Analysis method for determining a functional parameter
of an organ using preferably an aqueous 13C-methacetin
solution
Description
The invention relates to an analysis method according
to the preamble of claim 1 for determining a functional
parameter of an organ, to an aqueous methacetin
solution according to the preamble of claim 19,
suitable for this analysis method, to the use of this
methacetin solution in accordance with the preambles of
claims 32 and 33, to a respiratory mask according to
the preamble of claim 34, for use in an analysis method
according to the invention, and to a diagnostic method
according to the preamble of claim' 48, for determining
a functional parameter of an organ.
The determination of a functional parameter of an
organ, in particular the quantitative determination of
liver function, is of great importance in many areas of
medicine. Chronic liver diseases are widespread in
Europe, with 8.9 million people affected by hepatitis
C. As their disease progresses, these individuals or
patients find themselves in most cases under permanent
medical care. In the therapy and management of patients
with chronic liver diseases, quantifying the liver

- 2 -
function can greatly improve the therapy control, and
assessment of liver function is crucial in ensuring
that-the correct therapeutic decisions are made.
Partial liver resection is a common method used in
surgery today. It is performed as a segmental resection
or hemihepatectomy along the anatomical margins..
Extensive interventions in the parenchymatous organ
were made possible by the development of a wide variety
of operating technigues. The post-operative morbidity
and mortality after liver failure, however, is still a
considerable problem, due to inadeguate liver function
capacity resulting from previously damaged liver tissue
or from there being too little liver tissue remaining.
Many- of the surgical procedures, however, have to be
performed in previously damaged liver tissue, in most
cases where the liver has been transformed by
cirrhosis. It is therefore necessary to be able to
determine the functional liver capacity of a patient
before the partial liver resection, so as to ensure
that patients who no longer have sufficient functional
reserves of liver tissue are not subjected to what is
for them a high-risk operation or are not assigned to
other treatment methods.
Assessment of liver function is of particular
importance in liver transplantation, since here the
organ function has to be assessed without delay and a
treatment decision has to be made guickly. In many
clinical situations, it is also difficult to assess
whether there is a parenchymatous disturbance or
whether other causes are responsible for the clinical
symptoms presented by the patients. In summary,
therefore, there is a great need for a genuinely
quantitative liver function test for broad application
in medicine.
Efforts are therefore being made across the world to
develop simple tests that allow prognostic statements

- 2a -
to be made concerning the functional reserves of liver
cell tissue. Conventional laboratory parameters are
very, unreliable and therefore unsuitable for this
purpose. They are not sufficiently sensitive to permit
reliable evaluation of the complex biological processes
in the hepatocyte (biosynthesis, biotransformation,
catabolism of xenobiotics, etc.) and of the changes in
these processes in the presence of disease.
In addition, they are subject to a large number of
external influences and are distorted by these. For
example, they are to some extent distorted by the
required therapeutic intervention, by replacement of
human plasma, clotting factors or albumin, and can thus
not be used as liver function parameters. Many
different liver function tests have been described in
the literature (Matsumoto, K., M. Suehiro, et al.
(1987): "[13C]methacetin breath test for evaluation of
liver damage." Dig Pis Sci 32 (4): 344-8, 1987.;
Brockmoller, J.

- 3 -
and I. Roots (1994): "Assessment of liver metabolic
function. Clinical implications." Clin Pharmacokinet 27
(3):.216-48).
However, it has not hitherto been possible, with any
Lest method, to make valid and genuinely quantitative
statements on liver function. In all methods to date,
it was possible only to make a significant
differentiation between different disease groups with
already clinically detectable signs. Consequently, in
clinical practice, no liver function test is employed
in routine diagnostics, since these tests do not afford
any additional clinical benefit based on their present
accuracy.
The 13C-methacetin breath test used hitherto, with an
exclusively oral administration of the substance, is a
method which can distinguish between the liver function
capacity of healthy subjects and that of patients with
chronic hepatitis without cirrhosis and with cirrhosis
in the different Child-Pugh stages (Matsumoto, K., M.
Suehiro, et al. (1987): " [13C] methacetin breath test for
evaluation of liver damage." Dig Pis Sci 32 (4): 344-8,
1987), but does not permit a genuine quantification.
The aim of genuine quantification with an individual
The substance methacetin is demethylated to paracetamol
in a- rapid one-step reaction by the enzyme CYP1A2 in
the liver, with C02 subsequently being produced. By 13C-
iabeling of the methyl group bonded via the ether
bridge, 13C02 can then be measured in the exhaled air.
The following formula (I) represents the structural
formula of methacetin:


- 3a -
measurement result cannot be achieved using the
previous methods. There are two reasons for this:
1. The basis for statements derived from a breath test
i.s that the step to be evaluated in the cascade of
processes of absorption and metabolism has to be the
step that determines the reaction rate. In the previous
methods for evaluating the liver function (oral
administration of the test substance), however, the
rate-determining step is in most cases the absorption,
not; the conversion of the substrate in the liver.

