EP1210451A1 - Rapid method for measuring complex carbohydrates in mammalian tissue - Google Patents
Rapid method for measuring complex carbohydrates in mammalian tissueInfo
- Publication number
- EP1210451A1 EP1210451A1 EP00955193A EP00955193A EP1210451A1 EP 1210451 A1 EP1210451 A1 EP 1210451A1 EP 00955193 A EP00955193 A EP 00955193A EP 00955193 A EP00955193 A EP 00955193A EP 1210451 A1 EP1210451 A1 EP 1210451A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- complex carbohydrates
- measuring complex
- rapid method
- measuring
- measurement
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims abstract description 77
- 150000001720 carbohydrates Chemical class 0.000 title claims abstract description 40
- 235000014633 carbohydrates Nutrition 0.000 title claims abstract description 40
- 229920002527 Glycogen Polymers 0.000 claims abstract description 42
- 229940096919 glycogen Drugs 0.000 claims abstract description 42
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 33
- 239000008103 glucose Substances 0.000 claims abstract description 31
- 238000005259 measurement Methods 0.000 claims abstract description 30
- 239000002002 slurry Substances 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 claims abstract description 16
- 230000003301 hydrolyzing effect Effects 0.000 claims abstract description 9
- 238000003307 slaughter Methods 0.000 claims abstract description 9
- 230000007062 hydrolysis Effects 0.000 claims abstract description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 235000013372 meat Nutrition 0.000 claims description 26
- 210000001519 tissue Anatomy 0.000 claims description 23
- 210000003205 muscle Anatomy 0.000 claims description 18
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 108010015776 Glucose oxidase Proteins 0.000 claims description 4
- 239000004366 Glucose oxidase Substances 0.000 claims description 4
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 claims description 4
- 229940116332 glucose oxidase Drugs 0.000 claims description 4
- 235000019420 glucose oxidase Nutrition 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 3
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 3
- 102100024295 Maltase-glucoamylase Human genes 0.000 claims description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000006194 liquid suspension Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 239000003125 aqueous solvent Substances 0.000 claims 2
- 102000004139 alpha-Amylases Human genes 0.000 claims 1
- 108090000637 alpha-Amylases Proteins 0.000 claims 1
- 229940024171 alpha-amylase Drugs 0.000 claims 1
- 238000003556 assay Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 description 9
- 239000008351 acetate buffer Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 238000007792 addition Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- WQZGKKKJIJFFOK-DVKNGEFBSA-N alpha-D-glucose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-DVKNGEFBSA-N 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 238000013494 PH determination Methods 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 239000003041 laboratory chemical Substances 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
Definitions
- the present invention relates to a rapid method for measuring complex carbohydrates
- glycogen in mammalian tissue. More particularly the present invention relates
- glycogen in mammalian tissue.
- the measurement of glycogen includes, for example, the ability to use the results of
- glycogen measurement as a determination of the ultimate pH of meat. This in turn is a direct
- the iodine method The principle of this method is that glycogen will react with a mixture
- glycogen is extracted from the
- glycogen which is reacted with the iodine can also be by liquid nitrogen,
- amyloglucosidase method of Dreiling et al (Meat Science, Vol 20, p. 167) is one
- a muscle sample is homogenised with
- Amyloglucosidase is added, converting the glycogen to glucose, for
- the first part of the method takes approximately 30 minutes at 37°C.
- the method also utilise mild or non-aggressive reagents.
- low glycogen levels in the liver of bobby calves are an
- rapid is used to refer to times of less than
- said complex carbohydrate is glycogen.
- solvent is water.
- said solvent may include at least one agent intended to
- steps (b) and (c) are performed simultaneously.
- steps (c) and (d) are performed simultaneously.
- the hydrolysing enzyme may be any enzyme, or combination of enzymes,
- hydrolysing enzyme is selected from the group: amyloglucosidase; -amylase;
- ⁇ -glucosidase ⁇ -glucosidase
- step (c) measuring complex carbohydrates in mammalian tissue, substantially as described above, wherein the amyloglucosidase added in step (c) is in a form selected from: a powder; a liquid
- said method further includes a step (e): measuring the
- steps (d) and (e) are performed simultaneously.
