NZ630182B2 - Method for measurement of peptidic degradation products of a proteolytic cascade in blood samples - Google Patents
Method for measurement of peptidic degradation products of a proteolytic cascade in blood samples Download PDFInfo
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- NZ630182B2 NZ630182B2 NZ630182A NZ63018212A NZ630182B2 NZ 630182 B2 NZ630182 B2 NZ 630182B2 NZ 630182 A NZ630182 A NZ 630182A NZ 63018212 A NZ63018212 A NZ 63018212A NZ 630182 B2 NZ630182 B2 NZ 630182B2
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- New Zealand
- Prior art keywords
- angiotensin
- ang
- steady state
- bradykinin
- sample
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Abstract
Disclosed is a method for measurement of peptidic degradation products of a proteolytic cascade comprising at least two consecutive proteolytic reactions in a biological sample, wherein the sample is incubated until the actual overall degradation rate of at least one peptidic degradation product is equal to the actual overall formation rate of said at least one peptidic degradation product and a steady state equilibrium is reached for said at least one peptidic degradation product involved in said proteolytic cascade and wherein said at least one peptidic degradation product in a steady state equilibrium concentration is quantified in the sample. equal to the actual overall formation rate of said at least one peptidic degradation product and a steady state equilibrium is reached for said at least one peptidic degradation product involved in said proteolytic cascade and wherein said at least one peptidic degradation product in a steady state equilibrium concentration is quantified in the sample.
Description
Method for measurement of peptidic degradation products of a
proteolytic cascade in blood samples
The present invention concerns the measurement of peptid:
degradation products of a proteolytic cascade in blood samples.
The qualitative and quantitative occurrence of peptid;
degradation products of proteolytic cascades in bloc
significantly varies with the physiologic or biochemical stati
of the subject. It may e.g. depend on the health status of tl
subject in relation to a given or suspected disease, and/or (
medication, and/or other factors. The proteolytic degradatic
products mainly depend on the regulation and activity c
proteolytic enzymes in the blood system which itself j
dependent on the physiological status of the patient.
The renin-angiotensin system (RAS) is a proteolytic cascade
which is constituted by multiple enzymes and peptides. T]-
cascade starts when angiotensin I (angiotensin 1-10 or Ang 1-1C
is released from the pro-peptide angiotensinogen (AGT) by kidne
secreted renin. AGT is abundantly expressed in the liver i
humans and secreted into the circulation to constitute
virtually inexhaustible plasma pool of AGT. The peptic
metabolites produced from Ang 1-10 by a variety of proteases ac
as ligands for angiotensin receptors in different tissue
leading to a diversified panel of physiological function
mediated by angiotensin peptides.
The regulation of blood pressure is a well-studie
physiological response affected by angiotensin peptides
Angiotensin II (angiotensin 1-8 or Ang 1-8) is produced by th
proteolytic action of Angiotensin Converting Enzyme (ACE) b
removing the two C-terminal amino acids from Ang 1-10. Ang 1-
binds to cellular receptors leading to vasoconstriction and
consecutive increase in blood pressure.
This crucial role of ACE in the production of Ang 1-8
renders it a favourable pharmacologic target with the main focu
of anti-hypertensive treatments set onto the reduction of Ang 1
8 levels leading to a decrease in blood pressure. This is als
reflected by the broad panel of ACE inhibitors being in use fo
the treatment of hypertension. More recently other targetabl'
RAS-enzymes including renin or neutral endopeptidase (NEP) were
identified as pharmacologic targets for anti-hypertensive
treatments while ACE inhibitors have been further optimised to
decrease the frequency of side effects which are thought to be
caused by a lack of ACE domain selectivity. Frequent side
effects occurring during administration of ACE inhibitors
include dry cough and angioedema and are thought to be caused by
the inhibition of bradykinin (BK) degradation by these
inhibitors as not only Ang 1-10 but also certain BK peptides are
described to be cleaved by ACE.