- 4 -
2. To be able to make quantitative statements on the
basis of an enzyme system (in the present case: to be
able to determine the maximum liver function capacity,
that is to say the functional liver capacity), the
enzyme system to be tested has to be fully utilized at
least in the short term. Only in this case does the
reaction proceed independently of the substrate
concentration.
For a genuine quantification, therefore, it is
imperative to reach substrate surplus. If this is not
achieved, the reaction rate is directly proportional to
and therefore dependent on the substrate concentration,
which for its parts drops non-linearly. A quantitative
statement on functional capacity is impossible. In all
studies using oral test substances, no genuine
quantification could therefore take place, because full
enzyme utilization is not achieved with the previous
methods. This has the following causes:
1. When used orally, methacetin must first pass through
the stomach and be transported as far as the duodenum
and the proximal jejunum in order to be absorbed. Only
then can the substance reach the liver by way of the
portal vein. In principle, this process costs time and
results in delayed and incomplete inundation in the
liver. This is extremely variable and is influenced by
numerous physiological and pathological conditions. For
example, in cirrhosis of the liver, in which liver
function tests could be used for staging and for
therapy management, the intestinal transit and
absorption is greatly changed (Castilla-Cortazar, I..,
J. Prieto, et al. (1997): "Impaired intestinal sugar
transport in cirrhotic rats: correction by low doses of
insulin-like growth factor I". Gastroenterology 113
(4): 1180-7). In the period following abdominal
operations too (e.g. liver resections or liver
transplants), intestinal atony (paralytic ileus) means
that no reliable statement can be made at all.

- 4a -
2. A sufficient dose of the test substance is
necessary. With too low a dose, as in most methods for
carrying out the oral methacetin breath test, full
utilization of the enzyme system per se is not
achieved.
It should also be noted that methacetin is extremely
sparingly soluble in water or in an aqueous buffer. It
crystallizes out of a usually aqueous solution within a
period of hours to days. Such a solution can be used
only, if indeed at all, for oral administrations of
methacetin. Other administration forms are not
possible.

- 5 -
Moreover, in the previous methods, the percentage
recovery rate of the applied dose (dose%/h) and the
cumulative dose are analyzed at specific times or time
intervals in order to determine the liver function. The
calculation of the dose%/h does not absolutely define
the reacted substrate quantities and also does not take
account of the individual bodyweight of the patient. It
is not possible in this way to individualize and thus
standardize the results in order to class the maximum
functional liver capacity into a standard population.
The previous determination of the metabolized
cumulative dose Dkum over a defined period of time is
equally inexpressive in respect of functional liver
capacity. For a reliable statement concerning the
maximum conversion of the enzyme system over time, said
system would have to be fully utilized over the entire
period. For the reasons mentioned above, this is not
the case. Consequently, the presently used calculation
of the cumulative dose cannot be used for quantifying
the functional liver capacity.
To transfer the air exhaled by an individual into a
measurement device, it is recommended to use a
respiratory mask which is placed onto the face of the
individual. For the subsequent reliable conduct of an
analysis method, it is critically important that the
exhaled air is safely separated from the inhaled air
and, in addition, that unforced breathing by the
individual is permitted by a low airway resistance of
the respiratory mask.
Various types of respiratory masks are in common use in
medicine, in occupational safety and also in diving. In
medicine, this is the case in the induction and
performance of anesthesia or also for respiratory
therapy and noninvasive ventilation. Masks with a good
matching shape and a tight fit are preferred, and the
required valves are fitted outside the masks, in the

- 5a -
tube systems or in the other connected appliances.
Valves are installed in some masks used in occupational
safety and also in masks used in diving, but the focus
here lies in the delivery of respiratory gas and in the
secure sealing of the system. High airway resistance
generally arises in these cases, with the result that,
for example, a medical test is needed to ensure
suitability before occupational use of such a system.

- 6 -
To analyze certain constituents in the exhaled air, it
is necessary to separate the respiratory gas path as
close as possible to the site of origin of the exhaled
substances, i.e. as close as possible to the pulmonary
alveoli. Otherwise, the inhaled air and the exhaled air
mix together. Moreover, the separation must not cause
any substantial increase in airway resistance,
especially not in the case of patients whose pulmonary
function is compromised for whatever reason. The
inhalation resistance specifically should not
substantially increase, since the respiratory work or
the supply of gas cannot be mechanically assisted as it
is, for example, in anesthesia, ventilation, or in
diving equipment or occupational safety equipment.
Moreover, it is of great importance to establish,
during the analysis, whether the respiratory mask is
sitting tightly on the face or has possibly just been
taken off.
The object of the invention is to make available a
reli able analysis method for determining a functional
parameter of an organ of an individual and also a
corresponding diagnostic method, and to make available
a mothacetin solution in which dissolved methacetin
remains stably dissolved over a period of weeks or
months and can thus be used as a substrate in the
methods according to the invention.
This object is achieved by an analysis method, with the
features of claim 1, for determining a functional
parameter of an organ of a human or animal individual.
According to this analysis method, the 13CO2 content in
the air exhaled by the individual is measured, the 13CO2
in the body of the individual being formed
enzymatically from a substrate that has been
administered beforehand to the individual, and then
being exhaled by the individual. The measurement of the
13CO2 content in the air exhaled by the individual is
carried out using a suitable measurement device. The

- 6a -
maximum reaction rate of the substrate in the body of
the individual is determined via a change of the
measured 13CO2 content in the air exhaled by the
Individual using zero-order enzyme kinetics. The
analysis method according to the invention therefore
proceeds from a consideration of enzyme kinetics.
The functional parameter of an organ that is to be
determined is preferably the liver function capacity
and/or the microcirculation in the liver. Thus, the
analysis method is suitable in particular for
quantifying the functional liver capacity of the
individual. The function of the liver, as the central
organ of metabolism, is extremely complex. Many
biochemical synthesis and degradation processes take
place in the liver. A common feature, however, is that
almost all of them function on the basis of an
enzymatic metabolism.