- steps (c), (d) and (e) are performed simultaneously.
- the method is conducted at approximately room temperature, although it is not
- concentration of glucose is achieved by the construction of sensors incorporating said
- hydrolysing enzyme and glucose oxidase hydrolysing enzyme and glucose oxidase.
- lactate as catalysed by lactate oxidase.
- This latter compound can be incorporated into any combination
- Figure 1 is a graph of glucose concentration and glucometer reading of a glucose
- Figure 2 is a graph of the glucometer reading for glucose and concentration of glucose added to meat slurry samples
- Figure 3 is a graph of the kinetics of glucose formation from glycogen in acetate buffer
- Figure 4 is a graph of glucometer readings and the concentration of glucose added to
- Figure 5 is a graph of glucose value and glycogen added to a post rigor meat slurry, at
- Figure 6 is a graph of muscle glycogen concentration in post slaughter samples, using
- Figure 7 is a graph showing the kinetics of glycogen loss, pH fall and lactate increase
- Figure 8 is a graph of the repeatability of glycogen determination by the method of the
- solution can be obtained by the use of 25 mg of powder, dissolved in 5 ml of 0.2M sodium acetate at a pH of 4.5. ⁇ -D-Glucose of a standard analytical grade was used.
- Glucose measurement was made with an Esprit glucometer (Bayer). Test sensors used in the
- Esprit were from Bayer New Zealand Limited. These were used for one reading only then
- the meat samples tested were obtained from the longissimus lumborum muscle of a beef
- test medium for all experiments was 0.2M sodium acetate buffer at a pH of 4.4 and at
- Polytron shearing head This was usually set at 25,000 ⁇ m. Alternatively a lower speed
- the homogeniser may be used, if so desired.
- the homogeniser may be a stainless steel paddle ⁇
- lactate concentration was also measured. At various times after
- amyloglucosidase was centrifuged in a micro-centrifuge (at 10, 000 ⁇ m for 30 seconds).
- the density of meat is about 1 g/ml.
- Example 1 The method of Example 1 was repeated over a range of samples and concentrations in which
- glycogen between 0 - 14 mg were added to 5 ml of acetate buffer and
- Meat samples ranged from between 0.97 - 1.07 g in six tests. The results are as
- gluteus maxims muscle sample contained 13.1 mg/g at 2.3 hours post slaughter
- This muscle is frequently used as an indicator of high pH condition. Lactate concentrations were also measured.
- NADH-linked method rather than the use of a sensor based on lactate oxidase.
- glycogen were added to three replicate rigor meat samples weighing 1.0 +/- 0.05 g.
- tissue samples may be equally treated in like manner to produce glycogen
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a rapid method of measuring complex carbohydrates in mammalian tissue, said method comprising the steps of: extracting a sample of tissue to be tested; forming a homogenous slurry of the sample with an aqueous solution; adding sufficient hydrolysing enzyme for ensuring complete hydrolysis of glycogen in the slurry; and measuring the concentration of glucose in the slurry. The method can be conducted post-mortem to assay the concentration at the time of slaughter. The invention further discloses the measurement of lactate concentrations. The method of the invention can be conducted in thirty minutes or less.
Description
TITLE: RAPID METHOD FOR MEASURING COMPLEX CARBOHYDRATES
IN MAMMALIAN TISSUE
TECHNICAL FIELD
The present invention relates to a rapid method for measuring complex carbohydrates,
particularly glycogen, in mammalian tissue. More particularly the present invention relates
to the rapid measurement of glycogen in non-living mammalian tissue.
BACKGROUND ART
There are presently a number of methods of measuring complex carbohydrates, and
particularly glycogen, in mammalian tissue. The discussion of these follows. The relevance
of the measurement of glycogen includes, for example, the ability to use the results of
glycogen measurement as a determination of the ultimate pH of meat. This in turn is a direct
measure of many of the qualities of meat.
