WO2001012844A1 - Rapid method for measuring complex carbohydrates in mammalian tissue - Google Patents

Rapid method for measuring complex carbohydrates in mammalian tissue Download PDF

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Publication number
WO2001012844A1
WO2001012844A1 PCT/NZ2000/000159 NZ0000159W WO0112844A1 WO 2001012844 A1 WO2001012844 A1 WO 2001012844A1 NZ 0000159 W NZ0000159 W NZ 0000159W WO 0112844 A1 WO0112844 A1 WO 0112844A1
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WO
WIPO (PCT)
Prior art keywords
complex carbohydrates
method
measuring complex
rapid method
measuring
Prior art date
Application number
PCT/NZ2000/000159
Other languages
French (fr)
Inventor
John West
Alan Leedham Hart
Owen Archibald Young
Original Assignee
Agresearch Limited
New Zealand Meat Board
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority to NZ337276 priority Critical
Priority to NZ33727699 priority
Application filed by Agresearch Limited, New Zealand Meat Board filed Critical Agresearch Limited
Priority claimed from NZ51744200A external-priority patent/NZ517442A/en
Publication of WO2001012844A1 publication Critical patent/WO2001012844A1/en

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/40Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase

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.
PCT/NZ2000/000159 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue WO2001012844A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
NZ337276 1999-08-17
NZ33727699 1999-08-17

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
BR0013395-7A BR0013395A (en) 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue
NZ51744200A NZ517442A (en) 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue
EP00955193A EP1210451A4 (en) 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue
CA 2382155 CA2382155A1 (en) 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue
AU67425/00A AU779415C (en) 1999-08-17 2000-08-17 Rapid method for measuring complex carbohydrates in mammalian tissue

Publications (1)

Publication Number Publication Date
WO2001012844A1 true WO2001012844A1 (en) 2001-02-22

Family

ID=19927450

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Country Status (6)

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EP (1) EP1210451A4 (en)
AR (1) AR025303A1 (en)
AU (1) AU779415C (en)
BR (1) BR0013395A (en)
CA (1) CA2382155A1 (en)
WO (1) WO2001012844A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9475223B2 (en) 2002-08-14 2016-10-25 Muhammed Aslam Nasir Method of manufacturing an airway device
USD842456S1 (en) 2015-12-15 2019-03-05 Intersurgical Ag Airway device

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4467811A (en) * 1979-08-02 1984-08-28 Children's Hospital Medical Center Method of polarographic analysis of lactic acid and lactate
US5262305A (en) * 1991-03-04 1993-11-16 E. Heller & Company Interferant eliminating biosensors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CALDER P.C. AND GEDDES R.: "Post mortem glycogenolysis is a combination of phosphorolysis and hydrolysis", INT J. BIOCHEM., vol. 22, no. 8, 1990, pages 847 - 856, XP002953344 *
See also references of EP1210451A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9475223B2 (en) 2002-08-14 2016-10-25 Muhammed Aslam Nasir Method of manufacturing an airway device
USD842456S1 (en) 2015-12-15 2019-03-05 Intersurgical Ag Airway device

Also Published As

Publication number Publication date
AU779415C (en) 2006-02-09
AU6742500A (en) 2001-03-13
EP1210451A1 (en) 2002-06-05
EP1210451A4 (en) 2004-08-25
AR025303A1 (en) 2002-11-20
CA2382155A1 (en) 2001-02-22
BR0013395A (en) 2002-07-09
AU779415B2 (en) 2005-01-20

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