- 7 -
The 13CO2/12CO2 ratio in the air exhaled by the
individual is preferably determined. This value can be
used as the 13CO2/12CO2 ratio in the formula (1) below.
In a particularly preferred embodiment of the
invention, the maximum reaction rate (LiMAx) is
calculated through the converted quantity of substrate
per unit of time in (μg/h/kg bodyweight at variable
times at which the maximum value is reached, such that
genuine quantification of the maximum functional liver
capacity can be made. The calculation is carried out
according to the following formula (1), which describes
zero-order enzyme kinetics:

Here, 513C is the difference between the 13CO2/12CO2 ratio
of the sample and the Pee Dee Belmite (PDB) standard in
delta per mil, RPDB is the 13CO2/12CO2 ratio of the PDB
standard (0.0112375), P is the CO2 production rate
(300 mmol/h x surface area of body in m2) , M is the
molecular weight of the substrate, and KG is the actual
bodyweight of the individual in kg.
In another preferred embodiment of the invention, it is
not the 13CO2/12CO2 ratio, but the absolute 13CO2 content
that is determined in the air exhaled by the
individual. This is possible, for example, by means of
isotope-selective infrared spectroscopy. In the formula
(1), the absolute 13CO2 volume concentrations integrated
over time are then used directly instead of the
13CO2/12CO2 ratios, and it is therefore also possible to
omit the factors RPDB and P and, consequently, the
dependence of the merely generally estimated CO2 -
production rate. The 13CO2 volume concentration
represents the concentration of the 13CO2 in the whole

- 7a -
of the exhaled air, that is to say that, in the
preferred use of the 13CO2 content in the air exhaled by
the individual, the volume of the entire respiratory
gas stream is determined in addition to the 13CO2
concentration. This also yields the rate of metabolism
(μg/h/kg) , that is to say the converted quantity of
substrate per unit of time, standardized to the
bodyweight of the individual.
The formula (1) is simplfied to the formula (2) when
determining the absolute 13CO2 concentration:


- 8 -
Here, [13CO2] is the absolute concentration of the 13CO2
per unit of volume in the air exhaled by the
individual, & is the volume per unit of time, t is the
time, traax is the time of maximum metabolism, i is the
smallest possible time resolution according to the
measurement method, M is the molecular weight of the
substrate, and KG is the current bodyweight of the
individual in kg.
An infrared spectrometer is preferably used to
determine the 13CO2 content in the exhaled air, since
13CO2 has an absorption band easily separated in the
infrared range.
In NDIRS measurement devices (NDIRS = nondispersive
infrared spectroscopy), the water vapor contained in
the exhaled air is removed before the measurement,
advantageously by a humidity exchanger connected
upstream of the measurement device, in order to avoid
undesired absorption of the water vapor in the infrared
range. A particularly suitable humidity exchanger is,
for example, a Nation humidity 'exchanger. However,
other humidity exchangers that are able to effectively
dry the air exhaled by the individual are also
similarly suitable.
In i sotope-selective determination of the absolute 13CO2
content in the air exhaled by an individual, this
drying of the exhaled air to be analyzed is preferably
omitted, since the bands of the water vapor are not
superposed with the 13CO2 band or bands to be observed.
In a preferred embodiment of the invention, not only is
the maximum reaction rate of the substrate in the body
of the individual determined, but also the inundation
time, that is to say the time necessary to reach the
maximum reaction rate.
The inundation time is preferably used to assess the

- 8a -
microcirculation in the liver, so as to be able to
detect microcirculation disturbances. Assuming the
quickest possible inundation by i.n particular
intravenous bolus injection and substrate excess and
the high first-pass effect of methacetin or another
substrate, the hepatic microcirculation can be assessed
particularly advantageously. The inundation time needed
to reach the maximum reaction rate tvmax is determined,
said inundation time lengthening in the presence of
microcirculation disturbances, since in this case the
substrate inundation is not uniformly or completely
delayed in all areas of the liver. Liver perfusion can
thus be assessed.

- 9 -
The microcirculation and the liver perfusion are
preferably not just assessed in isolation, but also
assigned to a standard population. To do so, the
inundation time is standardized to a normal, population
taking into account the bodyweight of the individual.
The same applies also to the maximum reaction rate. By
the reference to the individual bodyweight, it is
possible to eliminate an interindividual variability
and thus achieve a standardization. Only in this way is
it possible to class the individual functional liver
capacity into a comparison population. With the method
according to the invention, it is not only possible to
differentiate between limited liver function (e.g. in
manifest cirrhosis of the liver) and healthy liver
performance; instead, as a result of the rapid and
complete utilization of the enzyme system, very slight
differences in the maximum reaction rate (functional
liver capacity) can now be determined over a wide
measurement range.
To be able to detect the time of the maximum reaction
rate, it is necessary to analyze the 13CO2/12CCO2 ratio
and the relative or absolute 13CO2 content in the
exhaled air over time.
Therefore, samples of respiratory gas are preferably
collected discontinuously at defined times and are
analyzed with a respiratory gas analyzer (measurement
device) according to one of the techniques described
above. For example, the respiratory gas samples can be
collected at the times 0 min and 2, 5, 10, 15, 20, 30,
40, .50 and 60 min after the application of the test
substance (substrate). This measurement method is also
referred to as discontinuous offline measurement. The
measurement of the respiratory gas. samples, that is to
say of the exhaled air to be analyzed, can be carried
out directly when the samples are collected or after a
delay time. In other words, the collected samples of
respiratory gas can be temporarily stored prior to a