There are a number of known methods of measuring ultimate pH in meat: including use of
liquid nitrogen in a freeze/thaw process and the use of a pH electrode for pH determination.
There are a number of variations of this method, also. However the maintenance and use
of liquid nitrogen in the quantities needed for the measurement on a continuous series of
carcasses reveals hazards for the work environment. Also there is some doubt as to the
accuracy and consistency of such measurement methods.
Methods of Measurement of Glycogen or Metabolites in Meat Samples
The iodine method: The principle of this method is that glycogen will react with a mixture
of iodide, iodine and calcium chloride, forming an amber pigment in acid solution that has
a linear absorption at least over a small, specified range. The glycogen is extracted from the
meat with perchloric acid that is then filtered and centrifuged to recover a solution of
glycogen which is reacted with the iodine. The extraction can also be by liquid nitrogen,
potassium hydroxide, ethanol and ammonium chloride.
However, methods of extraction and then assay are time-consuming and employ aggressive
chemical reagents.
Hydrolysis of glycogen with enzymes: The principle of this method is that glycogen
hydrolyses to glucose, after which standard methods of measurement of free glucose may be
used. The amyloglucosidase method of Dreiling et al (Meat Science, Vol 20, p. 167) is one
such method, although other enzymes may be used. A muscle sample is homogenised with
perchloric acid, and centrifuged, The supernatant, containing dissolved glycogen, is
neutralised. Amyloglucosidase is added, converting the glycogen to glucose, for
measurement. The first part of the method takes approximately 30 minutes at 37°C.
However there are some instances with the processing of meat in which a test of some 30
minutes or more is too long a time to wait for the test results, and some reagents are
aggressive.
It is an object of the present invention to provide a rapid method for glycogen measurement
in mammalian tissues, and in particular non-living tissue. It is a further object of the present
invention that in addition to the provision of a rapid accurate measurement of glycogen in
tissue, the method also utilise mild or non-aggressive reagents.
Whilst the prior art and uses of glycogen measurement have been described with reference
to a determination of the ultimate pH in meat, it will be appreciated that this method is not
limited thereto. For example, low glycogen levels in the liver of bobby calves are an
indicator of inadequate feeding before slaughter. Thus such rapid method of assessment of
these levels could assist in ensuring good animal welfare.
It is an object of the present invention to address the foregoing problems or at least to provide
the public with a useful choice.
Further aspects and advantages of the present invention will become apparent from the
ensuing description which is given by way of example only.
DISCLOSURE OF INVENTION
For the purposes of this specification, the term "rapid" is used to refer to times of less than
30 minutes and, more preferably, significantly less than 30 minutes
According to one aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, said method comprising the steps
of:
(a) extracting a sample of tissue to be tested;
(b) forming a homogenous slurry of the sample with an aqueous solution;
(c) adding sufficient hydrolysing enzyme for ensuring complete hydrolysis of
glycogen in the slurry; and
(d) measuring the concentration of glucose in the slurry.
Advantageously, said complex carbohydrate is glycogen. Advantageously, said aqueous
solvent is water. Optionally said solvent may include at least one agent intended to
standardise ionic conditions and/or facilitate the steps of the above method.
Preferably the formation of the homogenous slurry is effected with a high or a low speed
homogeniser, or with an ultrasonic apparatus.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above,
wherein steps (b) and (c) are performed simultaneously.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above,
wherein steps (c) and (d) are performed simultaneously.
Advantageously, the hydrolysing enzyme may be any enzyme, or combination of enzymes,
capable of hydrolysis of glycogen to glucose.
According to a still further aspect of the present invention there is provided a rapid method
of measuring complex carbohydrates in mammalian tissue, substantially as described above,
in which said hydrolysing enzyme is selected from the group: amyloglucosidase; -amylase;
α-glucosidase, and a combination thereof.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above,
wherein the amyloglucosidase added in step (c) is in a form selected from: a powder; a liquid
suspension; and a solution.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above, in
either embodiment, wherein said method further includes a step (e): measuring the
concentration of lactate in the sample.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above,
wherein steps (d) and (e) are performed simultaneously.