Such limited substrate selectivity is a very common feature
observed among RAS enzymes. Beside ACE, which is slightly
different to other RAS enzymes because of its two-domain
structure, also Angiotensin Converting Enzyme 2 (ACE2) is known
to be able to cleave Ang 1-8 and Ang 1-10 to yield angiotensin
1-7 (Ang 1-7) and angiotensin 1-9 (Ang 1-9) respectively.
Furthermore, different RAS proteases can share their
substrates leading to a competition of enzymes for a given
substrate. For example, Ang 1-10 is known to be cleaved by ACE,
ACE2, NEP and different aminopeptidases. All these
considerations culminate in a very complex picture of the RA.S,
appearing as a meshwork of different enzymes, receptors and
peptides leading to peptide concentrations affected by multiple
factors.
Comparing published plasma levels of soluble ACE with Ang 1-
plasma concentrations, it becomes clear that the ACE enzyme
is present in significant molar excess compared to the Ang 1-10
peptide in circulation. This leads to a fail in describing the
RAS by means of classic enzyme kinetics, which are based on the
presence of a vast excess of substrate. At physiological
conditions where angiotensin peptide levels in circulation do
not exceed 10-200 fmol/ml, most enzymes are present in molar
excess compared to their substrates forcing the investigator to
step away from classical Menten-Kinetics to get a reliable view
about biochemical processes at physiological conditions in the
original sample matrix.
For example, Ang 1-10 has been described to be degraded by
ACE2 with a much lower catalytic efficiency than Ang 1-8.
Experiments showing this were performed in an artificial matrix
using a vast excess of the substrates compared to the respective
enzymes yielding maximal ACE2 conversion rate for Ang 1-8 to Ang
1-7 which is 65 times higher than the conversion rate for Ang 1-
to Ang 1-9 [Rice et al., Biochem J., 383 (2004),45-51].
Taking together previous considerations with the fact that
multiple angiotensin peptides show biologic activity, it becomes
obvious that there is a big need for the evaluation of the RAS
in biological samples ensuring the maintenance of physiological
substrate concentrations in the sample. Manipulation of the RA.S
by pharmacologic means alters enzyme activities and peptide
conversion rates at multiple levels of the system, which clearly
favours the comprehensive assessment of the RAS for monitoring
drug efficacy and inhibitor selectivity for defined enzyme
reactions.
For example ACE inhibitors, which are described to inhibit
both ACE domains to an unknown extent at physiological substrate
concentrations/ could be investigated for their inhibition of
the conversion of Ang 1-10 to Ang 1-8, but also for their
inhibitory potential regarding the conversion of Ang 1-7 to
angiotensin 1-5 (Ang 1-5) or bradykinin 1-9 (BK 1-9) to
bradykinin 1-7 (BK 1-7) or BK 1-7 to bradykinin 1-5 (BK 1-5) in
order to optimise their side effect profiles.
Taking together published data for concentrations of
angiotensin peptides and metabolising enzymes in human plasma, a
great variance among different groups becomes obvious pointing
to a low reproducibility of the used methods. Nevertheless,
there is a vast molar excess reported for the pre-hormone AGT in
human plasma compared to renin meaning that it serves as a very
long-living source of Ang 1-10 which is produced by a constant
renin activity in the plasma sample. This fact is also used in
state-of-the-art PRA-Assays where the produced amount of Ang 1-
over a defined time period is used to calculate back to the
renin activity in the sample. Unfortunately, these values are
frequently too low due to a lack of stabilisation (incomplete
inhibition of degradation) of produced Ang 1-10, which requires
a well-defined set of protease inhibitors to ensure that the
degradation rate of Ang 1-10 is near zero [Bystrom et al., Clin.
Chem. 56(2010), 1561-1569]. These pitfalls together with
significant variances among donors result in a poorly
reproducible procedure with only limited diagnostic value.
It is therefore an object of the present invention to
provide a new and improved tool for analysing a subjecfs
physiologic or biochemical status, especially the physiologic or
biochemical status of a subject or human patient before, during
and after a certain therapeutic treatment, including but not
limited to drug administration, surgery or haemodialysis, by use
of a blood sample of such subject.