- 9a -
measurement, for example if no measurement device is
immediately available for use.
It is ideal, and preferable, to perform a continuous
analysis of the exhaled air using a respiratory gas
analyzer as measurement device (online measurement).
The measurement preferably extends over a time interval
in which the enzyme kinetics proceed or the shortest
time interval within which a reliable determination of
the enzyme kinetics takes place. In diseased
individuals, an analysis for a time interval of about
60 minutes is necessary. In healthy individuals, in
whom the maximum reaction rate of the substrate in the
liver is reached after just

- 10 -
a few minutes, the analysis method can be terminated
once the maximum reaction rate has been reached, that
is to say after a few minutes (for example 5 minutes).
The continuous online measurement of the 13CO2 content
in the air exhaled by the individual results in
particular in a considerable saving in time in the
examination of healthy individuals since, in the
offline measurement, the respiratory samples are first
of all collected and thereafter analyzed, such that
early termination of the analysis method is not really
possible. In diseased individuals 'too, however, there
is still a distinct saving in time, since the result of
the measurement is available immediately upon
conclusion of the measurement, and forwarding to a
laboratory, or a renewed intervention by the operator,
is not needed.
As a result of an increased time resolution in
continuous online measurement compared to the
discontinuous offline measurement, more data points are
obtained, which results in greater precision of the
analysis of the curves.
Since, in continuous online measurement, no respiratory
gas bag has to be inflated at set times, there is no
dependency on the operator in respect of said times;
the data points can thus be assigned, with a
significantly reduced error, to a point in time within
the proceeding enzyme kinetics.
The continuous online measurement is also preferred for
the reason that it permits fully automatic measurement,
especially if, by suitable measures, the gas delivery
(i.e. the correct fit of a respiratory mask on the face
of the individual) is guaranteed.
The air exhaled by the individual is preferably
collected at the individual's face by means of a

- 10a -
respiratory mask, in particular 'a respiratory mask,
having the features explained further below in the
description, and is transferred from here through a
tube or other connecting line into the measurement
device, in order to then perform the analysis method.
In a particularly preferred embodiment of the
invention, the substrate used is 13C-labeled methacetin.
Thi s 13C-methacetin has a molecular weight of
166.19 g/mol.

- 11 -
In the reaction of 13C-methacetin -in the body of the
individual, a liver-specific enzyme system is tested
which, however, does not occur in such great ratio that
complete utilization of the enzyme could be achieved at
any time. The cytochrome p-450 isoenzyme CYP1A2 is
therefore suitable. Only relatively few substances are
metabolized by CYP1A2, such that the factors
influencing and disturbing it are small. Nevertheless,
it is to be regarded as representative of the liver
function.
The substance methacetin is demethylated to paracetamol
by CYP1A2 in a rapid one-step reaction, with CO2 then
resulting. A rapid and complete reaction is ensured by
the high first-pass effect. 13C-methacetin is thus
eminently suitable. By 13C-labeling of the methyl group
bonded via the ether bridge, 13CO2 can then be measured
in the exhaled air. This can be done by analyzing the
exhaled air using a suitable respiratory gas analyzer.
Suitable methods for this purpose are, for example,
isotope-selective nondispersive infrared spectroscopy
or isotope-selective mass spectroscopy. With both
methods, the measured value provided is the difference
of the 13CO2/12CO2 ratio of the sample and the Pee Dee
Belmite (PDB) standard in delta per'mil (513C).
The object of the invention is also achieved with an
aqueous methacetin solution having the features of
claim 19. Accordingly, the pH value of the solution is
set such that the solution is basic, with a pH value of
7.5 to 9.5 and in particular of 8.0 to 8.5 being
particularly preferred. Thus, for example, a pH value
of 8.2 has proven particularly advantageous.
To better dissolve the methacetin, the methacetin
solution preferably contains a solubilizer.
Propylene glycol has particui arly advantageous
solubilizing properties, and it is at a concentration

- 11a -
of preferably 10 to 100 mg/ml, more preferably of 20 to
bO mg/ml, particularly preferably of 25 to 35 mg/ml,
and very particularly preferably of 30 mg/ml, that said
advantageous solubilizing properties of the propylene
glycol come to the fore.
In a preferred variant of the invention, the methacetin
solution is sterile and/or pyrogen-free, such that the
methacetin solution can be administered to a human or

- 12 -
animal individual or patient without fear of health-
related complications.
The methacetin in the methacetin solution preferably
has a concentration of 0.2 to 0.6% (w/v) , particularly
preferably of 0.3 to 0.5%, and very particularly
preferably of 0.4%. At this concentration, the
methacetin in the methacetin solution according to the
invention is readily soluble. At lower concentrations,
preferred in respect of solubility, the volume of the
methacetin solution that has to be administered to a
test individual increases significantly, which is
undesirable. At a higher methacetin concentration, by
contrast, there is a danger -of the methacetin
precipitating or crystallizing out of the solution.
In order to advantageously use the methacetin solution
for performing a breath test for determining functional
parameters of an organ, the dissolved methacetin is
preferably labeled with the carbon isotope 13C. This
labeling is preferably restricted only to those areas
of the molecule which are released as CO2 upon reaction
in the body of an individual. This is the methyl group
shown at the left margin in formula (I) . By limiting
the 13C labeling to this methyl group, products of the
breakdown of methacetin other -than CO2 have no
labeling, such that these breakdown products do not
interfere with measurements that are based on the
determination of the content of 13CO2
The invention also relates to the use of a methacetin
solution according to the invention in an analysis
method as claimed in one of claims 1 through 18, for
determining the functional parameter of an organ of a
human or animal individual.
A further aspect of the invention is the use of a
methacetin solution according to the invention in an
analytical or diagnostic method for determining the