According to another aspect of the present invention there is provided a rapid method of
measuring complex carbohydrates in mammalian tissue, substantially as described above,
wherein steps (c), (d) and (e) are performed simultaneously.
The measurement of both metabolites gives a good post-mortem estimate of the
concentration of glycogen present in tissue at the time of death, no matter when the
measurement is made.
Advantageously, the method is conducted at approximately room temperature, although it
may be conducted at a temperature in the range 0°C to 100°C.
Measurement of Glucose
There are a range of methods for measuring glucose. For the present invention the most
useful are those adapted from known technologies to measure glucose in blood. These are
usually based on the generation of hydrogen peroxide in stoichiometric proportion to glucose,
as catalysed by glucose oxidase.
Therefore, according to a still further aspect of the present invention there is provided a rapid
method of measuring complex carbohydrates in mammalian tissue, substantially as described
above, and in which glycogen levels are measured and in which said measurement of the
concentration of glucose is achieved by the construction of sensors incorporating said
hydrolysing enzyme and glucose oxidase.
Measurement of Lactate
There are a range of methods for measuring lactate, including the standard NADH-linked
method and those based on the generation of hydrogen peroxide in stoichiometric proportion
to lactate, as catalysed by lactate oxidase. This latter compound can be incorporated into any
sensor which may already incorporate glucose oxidase.
BRIEF DESCRIPTION OF THE DRAWINGS
Further aspects of the present invention will become apparent from the following example,
which is given by way of example only, and with reference to the accompanying drawings
in which:
Figure 1 is a graph of glucose concentration and glucometer reading of a glucose
sample in acetate buffer;
Figure 2 is a graph of the glucometer reading for glucose and concentration of glucose
added to meat slurry samples;
Figure 3 is a graph of the kinetics of glucose formation from glycogen in acetate buffer
in the present of amyloglucosidase;
Figure 4 is a graph of glucometer readings and the concentration of glucose added to
a meat/acetate buffer slurry;
Figure 5 is a graph of glucose value and glycogen added to a post rigor meat slurry, at
5 minutes incubation at 55 °C;
Figure 6 is a graph of muscle glycogen concentration in post slaughter samples, using
the method of measurement of the present invention;
Figure 7 is a graph showing the kinetics of glycogen loss, pH fall and lactate increase
in a bovine muscle sampled after slaughter; and
Figure 8 is a graph of the repeatability of glycogen determination by the method of the
present invention.
BEST MODES FOR CARRYING OUT THE INVENTION
Chemicals, Equipment and Meat.
Amyloglucosidase from the fungus Aspergillus niger, in powder form, was added to meat
slurries as a liquid suspension or solution. In suspension, 25 mg amyloglucosidase was
dissolved in 5 ml of 3.2M ammonium sulphate and adjusted to pH 6.0 with ammoma. This
particular solvent is known to be one in which the enzyme is stable. As an alternative, a clear
solution can be obtained by the use of 25 mg of powder, dissolved in 5 ml of 0.2M sodium
acetate at a pH of 4.5. α-D-Glucose of a standard analytical grade was used.
Glucose measurement was made with an Esprit glucometer (Bayer). Test sensors used in the
Esprit were from Bayer New Zealand Limited. These were used for one reading only then
discarded.
The meat samples tested were obtained from the longissimus lumborum muscle of a beef
carcass, obtained from a butcher. Pre-rigor muscle, variously gluteus medius, semitendinosus
and longissimus lumborum, was obtained from an abattoir. These muscles were dissected
from unstimulated carcasses approximately 25 minutes after slaughter and tested very shortly
thereafter. The muscles were held at room temperature while measurements were made.