Therefore, the present invention provides a method for
measurement of peptidic degradation products of a proteolytic
cascade in a biological sample, especially a blood sample,
wherein the sample is incubated until a steady state equilibrium
is reached for at least one peptidic degradation product
involved in said proteolytic cascade and wherein said at least
one peptidic degradation product in a steady state equilibrium
concentration is quantified in the sample.
The term "steady state equilibrium" (SSE) as used herein
means that the actual overall degradation rate of at least one
peptidic degradation product involved in the proteolytic cascade
is equal to the actual overall formation rate of said peptidic
degradation product, thereby leading to a stable concentration
of said peptidic degradation product, i.e. a steady state
equilibrium peptide concentration which does not substantially
vary over a certain time period, as further specified below. The
actual overall formation rate of a peptidic degradation product
is defined by the sum of the actual turnover rates of all
enzymes involved in the formation of said peptidic degradation
product, i.e. said peptidic degradation product is a direct
product of said enzyme(s). The actual overall degradation rate
of a peptidic degradation product is defined by the sum of the
actual turnover rates of all enzymes involved in the degradation
of said peptidic degradation product, i.e. said peptidic
degradation product is a direct substrate of said enzyme(s).
The term "proteolytic cascade" as used herein shall mean a
cascade comprising at least two consecutive proteolytic
reactions. In one embodiment, the proteolytic cascade is a
cascade of at least two, three, four, or five consecutive
proteolytic reactions. Such proteolytic cascade may comprise at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17
consecutive or non-consecutive proteolytic reactions, or
combinations thereof, which may be branched or multiply
branched. For example, proteolytic cascades according to the
invention include the renin-angiotensin system (RAS), also
called renin-angiotensin-aldosteron system (RAAS) , the
bradykinin system, the kinin-kallikrein system, the blood-
clotting cascade, the complement system, apoptosis pathways,
neuropeptide cascades (e.g. the oxytocin system), endothelin
peptide cascades, and the natriuretic peptide cascades.
The terms "peptidic degradation product involved in the
proteolytic cascade" or "peptidic degradation product of the
proteolytic cascade" as used herein shall mean a peptide, which
is part of said given proteolytic cascade, as substrate
(precursor peptide) or product of at least one enzyme involved
in the proteolytic cascade, or as substrate and product of at
least two enzymes involved in the proteolytic cascade.
Accordingly, for the RAS proteolytic cascade, peptides (or
peptidic degradation products) involved in the proteolytic
cascade may be selected from angiotensinogen, angiotensin I (Ang
1-10) and its biological degradation products, especially
angiotensin I (Ang 1-10), angiotensin 2-10 (Ang 2-10),
angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin III
(angiotensin 2-8 or Ang 2-8), angiotensin IV (angiotensin 3-8 or
Ang 3-8), angiotensin 1-9 (Ang 1-9), angiotensin 1-7 (Ang 1-7),
angiotensin 2-7 (Ang 2-7), angiotensin 3-7 (Ang 3-7) and
angiotensin 1-5 (Ang 1-5). For the bradykinin proteolytic
cascade, peptides (or peptidic degradation products) involved in
the proteolytic cascade may be selected from kallidin (KD or
Lys-bradykinin) and its biological degradation products,
especially bradykinin 1-9 (BK 1-9), bradykinin 2-9 (BK 2-9),
bradykinin 1-8 (BK1-8), bradykinin 1-7 (BK 1-7) and bradykinin
1-5 (BK 1-5). In one embodiment, the one or more peptidic
degradation products may be selected from peptides involved in
one or more proteolytic cascades, e.g. from two or more
proteolytic cascades, being either separate cascades or cascades
that may be related or linked, e.g. by one or more identical or
similar enzymes or peptides. In one embodiment, the proteolytic
cascade is the renin-angiotensin system (RAS) or the bradykinin
system/ or both. For example, the one or more peptidic
degradation products may be selected from the RAS and the
bradykinin system. In one embodiment, the peptidic degradation
product is selected from angiotensinogen/ angiotensin I (Ang 1-
), angiotensin 2-10 (Ang 2-10), angiotensin II (angiotensin 1-
8 or Ang 1-8), angiotensin III (angiotensin 2-8 or Ang 2-8),
angiotensin IV (angiotensin 3-8 or Ang 3-8), angiotensin 1-9
(Ang 1-9), angiotensin 1-7 (Ang 1-7), angiotensin 2-7 (Ang 2-7),
angiotensin 3-7 (Ang 3-7), angiotensin 1-5 (Ang 1-5), kallidin
(KD or Lys-bradykinin), bradykinin 1-9 (BK 1-9), bradykinin 2-9
(BK 2-9), bradykinin 1-8 (BK1-8), bradykinin 1-7 (BK 1-7) and
bradykinin 1-5 (BK 1-5).