- 12a -
dynamic distribution of methacetin in an organ of an
individual by means of nuclear magnetic resonance
spectroscopy (NMR) or magnetic resonance therapy (MRT).
Since 13C is NMR-active, it is recommended to
investigate the dynamic distribution of methacetin in
the liver, for example, in order to be able to draw
conclusions regarding liver damage. In such a method,
dynamic processes in the liver can be investigated with
relatively high time resolution (in the minute range).
Liver areas not accessible to a through-flow of
methacetin are only inadequately supplied, if at all,
with other substances too, such that a correlation to
(partial) liver damage can be drawn in this way.

- 13 -
The object is also achieved by a respiratory mask,
having the features of claim 34, for separating the
exhaled air from the inhaled air of an individual,
since such a respiratory mask is particularly suitable
for conveying the air exhaled by an individual into a
measurement device, which air is analyzed in the latter
in an analysis method according to the invention. Such
a respiratory mask has a respiratory mask body and a
gas cushion, which extends around the respiratory mask
body and is arranged between the face of the individual
and the respiratory mask body during operation.
The gas cushion is filled with gas and permits a
substantially gas-tight contact between the face of the
individual and the respiratory mask, which is arranged
on the face of the individual. This gas-tight contact
ensures that inhaled air required by the individual and
exhaled air exhaled by the individual is guided
substantially completely through the respiratory mask.
Inhaled air or exhaled air does not in practice flow
under the gas cushion into the area surrounded by the
respiratory mask, but only through inhalation and
exhalation valves, specifically at least one exhalation
valve and at least , one inhalation valve that are
integrated directly into the respiratory mask.
The basic shape of the respiratory mask body is
preferably produced in different sizes to match the
size of face of the individual who is to wear the mask
(e.g. for children, small adults, medium-sized adults,
large adults, very large adults, etc.), so as to permit
a secure fit of the respiratory mask on a large number
of face shapes.
The gas cushion preferably has a valve, which can have
a Luer connector for example, or a similar closure
piece. By means of this valve or closure piece, the
degree of filling of the gas cushion can be adjusted in
order to further optimize the 'secure fit of the

- 13a -
respiratory mask on the face of the individual.
Air in particular is recommended as the gas with which
the gas cushion is filled. However, other gases can
equally be used as the gas for filling the gas cushion-,
but these gases should be noncombustible and nontoxic,
so as to minimize the risk of injury to the individual
wearing the mask.
The solid plastic housing of the respiratory mask body
add i tionally comprises a solid, conical attachment for
an oxygen tube, so as to be able, when necessary, to
supply the individual wearing the mask additionally
with oxygen or other added gas mixtures.

- 14 -
In a preferred embodiment of the invention, the
inhalation valve and exhalation valve are installed
fixedly in the respiratory mask body.
In an alternative embodiment of the invention, the
valves can be separately removed from the respiratory
mask, body and are clipped into a matching conical
fixture in the respiratory mask body, as a result of
which a gas-tight connection is established between the
inhalation/exhalation valve and the respiratory mask
body.
The -inhalation and exhalation valves are preferably
composed of a thin and flexible, but sufficiently stiff
membrane that covers through-openings formed in the
valve for the through-flow of gas or air. The membrane
can preferably be made of silicone, but can also be
made of other materials that satisfy said criteria.
for conveying the separated exhaled air to an analysis
or measurement device, for example via a special tube,
the exhalation valve preferably has an attachment for
carrying off the separated exhaled air.
To ensure that the separated exhaled air is carried off
from the exhalation valve in a uniform manner, the
attachment for carrying off the exhaled air is
particularly preferably arranged centrally on the
exhalation valve.
The attachment is preferably a conical attachment with
an internal diameter of 20 to 30 mm at its narrower
opening, an internal diameter of 22 mm being
particularly preferred. Such a (standardized)
attachment permits uncomplicated connection of a tube
or other withdrawal device to the attachment of the
exhalation valve.
The main body of the respiratory mask preferably has a

- 14a -
securing device for securing at least one holding
element, by means of which the respiratory mask can be
securely held on the face of the individual. The
securing device can, for example, be a hole that is
suitably sealed off to ensure that no air passes
through the hole into the area surrounded by the
respiratory mask. The securing device is advantageously
connected only to the outside wall of the respiratory
mask body and has the shape of a ring, a nipple or an
eyelet. The securing device can, for example, be
injection molded onto the respiratory mask body.