Glycogen Test Procedure
The test medium for all experiments was 0.2M sodium acetate buffer at a pH of 4.4 and at
a temperature of 55°C. In the experiments the muscle or meat (in samples of accurately
known weight, but approximately 1 g) was homogenised in 5 ml of buffer, with a high speed
Polytron shearing head. This was usually set at 25,000 φm. Alternatively a lower speed
homogeniser may be used, if so desired. The homogeniser may be a stainless steel paddle¬
like blade rotating at 2000 φm in a steel cup within an interior shaped like a standard
domestic Waring blender.
After homogenisation the enzyme solution was added. The volume of this solution was
usually 200 μl, containing 1 mg of amyloglucosidase. The mixture was briefly shaken, then
held at 55°C.
Small aliquots, of approximately 20 μl, were withdrawn at intervals with disposable pipettes,
and spotted onto plastic film. The glucometer sensor sampled these drops and returned a
meter reading for glucose concentration in 30 seconds.
As a control, tests were also carried out with a range of glucose and glycogen concentrations
in an acetate buffer to which no meat or meat samples had been added.
Lactate Measurement
In one range of experiments lactate concentration was also measured. At various times after
slaughter, a crude aliquot of the slurry, containing homogenised muscle and
amyloglucosidase was centrifuged in a micro-centrifuge (at 10, 000 φm for 30 seconds).
The clear supernatant was recovered and analysed for lactate concentration by the NADH-
linked method.
Results
The results from the glucometer were in mg of glucose/dl. These results are tabulated in Fig.
1 of the attached drawings. It is noted that the relation was linear but that the readings were
approximately double the glucose concentration in fact present and did not pass through the
origin.
The exact reason for the approximate doubling of the readings is not known. However it is
understood that this might relate to the characteristics of glucose in blood. α-D-glucose as
a laboratory chemical dissolved in acetate at a pH of 4.5 may require a different calibration
from that in blood. However, the failure of the straight line to pass through the origin
suggests that the acetate medium affected the sensor performance.
Example 1
Various samples of meat (the samples being as described above) between 0.90 and 1.16 g
were homogenised in the high speed homogeniser in 5 ml of acetate buffer containing up to
16 mg of added glucose. This addition rate translates to approximately 267 mg/dl, assuming
the density of meat is about 1 g/ml.
As can be seen from Fig. 2 of the attached drawings the relationship, the relationship between
added glucose and the average meter reading is linear. The positive value of detected glucose
with zero added glucose may be explained by the small quantities of glucose left over from
glycolysis in the meat samples.
Example 2
The method of Example 1 was repeated over a range of samples and concentrations in which
various quantities of glycogen (between 0 - 14 mg) were added to 5 ml of acetate buffer and
the reactions were started by amyloglucosidase addition.
The results are as set out in Fig. 3 of the attached drawings, in which can be seen the glucose
values peaked and declined slightly. The reason for the decline is not understood but it is
possible that this may result from a contaminating activity in the enzyme preparation or from
isomerisation reactions of glucose liberated from glycogen.
The data tabulated in Fig. 3 suggest that at even the highest concentrations of glycogen,
around 40 activity units of amyloglucosidase, are sufficient to fully hydro lyse the glycogen
within approximately 5 minutes.
Example 3
The experiment the results of which are tabulated in Fig. 3 was repeated in the presence of
rigor meat. Meat samples ranged from between 0.97 - 1.07 g in six tests. The results are as
set out in Fig. 4 of the attached drawings.
Individual data points were abstracted to plot glycogen added against meter readings for 5
minutes after the amyloglucosidase addition. The results of this are shown in Fig. 5. A
quadratic equation was fitted, but the shape was close to a straight line.
Example 4
Samples of gluteus medius and a semitendinosus muscle were removed from pre-rigor meat,
cut to samples in the size between 0.95 - 1.05 g, and homogenised in 5 ml of acetate buffer.
This slurry was then treated with amyloglucosidase. The tests began 1.3 hours after
slaughter, first with the semitendinosus muscle and at that time the muscle contained only
3.8 mg glycogen /g. By 2 hours the glycogen level declined to 1 mg/g.