In one embodiment, the peptidic degradation product is
selected from angiotensin I (1-10), angiotensin II (angiotensin
1-8 or Ang 1-8), angiotensin 1-7 (Ang 1-7), and angiotensin 1-5
(Ang 1-5) . In an embodiment, the peptidic degradation products
that are quantified are angiotensin I (1-10), angiotensin II
(angiotensin 1-8 or Ang 1-8), angiotensin 1-7 (Ang 1-7), and
angiotensin 1-5 (Ang 1-5).
In another embodiment, the peptidic degradation product is
selected from angiotensin II (angiotensin 1-8 or Ang 1-8),
angiotensin 1-7 (Ang 1-7), and angiotensin 1-5 (Ang 1-5) . In an
embodiment, the peptidic degradation products that are
quantified are angiotensin II (angiotensin 1-8 or Ang 1-8),
angiotensin 1-7 (Ang 1-7), and angiotensin 1-5 (Ang 1-5).
In still another embodiment, the peptidic degradation
product is selected from bradykinin 1-9 (BK 1-9), bradykinin 1-8
(BK 1-8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5).
In an embodiment, the peptidic degradation products that are
quantified are bradykinin 1-9 (BK 1-9), bradykinin 1-8 (BK 1-8),
bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5).
In still another embodiment, the peptidic degradation
product is selected from bradykinin 1-8 (BK 1-8), bradykinin 1-7
(BK 1-7) and bradykinin 1-5 (BK 1-5) . In an embodiment, the
peptidic degradation products that are quantified are bradykinin
1-8 (BK 1-8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-
) .
The terms "enzyme involved in the proteolytic cascade" or
"enzyme of the proteolytic cascade" as used herein shall mean an
enzyme which is part of said given proteolytic cascade, forming
or degrading at least one peptide involved in the proteolytic
cascade. Accordingly, for the RAS proteolytic cascade, enzymes
involved in the proteolytic cascade may be selected form renin,
aminopeptidases (AP, especially aminopeptidase A (APA) and
aminopeptidase N (APN)), dipeptidyl aminopeptidases (DAP)/
carboxypeptidases (especially ACE2), dipeptidyl
carboxypeptidases (especially ACE), and endopeptidases
(especially neutral endopeptidase, also called neprilysin). For
the bradykinin proteolytic cascade, enzymes involved in the
proteolytic cascade may be selected from kallikrein,
aminopeptidases (AP, especially aminopeptidase A (APA) and
aminopeptidase N (APN)), dipeptidyl aminopeptidases (DAP),
carboxypeptidases (especially ACE2), dipeptidyl
carboxypeptidases (especially ACE), and endopeptidases
(especially neutral endopeptidase, also called neprilysin).The
term "actual" as used herein means the actual (or effective)
formation or degradation rate of a peptide, or the actual or
effective turnover rate of an enzyme, under the conditions as
present in the sample.
The term "equal to// as used herein means that the peptide
concentration resulting from any such equal formation or
degradation rate(s) of said peptide (the "steady state
equilibrium peptide concentration" or "steady state equilibrium
peptide level"), or resulting from any such equal turnover rates
of at least two enzymes involved in the formation or degradation
of said peptide (the "steady state equilibrium enzyme turnover
rate"), does not vary more than 15%, 14%, 13%, 12%, 11%, 10%,
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% over a time period of at
least 30 minutes (min), 60 minutes, 90 minutes, 120 minutes, 150
minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, or
300 minutes. Accordingly, the actual overall turnover rates of
the enzymes involved in degradation of said peptide are
determined by the actual overall formation rates of their
substrate peptide(s),. so that any newly or additionally formed
substrate is degraded. However, this does not necessarily mean
that the net concentration of said peptide is zero, but the net
concentration as present in the sample in the steady state
equilibrium does not significantly vary as further described
above.