- 15 -
The holding element is preferably a rubber band, the
length of which can be varied if appropriate in order
to ensure a secure fit of the respiratory mask on the
face of the individual. A rubber band for holding the
respiratory mask is fitted particularly preferably in
each case in the mouth area and nose area.
A respiratory mask with the features discussed above is
particularly suitable for use in an analysis method or
in a method for determining the functional parameter of
an organ of a human or animal individual.
A pressure sensor is preferably integrated into the gas
cushion and is connected via a cable to a plug
conenction in the area of the attachment on the
exhalation valve. By means of this- pressure sensor, it
is possible to reliably detect whether the respiratory
mask is fitted in place or not. The contact pressure
provided by the rubber bands leads to an increase in
pressure in the air cushion. By means of this change in
pressure, and the time profile, it is possible to
determine the times at which the respiratory mask is
fitted tightly in place. Similarly, removal of the
respiratory mask, for example by the individual, can be
automatically registered. This control possibility
afforded by the integrated pressure sensor is also
conceivable in many situations and in other respiratory
masks with air cushions (e.g. -in intensive care
med i.eine) .
The data measured by the pressure sensor integrated in
the respiratory mask can be evaluated by an
interconnected online measurement device, that is to
say a measurement device suitable for a continuous
online measurement.
The object of the invention is also achieved by use of
the respiratory mask according to the invention in an
analysis method according to the invention, since the

- 15a -
respiratory mask ensures a safe and reliable provision
of the exhaled air that is to be analyzed at the
measurement device, such that the analysis method can
be reliably performed.
The object of the invention is also achieved by a
diagnostic method, having the features of claim 48, for
determining the functional parameter of an organ of a
human or animal individual or patient. This method
involves an intravenous injection of a 13C-labeled
methacetin solution according to the invention into the
body of the individual, and thereafter an analysis of
the relative and/or absolute 13CO2 content in the air
exhaled by the patient, using an analysis method
according to the invention that has already been
explained above.

- 16 -
The functional parameter of an organ to be determined
is preferably the liver function capacity and/or the
microcirculation in the liver.
The rapid substrate inundation required for the enzyme-
kinetic evaluation is ensured by the fact that C
methacetin is administered intravenously in a bolus.
The Intravenous bolus injection ensures an immediate
and complete substrate inundation in the liver. The
absorption of the test substance 13C-methacetin is no
longer the step that determines the reaction rate. With
the preferred dose of 2 mg methacetin per kg
bodyweight, a temporary enzyme utilization is also
permitted in patients with a healthy liver by a short-
term substrate excess. This permits the actual
quantitative expressiveness, since zero-order enzyme
kinetics are achieved, and only this permits a
statement concerning the maximum functional capacity of
an enzyme system.
The stable intravenous administration of the sparingly
water-soluble 13C-methacetin in a concentration
sufficient for bolus injection is preferably achieved
by the 13C-methacetin being dissolved in a concentration
of A mg/ml in water for injection and the addition of
30 mg propylene glycol per ml and the setting of a
basic pH value of 8.5. The solution is prepared sterile
and pyrogen-free, and its osmolarity is set such that
its administration to the individual to be tested can
be performed without difficulty through a peripheral
vein.
The invention will be explained in more detail on the
basis of the following figures and of an illustrative
embodiment, without these explanations limiting the
scope of protection of the invention.
Fig..1 is a graph showing the mean delta-over-baseline
values over time in a liver function study on

- 16a -
patients who had undergone a partial liver
resection;
Fig. 2 is a graph showing the actual functional mean
liver capacities (LiMAx), .calculated from the
data in Figure 1, as a function of time;
Fig. 3 is a graph showing the mean inundation time of
the substrate 13C-methacetin in the liver as a
function of time, for determining the micro-
circulation in the liver;

- 17 -
Fig. 4 is a side view of an illustrative embodiment of
a respiratory mask according to the .invention,
and
Fig. 5 is a plan view of the respiratory mask from
Figure 4.
Figures 1 to 3 will be explained in more detail on the
basis of the following illustrative embodiment.
Kxample 1
In the context of a prospective study, a method, which
is discussed in more detail below, was used to evaluate
liver function before and after partial liver resection
in a number of patients:
1. Method and means for enzyme-kinetic quantification
of liver function capacity and for assessment of
microcirculation disturbances of the liver,
characterized in that
a) A special intravenous ready-to-use solution of
13C-labeled methacetin is injected intravenously
in a sufficient dose to ensure rapid inundation
of the substance in the liver, with substrate
surplus on the metabolizing enzyme system.
b) By analyzing the 13CO2/12CO2 'ratio in the exhaled
air over a period of time of one hour following
injection, the maximum reaction rate of the Re-
labeled methacetin is determined using zero-
order enzyme kinetics.
c) By standardizing to the individual bodyweight,
comparability with a normal population is
provided.
d) Microcirculation disturbances in the liver can

- 17a -
be assessed by determining the inundation time
until the maximum reaction rate is reached.

- 18 -
2. Method according to point 1, characterized in that
a 13C-labeled methacetin solution is used in a
concentration of 0.4%.
3. Method according to points 1 and 2, characterized
in that the 13C-labeled methacetin solution
contains 30 mg/ml of propylene glycol for
stabilization, and the pH value is basic.
4. Method according to points 1 to 3, characterized
in that the 13C-labeled methacetin solution is
sterile and pyrogen-free.
5. Method according to points 1 to 4, characterized
in that the 13CO2/12CO2 ratio is ideally determined
continuously, or otherwise at the defined times of
0 min and 2%, 5, 10, 15, 20, 30, 40, 50 and 60 min
after injection of the 13C-labeled methacetin.
From the prospective study, Figure 1 shows the curves
of the mean delta-over-baseline (DOB) values of the
13CO2/12CO2 ratio in the air exhaled by the patients at
the individual measurement times, plotted over time.
In the pre-operative stage, where there is a normal and
maximum functional liver capacity, the curve rises
steeply, then falls again after the maximum value is
reached. After partial liver resection, the curve
changes drastically on post-operative day 1 (POD1) to
saturation kinetics with markedly reduced maximum
function. As liver regeneration increases after the
partial liver resection, the curve changes back again
toward the pre-operative curve (POD3-10).
The actual functional liver capacity calculated from
this in μg/h/kg is shown in Figure 2. On POD1, the
maximum functional liver capacity (LiMAx) falls from
301 + 24 μg/h/kg to 141 ± 13 μg/h/kg, thereafter slowly
regenerating back to a value of 24 9 + 17 μg/h/kg on