In contrast, gluteus medius muscle sample contained 13.1 mg/g at 2.3 hours post slaughter,
and the value declined steadily with time. The results are as shown in Fig. 6.
This experiment was repeated using longissimus lumborum muscle from another animal.
This muscle is frequently used as an indicator of high pH condition. Lactate concentrations
were also measured.
The above tests as described were carried out and the results are as shown in Fig. 7 of the
attached drawings. In this method the measurement of lactate was as per the standard
NADH-linked method rather than the use of a sensor based on lactate oxidase.
Example 5
The robustness of the preferred embodiment of the test of the present invention was further
determined by the following: meter readings were recorded in which triplicate aliquots of
glycogen were added to three replicate rigor meat samples weighing 1.0 +/- 0.05 g. The
slurry was treated in the same manner as described above for Example 2. The results of this
are set out in Fig. 8 of the attached drawings.
Whilst the Examples given above to show the best method of performing the invention are
all with reference to meat samples (beef) it would be appreciated by those skilled in the arts
that other tissue samples may be equally treated in like manner to produce glycogen
measurement as a result.
Aspects of the present invention have been described by way of example only and it should
be appreciated that modifications and additions may be made thereto without departing from
the scope thereof.
Throughout this specification and the claims which follow, unless the context requires
otherwise, the word "comprise", or variations such as "comprises" or "comprising", will
be understood to imply the inclusion of a stated integer or step or group of integers or
steps but not the exclusion of any other integer or step or group of integers or steps.
Claims
1. A rapid method of measuring complex carbohydrates in mammalian tissue, said method
comprising the steps of:
(a) extracting a sample of tissue to be tested;
(b) forming a homogenous slurry of the sample with an aqueous solution;
(c) adding sufficient hydrolysing enzyme for ensuring complete hydrolysis of
glycogen in the slurry; and
(d) measuring the concentration of glucose in the slurry.
2. A rapid method of measuring complex carbohydrates as claimed in claim 1 wherein
said method is performed in less than 30 minutes.
3. A rapid method of measuring complex carbohydrates as claimed in either claim 1 or
claim 2 wherein said complex carbohydrate is selected from glycogen, lactate and a
combination of these.
4. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said aqueous solvent is water.
5. A rapid method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein said aqueous solvent includes at least one agent to standardise
ionic conditions obtaining for the method.
6. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein the formation of the homogenous slurry is effected with apparatus
selected from: a high speed homogeniser; a low speed homogeniser and an ultrasonic
apparatus.
7. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said hydrolysing enzyme is selected from the group:
amyloglucosidase; α-amylase; α-glucosidase, and a combination thereof.
8. A rapid method of measuring complex carbohydrates as claimed in claim 7 wherein
said hydrolysing enzyme is amyloglucosidase which is in a form selected from: a powder;
a liquid suspension; and a solution.
9. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said method further includes a step (e): measuring the
concentration of lactate in the sample.
10. A rapid method of measuring complex carbohydrates as claimed in any one of the preceding claims wherein steps (b) and (c) are performed simultaneously.
11. A rapid method of measuring complex carbohydrates as claimed in any one of claims
1 to 9 wherein steps (c) and (d) are performed simultaneously.
12. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein
steps (d) and (e) are performed simultaneously.
13. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein
steps (c) to (e) are performed simultaneously.
14. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein
said measurement of lactate concentration is by the NADH-linked method.
15. A rapid method of measuring complex carbohydrates as claimed in claim 9 wherein
said measurement of lactate concentration is based on the generation of hydrogen peroxide
in stoichiometric proportion to lactate, as catalysed by lactate oxidase.
16. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said glycogen measurement is effected by use of sensors which incoφorate said hydrolysing enzyme and glucose oxidase.
17. A rapid method of measuring complex carbohydrates as claimed in claim 16 wherein
said sensor further includes lactate oxidase.
18 A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said method is carried out at room temperature.
19. A rapid method of measuring complex carbohydrates as claimed in any one of the
preceding claims wherein said method is performed post-mortem, providing a measurement
of concentrations of complex carbohydrates at the time of death.