Accordingly, in an embodiment of the invention, the
concentration of said at least one peptidic degradation product
of the proteolytic cascade remains within a constant range over
the time period of the steady state equilibrium, despite a
continuous flow of formation and degradation. In one embodiment
of the invention, the concentration of said at least one
peptidic degradation product in steady state equilibrium does
not vary more than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,
%, 4%, 3%, 2%, or 1% over a time period of at least 30 minutes,
60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes,
210 minutes, 240 minutes, 270 minutes, or 300 minutes. In one
embodiment, the concentration of said at least one peptidic
degradation product in steady state equilibrium does not vary
more than 15%, or not more than 10%, within 60 minutes.
Accordingly, said peptide does neither significantly accumulate
nor significantly diminish during the above specified time
periods.
In an embodiment, the sample is incubated for up to 15
minutes, 20 minutes, 25 minutes, 30 minutes, 60 minutes, 90
minutes, 120 minutes, 150 minutes, 180 minutes/ 210 minutes, 240
minutes, 270 minutes, or up to 300 minutes, before the at least
one peptidic degradation product in steady state equilibrium
concentration is quantified in the sample. In another
embodiment, the sample may be incubated for more than 6 hours
(h) , especially for up to 8 h, 12 h, 18 h, 24 h or up to 48 h.
Suitable incubation time periods mainly dependent on the given
proteolytic cascade, on the peptidic analytes to be quantified,
on the nature of the sample and on the incubation parameters.
Such incubation time periods can easily be determined by a
person skilled in the art. In one embodiment, the steady state
equilibrium is conserved (or stabilised or frozen or quenched)
after incubation. The terms "conserved", "stabilised", "frozen",
and "quenched" as used herein shall mean the conservation of a
biochemical status, e.g. the conservation of peptide levels,
e.g. by inhibition of proteolytic degradation. The stabilisation
of the steady state equilibrium peptide levels (or the in vivo
peptide levels) can be done by addition of one more protease
inhibitors, especially by addition of a protease inhibitor
cocktail. Accordingly, one or more protease inhibitors may be
added after the incubation until a steady state equilibrium is
reached for at least one peptidic degradation product. Suitable
protease inhibitors or combinations thereof can be selected by a
person skilled in the art and may e.g. comprise a combination of
specific or non-specific enzyme inhibitors, or a combination
thereof. The one or more protease inhibitors or the protease
inhibitor cocktail ensure that especially the proteolytic steps
of the cascade which are of interest (i.e. the enzymes forming
and degrading the peptides to be measured) , or each enzyme of
the proteolytic cascade is completely inhibited.
In one embodiment, each step of the proteolytic cascade is
inhibited, i.e. each enzyme involved in the proteolytic cascade
is inhibited by at least one component of the protease inhibitor
cocktail. In another embodiment, the protease inhibitor cocktail
comprises at least one specific or non-specific inhibitor of
each class of proteases involved in the proteolytic cascade. The
protease inhibitor cocktail may comprise one or more inhibitors
inhibiting one or more enzymes involved in the proteolytic
cascade. Examples for such inhibitors of the RAS are Lisinopril
(ACE inhibitor) and Aliskiren (renin inhibitor). The protease
inhibitor cocktail may also comprise one or more inhibitors
inhibiting one or more groups of enzymes involved in the
proteolytic cascade, such as e.g. EDTA (inhibits
metalloproteases). Furthermore, the protease inhibitor cocktail
may comprise one or more non-specific inhibitors. In one
embodiment, the protease inhibitor cocktail comprises a
combination of at least two of the aforementioned classes of
inhibitors. In another embodiment, the protease inhibitor
cocktail comprises one or more inhibitors of the feeding enzyme,
especially specific inhibitors of the feeding enzyme.
