypoxanthine in a sample obtained from a corpse and the means for comparing the amount of hypoxanthine present in the sample with known values for amounts of hypoxanthine with time since death are provided in a single portable device as hereinbefore defined.
  • Preferred portable devices comprise a fiuorometer as the means for assessing the amount of hypoxanthine and a computer program as the means for comparing the amount of hypoxanthine present in the sample with known values for amounts of hypoxanthine with time since death.
  • the means for comparing the amount of hypoxanthine present in the sample with known values for amounts of hypoxanthine with time since death comprises a computer program, e.g. a program as hereinbefore defined.
  • the device of the invention may be programmed to utilise the amount of Hx found in a sample to automatically calculate the time since death.
  • the preferred means for assessing the amount of hypoxanthine in a sample are as discussed above in relation to kits and methods of the invention.
  • portable devices comprising a fiuorometer are particularly preferred. Such devices are small and light and therefore are easy to transport to, for example, the scene of a crime.
  • the present invention provides a portable device for assessing the amount of hypoxanthine comprising:
  • Such devices may be used, for example, to determine the freshness of food, especially meat and fish (e.g. fish).
  • the Hx levels of hospital patients e.g. those having undergone surgery, organ transplantation or child birth
  • Figures Ia and Ib show the reliability of Hx determination using a kit according to the present invention versus capillary electrophoresis;
  • Figures 2 and 3 are graphs for estimation of the time since death; and Figure 4 shows a systematic diagram of a kit of the invention.
  • Vitreous humor hypoxanthine (Hx) levels were measured by an enzymatic reaction, followed by fluorimetric analysis. This was done using the enzymes xanthine oxidase (XO), horseradish peroxidase (HRP) and Amplex Red. XO catalyzes the oxidation of the purine bases Hx and xanthine to uric acid and hydrogen peroxide (H 2 O 2 ). In the presence of HRP, H 2 O 2 reacts stoichiometrically with the Amplex Red reagent to generate the fluorescent product resorufin. This reaction has been performed using different conditions, as listed in methods 1-6 below.
  • the reagents used were the following: Amplex Red (supplied by Invitrogen) Xanthine Oxidase (supplied by Fluka) Horseradish peroxidase (supplied by Sigma) 0.1 M Tris-HCl, pH 7.5 20 mM standard solution of hypoxanthine
  • the reaction mix for each sample consisted of 0.0455 mg Amplex Red, 0.7 units HRP, 0.0525 units XO, in a total volume of 1 ml, with 0.1 M Tris-HCl as reaction buffer. This was mixed with 1 ml of vitreous humour diluted with 0.1 M Tris-HCl, pH 7.5 (dilution factor of 1 :200 or 1 :400) and incubated for 5 minutes at 37°C, in a dark environment. The fluorescence was measured using 2 ⁇ M Hx as a standard, excitation at 540 +/- 20 nm and emission at > 570 nm.
  • the reliability of each of methods 1-6 was determined by comparing the Hx values obtained with those determined by capillary electrophoresis.
  • the running conditions used for capillary electrophoresis were those described in Rognum et al, Scand J Forensic Sci 2002;l:17-22.
  • the columns were made of coated fiised-silica capillary tubing (50 ⁇ M ID x 30 cm total length for the separation of Hx), which was mounted in a user assembled cartridge (Bio-Rad). Injection of the sample was effected by applying a pressure (15 psi/sec).
  • a constant voltage of 10-13 kV (current ⁇ 100 ⁇ A) was applied and the temperature of the capillary cartridge and carousel were maintained at 2O 0 C and 12 0 C respectively.
  • Polarity was - to + for Hx.
  • Detection was effected at 254 nm.
  • the identity and purity of the peaks was established by high speed scanning (range 200-280 nm) of the sample and of pure standard. Biofocus integration software (version 5.0 Bio-Rad) was employed for data conversion and evaluation.
  • Capillary equilibration between runs were obtained by rinsing the capillary with distilled H 2 O for 15 sec, 1 mM NaOH for 60 sec, distilled H 2 O for 15 sec, and the working buffer for 60 sec.
  • a forensic pathologist arrives at a crime scene where an elderly lady is found dead with gun shot wounds.
  • the ambient temperature where the lady is found is 16.5 0 C.
  • Her brain temperature is 16.6 0 C and the rectal temperature is 20.5 0 C. Based on these temperature measurements, it may only be concluded that the lady died at least 24 hours before the measurements were performed.
  • the forensic pathologist also takes a sample of vitreous humor and by means of the kit hereinbefore described he determines that its hypoxanthine concentration is 205 ⁇ mol/1. By means of the diagrams for estimation of time since death (Figs 2 and 3), he calculates that the ⁇ ady died 29 hours ago (90 % confidence interval 23-41 hours).
  • hypoxanthine slope is 6.8 ⁇ mol/l/h.
  • time since death can be calculated by dividing the measured hypoxanthine concentration (205 ⁇ mol/1) minus normal hypoxanthine concentration (K 2 as hereinbefore defined, e.g. 7.6 ⁇ mol/1) by the temperature corrected hypoxanthine slope.
  • K 2 normal hypoxanthine concentration
  • the 90 % confidential interval can be calculated by performing the same procedure by means of the 10th and 90th percentiles (dotted lines).

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  • Life Sciences & Earth Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une trousse portative et un procédé permettant d'estimer l'heure du décès d'un cadavre. La trousse comprend : i) un moyen pour déterminer le taux d'hypoxanthine dans un échantillon prélevé sur le cadavre; et ii) un moyen pour comparer le taux d'hypoxanthine présent dans l'échantillon à des taux d'hypoxanthine connus corrélés au temps écoulé depuis le décès.
PCT/GB2007/000390 2006-02-06 2007-02-05 Trousse portative et procédé permettant d'estimer l'heure du décès d'un cadavre par détermination du taux d'hypoxanthine dans le cadavre relativement à la température et au temps écoulé Ceased WO2007091033A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0602329.5 2006-02-06
GB0602329A GB0602329D0 (en) 2006-02-06 2006-02-06 Portable kit

Publications (1)

Publication Number Publication Date
WO2007091033A1 true WO2007091033A1 (fr) 2007-08-16

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PCT/GB2007/000390 Ceased WO2007091033A1 (fr) 2006-02-06 2007-02-05 Trousse portative et procédé permettant d'estimer l'heure du décès d'un cadavre par détermination du taux d'hypoxanthine dans le cadavre relativement à la température et au temps écoulé

Country Status (2)

Country Link
GB (1) GB0602329D0 (fr)
WO (1) WO2007091033A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2012017300A1 (fr) * 2010-08-04 2012-02-09 University Of Cape Town Appareil permettant d'estimer le délai post-mortem d'un cadavre
CN104000601A (zh) * 2013-02-22 2014-08-27 谭唱 快速测血尿酸仪
CN113341043A (zh) * 2021-06-18 2021-09-03 中国医科大学 一种基于代谢组学标志物推断水中尸体早期死后淹没时间的方法及其应用

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
CN106305577A (zh) * 2016-08-23 2017-01-11 山东商业职业技术学院 一种用于水产品无水保活的智能储存箱

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012017300A1 (fr) * 2010-08-04 2012-02-09 University Of Cape Town Appareil permettant d'estimer le délai post-mortem d'un cadavre
CN104000601A (zh) * 2013-02-22 2014-08-27 谭唱 快速测血尿酸仪
CN113341043A (zh) * 2021-06-18 2021-09-03 中国医科大学 一种基于代谢组学标志物推断水中尸体早期死后淹没时间的方法及其应用

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