20. A rapid method of measuring complex carbohydrates as claimed in claim 19 wherein
said method is performed wherein said measurement is up to half an hour after slaughter.
21. Measurement of ultimate pH by use of the method of measuring complex
carbohydrates as claimed in any one of the preceding claims wherein said tissue is meat.
22. Measurement of ultimate pH by use of the method of measuring complex
carbohydrates as claimed in any one of the preceding claims wherein said tissue is muscle.
23. Measurement of ultimate pH by use of the method of measuring complex
carbohydrates as claimed in claim 22 wherein said muscle is selected from: the longissimus
lumborum; gluteus medius; semitendinosus and longissimus lumborum muscles.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| NZ33727699 | 1999-08-17 | ||
| NZ33727699 | 1999-08-17 | ||
| PCT/NZ2000/000159 WO2001012844A1 (en) | 1999-08-17 | 2000-08-17 | Rapid method for measuring complex carbohydrates in mammalian tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| EP1210451A1 true EP1210451A1 (en) | 2002-06-05 |
| EP1210451A4 EP1210451A4 (en) | 2004-08-25 |
Family
ID=19927450
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP00955193A Withdrawn EP1210451A4 (en) | 1999-08-17 | 2000-08-17 | Rapid method for measuring complex carbohydrates in mammalian tissue |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1210451A4 (en) |
| AR (1) | AR025303A1 (en) |
| AU (1) | AU779415C (en) |
| BR (1) | BR0013395A (en) |
| CA (1) | CA2382155A1 (en) |
| WO (1) | WO2001012844A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0218868D0 (en) | 2002-08-14 | 2002-09-25 | Nasir Muhammed A | Improved airway management device |
| GB2521375C (en) | 2013-12-17 | 2021-09-29 | Aslam Nasir Muhammed | Intubating Airway Device |
| USD842456S1 (en) | 2015-12-15 | 2019-03-05 | Intersurgical Ag | Airway device |
| USD1051359S1 (en) | 2015-06-15 | 2024-11-12 | Intersurgical Ag | Airway device |
| GB201720733D0 (en) | 2017-12-13 | 2018-01-24 | Ashkal Development Ltd | Airway device |
| USD1025348S1 (en) | 2020-04-16 | 2024-04-30 | Intersurgical Ag | Airway device |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0086108A2 (en) * | 1982-02-08 | 1983-08-17 | Children's Hospital Medical Center | Method of measuring lactic acid in a liquid |
| US5262305A (en) * | 1991-03-04 | 1993-11-16 | E. Heller & Company | Interferant eliminating biosensors |
-
2000
- 2000-08-17 AU AU67425/00A patent/AU779415C/en not_active Ceased
- 2000-08-17 BR BR0013395-7A patent/BR0013395A/en not_active IP Right Cessation
- 2000-08-17 CA CA002382155A patent/CA2382155A1/en not_active Abandoned
- 2000-08-17 AR ARP000104247 patent/AR025303A1/en unknown
- 2000-08-17 EP EP00955193A patent/EP1210451A4/en not_active Withdrawn
- 2000-08-17 WO PCT/NZ2000/000159 patent/WO2001012844A1/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0086108A2 (en) * | 1982-02-08 | 1983-08-17 | Children's Hospital Medical Center | Method of measuring lactic acid in a liquid |
| US5262305A (en) * | 1991-03-04 | 1993-11-16 | E. Heller & Company | Interferant eliminating biosensors |
Non-Patent Citations (5)
| Title |
|---|
| BURTON SHELLEY A ET AL: "Evaluation of a glucose oxidase/peroxidase method for indirect measurement of glycogen content in marine mussels (Mytilus edulis)" JOURNAL OF SHELLFISH RESEARCH, vol. 16, no. 2, December 1997 (1997-12), pages 435-439, XP001182147 ISSN: 0730-8000 * |
| BURTON SHELLEY A ET AL: "Evaluation of a glucose oxidase/peroxidase method for indirect measurement of glycogen content in oysters (Crassostrea virginica)" JOURNAL OF SHELLFISH RESEARCH, vol. 19, no. 2, December 2000 (2000-12), pages 841-844, XP001181700 ISSN: 0730-8000 * |