- 18a -
post-operative day 10 (POD10).
The analysis of the microcirculation, represented in
Figure 3, shows a marked disturbance after the partial
liver resection, in parallel with a prolongation of the
inundation time (tvmax) from 11.9 + 2.17 min to 53.5 ±
7.51 min, which subsequently recovers again.
Figure 4 shows an illustrative embodiment of a
respiratory mask 1 according to the invention, which
mask is particularly suitable for introducing the air
exhaled by an individual into a measurement device for

- 19 -
carrying out an analysis method according to the
invention. The respiratory mask 1 is a respiratory mask
to be placed centrally over the face.
The respiratory mask 1 has a housing 2 serving as the
main body of the respiratory mask, .and an air cushion 3
serving as a gas cushion which, on the side of the
respiratory mask 1 directed downward in Figure 4,
extends all round the housing 2. During the use of the
respiratory mask 1, this side is directed toward the
face of the individual wearing the respiratory mask 1.
The housing 2 of the respiratory mask 1 is made of a
firm plastic. The basic shape of the housing can be
configured in different ways to ensure that individuals
with different shapes of faces can apply a respiratory
mask 1 that provides the best possible fit.
Two inhalation valves 4 are arranged at the sides of
the upper end of the respiratory mask 1, which
represents the nose area N of the respiratory mask 1,
only one of these inhalation valves being seen in
Figure 4. Air flows through through-openings 40 formed
in these inhalation valves 4 and into the space between
the respiratory mask 1 and the face of the individual
wearing the respiratory mask 1, when the individual
breathes in.
An exhalation valve 5 is arranged on the front of the
respiratory mask 1, which is arranged at the top in
Figure 4 . Air exhaled by the individual f lows through
through-openings 50 arranged in the exhalation valve 5
when the individual is wearing the. respiratory mask 1
correctly and breathes out. Arranged at the center of
this exhalation valve 5, there is a minimally conical
attachment 6, which has an internal diameter of 22 mm.
Air exhaled by the individual is carried off through
this attachment 6. For this purpose, it is possible to
use a tube (not shown here) connected to the attachment

- 19a -
6.
By virtue of the specific arrangement of the inhalation
valves 4, on the one hand, and of the exhalation valve
b, on the other hand, and of the configuration of the
inhalation valves 4 and of the exhalation valve b, it
is possible to ensure separation of the inhaled air
from the exhaled air of the individual wearing the
respiratory mask 1. In addition, the mixing volumes and
dead volumes i.n the respiratory mask are minimized by
the arrangement of the inhalation valves 4 and of the
exhalation valve b.
The housing 2 further comprises a conical gas
attachment 7 through which oxygen or a gas mixture can,
if necessary, be introduced into the space between the
respiratory mask 1 and the face of the individual
wearing the respiratory mask 1. In this way, it is

- 20 -
possible, for example, to provide an additional supply
of oxygen to the individual wearing the respiratory
mask 1.
A rubber band (not shown in Figure 4) is secured on
holders 8 serving as securing elements, which can be in
the form of eyelets or nipples, for example, this band
ensuring a secure hold of the respiratory mask 1 on the
face of the individual wearing the mask. Instead of a
rubber band, other holding means could also be used.
The holders 8 are arranged in each case in pairs in the
nose area N at the top end and in the mouth area M at
the bottom end of the respiratory mask 1.
The air cushion 3 of the respiratory mask 1 has a valve
9 arranged in the mouth area M of the respiratory mask
1 with a Luer connector through which the quantity of
air in the air cushion 3 can be regulated. This
possibility of adaptation allows the respiratory mask 1
to be optimally adapted to the shape of the face of the
individual wearing the respiratory mask 1.
A pressure sensor 10 is also integrated into the air
cushion 3 in order to allow the pressure in the air
cushion 3 to be measured. For this purpose, a cable
connection 11 arranged on the front of the mask housing
extends from the pressure sensor 10 to the exhalation
valve 5. In the area of the exhalation valve b and of
the attachment 6 mounted on the exhalation valve, the
cable connection 11 has a plug connection 12 onto which
a further cable connection for controlling or reading
the pressure sensor can be attached.
By means of the pressure sensor 10, it is possible to
detect whether the respiratory mask 1 is sitting on the
face of an individual or not. The contact pressure
afforded by the rubber bands leads to a pressure
increase in the air cushion 3. Based on this change in
pressure and on the pressure profile over the course of

- 20a -
time, it is possible to determine the times at which
the respiratory mask 1 is firmly fitted in place.
Moreover, an (undesired) removal of the respiratory
mask by the individual can be automatically registered.
This control possibility via the pressure sensor 10
integrated into the air cushion 3 . is also conceivable
in many situations and in other respiratory masks with
air cushions (e.g. in intensive care medicine).
Figure 5 shows a top view of the front of the
respiratory mask 1 from Figure 4. For an explanation of
the respiratory mask 1, reference is made to the same
reference numbers for the elements already discussed in
Figure 4. In a view complementing that of Figure 4,

- 21 -
Figure 5 clearly illustrates the lateral arrangement of
the exhalation valves 4 and the paired arrangement of
the holders 8 in the nose area N and in the mouth area
M of the respiratory mask 1.