For example, at least two, at least three, or at least four
protease inhibitors are added to the sample. In one embodiment,
the protease inhibitor cocktail comprises Pepstatin A, 1,10-
Phenanthroline, EDTA, p-Hydroxymercuri-benzoic acid and Z-Arg.
Alternatively, or in addition to the use of one or more
protease inhibitors or a protease inhibitor cocktail, the steady
state equilibrium may be conserved by the addition of one or
more chaotropic agents, such as sodium iodide, sodium
perchlorate, lithium perchlorate, magnesium chloride, guanidine
thiocyanate (GTC), guanidinium chloride, phenol, propanol,
butanol, ethanol, sodium dodecyl sulfate, thiourea, urea or
others.
Alternatively, or in addition to the use of one or more protease
inhibitors or a protease inhibitor cocktail, the steady state
equilibrium may be conserved by other means of physical
inactivation of the enzymes in the sample, for example,
denaturation of the enzyme induced by heat, salt, pH, or
detergent; or by cooling, e.g. placing the samples on ice
directly after incubation. For one or more of the further
processing steps of the samples, e.g. the plasma or serum
separation and the separation by solid phase extraction (SPE;
e.g. for matrix depletion and/or peptide enrichment), an
according ambient temperature can be selected as well to ensure
that all enzymes in the sample are inactive. For example, any
sample pre-treatment or sample processing prior to sample
analysis may be done at 4°C ambient temperature (or lower), at
least up to the complete denaturation or inactivation of the
enzymes involved in the proteolytic cascade (e.g. until eluation
from the SPE column).
In contrast, classical PRA assays are aimed on complete
inhibition of the degradation pathways of Ang I immediately
after the sample has been taken, and the enzyme activity is
calculated based on the accumulation rate of the peptide formed
by said enzyme. Even if the inhibition of the Ang I degradation
pathways may be incomplete in such PRA assays, they are still
far from any steady state equilibrium according to the
invention, since the net concentration of Ang I significantly
changes over time. In the PRA assay as described e.g. in Bystrom
et al. (Clin. Chem. 56(2010), 1561-1569), the Ang I
concentration significantly increases over time. Moreover,
state-of-the-art assays are seeking to assess the overall
activity of the RA.S by measuring the conformational activated
form of renin in plasma samples by ELISA and RIA based methods
(DRG Diagnostics, http://www.drg-diagnostics.de/49
DRG+Renin+active+ELISA.html). In contrast to RSSE-
Fingerprinting, these assays critically depend on the
specificity of the used antibody and allow no conclusions about
the concentration of the effector peptides in the samples, which
are responsible for the physiologic effects of the RAS. The
reason for that is that there exist multiple enzymes affecting
the level of effector peptides. All these peptides are
simultaneously analysed by RSSE-Fingerprinting while state-of-
the-art assays focus on just one enzyme activity or
concentration per sample.
In an embodiment of the invention, in the steady state
equilibrium, the actual turnover rate of the feeding enzyme is
maximal, i.e. the feeding substrate is present in vast molar
excess compared to the feeding enzyme, and any addition of
external feeding substrate would not further increase the actual
turnover rate of the feeding enzyme. Accordingly, the feeding
enzyme of a proteolytic cascade is the enzyme, which is
responsible for the feeding conversion reaction, i.e. the rate-
limiting step of the subsequent proteolytic cascade (or the
bottleneck of the proteolytic cascade) . For the RAS proteolytic
cascade under physiologic conditions, the feeding enzyme is
renin, which is responsible for the conversion of
angiotensinogen to angiotensin I. In physiological systems (e.g.
in the body, in blood, plasma or serum samples), angiotensinogen
as the substrate for renin is present in vast molar excess of
renin. However, in one embodiment of the invention, one or more,
or all other enzymes of the RAS proteolytic cascade, such as
e.g. aminopeptidases (especially APA and/or APN), dipeptidyl
aminopeptidases, carboxypeptidases (especially ACE2), dipeptidyl
carboxypeptidases (especially ACE), and/or endopeptidases
(especially neutral endopeptidase, also called neprilysin), are
present in the sample at concentrations sufficient to degrade
any newly or additionally formed substrate and thus, allow the
establishment of a steady state equilibrium for said enzymes and
peptide(s) during incubation, i.e. their actual overall turnover
rates are determined by the actual overall formation rates of
their substrate peptide(s).