| CARR R S ET AL: "Quantitative semi-automated enzymatic assay for tissue glycogen." COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY. B, COMPARATIVE BIOCHEMISTRY. ENGLAND 1984, vol. 77, no. 3, 1984, pages 447-449, XP002286075 ISSN: 0305-0491 * |
| See also references of WO0112844A1 * |
| VOET AND VOET: "Biochemie" 1992 , VCH , WEINHEIM XP002286178 * page 480, column 1, paragraph 1 * * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2001012844A1 (en) | 2001-02-22 |
| AU6742500A (en) | 2001-03-13 |
| BR0013395A (en) | 2002-07-09 |
| CA2382155A1 (en) | 2001-02-22 |
| EP1210451A4 (en) | 2004-08-25 |
| AR025303A1 (en) | 2002-11-20 |
| AU779415B2 (en) | 2005-01-20 |
| AU779415C (en) | 2006-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Young et al. | A method for early determination of meat ultimate pH | |
| Dreiling et al. | Muscle glycogen: Comparison of iodine binding and enzyme digestion assays and application to meat samples | |
| Carr et al. | Quantitative semi-automated enzymatic assay for tissue glycogen | |
| US4127448A (en) | Amperometric-non-enzymatic method of determining sugars and other polyhydroxy compounds | |
| Weber et al. | Interferences in current methods for measurements of creatinine | |
| Cotlove | Determination of the True Chloride Content of Biological Fluids and Tissues. II. Analysis by Simple, Nonisotopic Methods. | |
| Guest et al. | A study of antifibrinolysin activity in the plasmas of various animal species | |
| Watanabe et al. | Determination of phosphate ions with an enzyme sensor system | |
| AU779415C (en) | Rapid method for measuring complex carbohydrates in mammalian tissue | |
| Jensen et al. | Determination of the fructosamine concentration in bovine serum samples | |
| JPH0425000B2 (en) | ||
| Denyer et al. | Effect of starvation and insulin in vivo on the activity of the pyruvate dehydrogenase complex in rat skeletal muscles | |
| CA1230807A (en) | Simplified method for determining the degree of freshness of fish and mollusks | |
| Jung et al. | Diagnostic value of low-molecular mass proteins in serum for the detection of reduced glomerular filtration rate | |
| Lüderitz et al. | A specific microdetermination of glucosamine and the analysis of other hexosamines in the presence of glucosamine | |
| CN118308457B (en) | Use of olfactory receptors for the recognition of trimethylamine and method for detecting trimethylamine | |
| Wilhelmi et al. | Some aspects of the nitrogen metabolism of liver tissue from rats in hemorrhagic shock | |
| Gnaedinger | THIAMINASE ACTIVITY IN FISH | |
| Arnold et al. | Determination of glutamine in cerebrospinal fluid with a tissue-based membrane electrode | |
| Campanella et al. | An enzyme sensor for determination of acetylcholine and choline in rat brain extracts | |
| TAGAMI et al. | An enzymatic method for the kinetic measurement of L-asparaginase activity and L-asparagine with an ammonia gas-sensing electrode | |
| US4096037A (en) | Arginase test | |
| Venturini et al. | Aluminium speciation studies in biological fluids. A new investigation of aluminium hydroxide equilibria under physiological conditions | |
| Troy et al. | The coulometric determination of uric acid in seruma and urine | |
| Hemker et al. | The evaluation of the two-stage prothrombin assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
| 17P | Request for examination filed |
Effective date: 20020311 |
|
| AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE |
|
| AX | Request for extension of the european patent |
Free format text: AL;LT;LV;MK;RO;SI |
|
| A4 | Supplementary search report drawn up and despatched |
Effective date: 20040712 |
|
| STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
| 18D | Application deemed to be withdrawn |
Effective date: 20070301 |