22-
Patent claims
1. An analysis method for determining a functional parameter
of an organ of a human or animal individual by measuring
the 13CO2 content in the air exhaled by the individual to
whom or to which a substrate has been administered whose
reaction in the body of the individual results in the
air exhaled by the individual containing 13CO2, the method
being carried out using a measurement device, wherein the
functional parameter of an organ is the liver function
capacity and/or the microcirculation in the liver and
wherein the maximum reaction rate of the substrate in the
body of the individual is determined via a change of the
measured 13CCO2 content in the air exhaled by the
individual using zero-order enzyme kinetics.
2. The analysis method as claimed in claim 1, wherein the
13CO2 content is determined relative to the 12CO2 content
in the air exhaled by the individual.
3. The analysis method as claimed in claim 2, wherein the
reaction rate of the substrate in the body of the
individual is determined by means of the following
formula:

where 513C is the difference between the 13CO2/12CO2 ratio
in the air exhaled by the individual and that of the Pee
Dee Belmite standard in delta per mil, RPDB is the
13CO2/12CO2 ratio of the Pee Dee Belmite standard, P is the
CO2 production rate, M is the molecular weight of the
substrate, and KG is the bodyweight of the individual.
4. The analysis method as claimed in one of the preceding
claims, wherein the absolute 13CO2 content in the air
exhaled by the individual is determined.

23>
5. The analysis method as claimed in claim 4, wherein the
reaction rate of the substrate in the body of the
individual is determined by means of the following
formula:

where [13CO2] is the absolute 13CO2 concentration per unit
of volume, & is the volume per unit of time, t is the
time, tmax is the time of maximum metabolism, i is the
smallest possible time resolution according to the
measurement method, M is the molecular weight of the
substrate, and KG is the bodyweight of the individual.
6. The analysis method as claimed in one of the preceding
claims, wherein the inundation time needed to reach the
maximum reaction rate of the substrate in the body of the
individual is determined.
7. The analysis method as claimed in one of the preceding
claims, wherein the maximum reaction rate and/or the
inundation time is standardized to the bodyweight of the
individual.
8. The analysis method as claimed in one of the preceding
claims, wherein the exhaled air to be analyzed is
collected discontinuously at fixed times and then
measured in the measurement device.
9. The analysis method as claimed in one of claims 1 through
7, wherein the exhaled air to be analyzed is measured
continuously in the measurement device.
10. The analysis method as claimed in one of the preceding
claims, wherein the measurement time is proportionate to
the shortest time interval needed for determining the
enzyme kinetics underlying the reaction of the substrate

24
in the body of the individual.
11. The analysis method as claimed in one of the preceding
claims, wherein the substrate is 13C-labeled methacetin;
12. An aqueous methacetin solution for use in the method as
claimed in one of claims 1 through 18, wherein the pH
value of the solution is 7.5 to 9.5..
13. The aqueous methacetin solution as claimed in claim 12,
wherein the methacetin solution contains a solubilizer
that promotes the dissolution of methacetin.
14. The aqueous methacetin solution as claimed in claim 13,
wherein the solubilizer is propylene glycol.
15. The aqueous methacetin solution as claimed in claim 14,
wherein the concentration of the propylene glycol is 10
to 100 mg/ml.
16. The aqueous methacetin solution as claimed in one of
claims 12 through 15, wherein the methacetin solution has
a concentration of 0.2 to 0.6% methacetin.
17. The aqueous methacetin solution as claimed in one of
claims 12 through 16, wherein the • methacetin is labeled
with the carbon isotope 13C.

25
18. A diagnostic method for determining the functional
parameter of an organ of a human or animal individual,
characterized by the following steps:
a) intravenous injection of a iJC-labeied methacetin
solution, as claimed in claim 17, into the body
of the individual, and
b) analysis of the relative and/or absolute 13CO2
concentration in the air exhaled by the
individual, using the analysis method as claimed
in one of the claims 1 through 11,
wherein the functional parameter of an organ is the
liver function capacity and/or the microcirculation
in the liver.
19. The diagnostic method as claimed in claim 18, wherein
the methacetin is injected in a concentration of 2 mg
per kg bodyweight of the individual.
Dated this 23rd day of JANUARY 2008

The invention relates to an analysis method for determining a functional parameter of an organ of a human or animal 13CO2 content in the air exhaled by the individual to which a
substrate has been administered the reaction of which in the body of the individual enriches the air exhaled by the individual 13CO2. The method is characterized by using a measuring device, the maximum reaction rate of the substrate in the body of the individual being determined
via a change of the measured 13CO2 content in the air exhaled by the individual using zero-order enzymes kinetics. The invention also relates to an aqueous methacetin solution
for use in said analysis method, the pH of the solution being greater 7.0. The invention also relates to a face mask (1) for use in the inventive method for separating the exhaled air from the air inhaled by an individual. Said
face mask comprises a face mask body (2) and an air cushion (3) encircling the face mask body. Said air cushion is filled with a gas and establishes a substantially gas-tight
contact between the face of the individual and the face mask (1) place on the face of the individual so that the air required by the individual for breathing and the air exhaled by the individual has to flow essentially
completely through the face mask (1). At least one exhalation valve (5) and at least one inhalation valve (4) are integrated into the face mask and allow a flow of inhaled and exhaled air through the face mask. The
invention finally relates to a diagnostic method for determining the functional parameters of an organ of a human or animal individual.