According to the present invention, the term "feeding
enzyme" shall mean an enzyme with a maximal actual turnover
rate, i.e. with an actual turnover rate that is the maximal
achievable turnover rate for said enzyme in the sample. The term
"maximal achievable turnover rate" shall mean the turnover rate
of an enzyme contained in the sample, which can be achieved
under the given conditions in the sample, if the substrate
peptide is (or would be) present in vast molar excess compared
to the enzyme (or a virtually inexhaustible amount) at least
until the steady state equilibrium is reached. Accordingly, the
actual turnover rate of the feeding enzyme cannot be further
increased by the addition of external substrate, since the
feeding substrate is already present in vast molar excess
compared to the feeding enzyme. If, for example, any external
substrate peptide (i.e. a peptide involved in the proteolytic
cascade) or an analogue of such substrate is added to a sample
before or during the incubation until a steady state equilibrium
is reached, this may -according to the definition of the present
invention- result in a change of the rate-limiting step(s) for
the proteolytic cascade, and thus, also of the feeding enzyme(s)
of the proteolytic cascade, if the amount of added substrate
peptide is sufficient to result in a maximal achievable turnover
rate of at least one enzyme involved in the degradation of said
substrate peptide (i.e. an enzyme other than the feeding enzyme
under physiologic conditions). For example, if the proteolytic
cascade under investigation is the RAS, and if a vast molar
excess of an Ang 1-10 (or an analogue thereof, e.g. a Ang 1-10
carrying a mass label or a any covalent modification including
amino acid exchanges) compared to one or more or all enzymes
involved in Ang 1-10 degradation is added to the sample before
or during incubation until a steady state equilibrium is
reached, at least one or even all enzymes involved in the
degradation of Ang 1-10 (e.g. ACE, ACE2, AP, and/or NEP) would
reach maximal achievable turnover rates and thus, would become
the rate-limiting feeding step(s) for the subsequent proteolytic
reaction(s) of the cascade.
In an embodiment of the invention, feeding enzyme may be
added to the sample. The addition of feeding enzyme increases
the flow-through of the enzyme cascade and thus, leads to
increased absolute levels of peptides, while the relative levels
(peptide ratios) remain unchanged. Accordingly, the steady state
equilibrium levels of the one or more peptides still reflect the
physiological situation, i.e. the enzyme activities, however,
the overall peptide levels are increased proportionally. This
would be useful, for example, if the peptide levels measured
without the addition of feeding enzyme would be below the
detection limit of the method used to quantify the peptide(s) .
Optionally, feeding substrate may be added as well. This may
ensure that feeding substrate is and remains in molar excess
compared to the feeding enzyme and that feeding substrate is
still present in virtually inexhaustible amounts leading to a
turnover rate of the feeding enzyme, which is stable for a
certain time defining the steady state equilibrium, even if
feeding enzyme is added.
In a further embodiment, in the steady state equilibrium the
actual overall degradation rate of the product of the feeding
enzyme (i.e. the peptide formed by the feeding enzyme) is equal
to its actual overall formation rate. In another embodiment, the
proteolytic cascade is the RA.S, and the product of the feeding
enzyme according to the above definition is Ang I. In said
embodiment, the actual turnover rate of said Ang I degrading
enzyme in the steady state equilibrium is equal to the actual
turnover rate of renin, which is the Ang I forming enzyme/ if
only one enzymatic Ang I degradation pathway is "open" in the
sample, i.e. only one Ang I degrading enzyme is active. If more
than one Ang I degradation enzymes are active in the sample, the
sum of the actual turnover rates of said active Ang I degrading
enzymes (i.e. the actual overall degradation rate of Ang I) is
equal to the actual turnover rate of renin (i.e. the actual
overall Ang I formation rate) in said embodiment.
another embodiment, in the steady state equilibrium the actual
overall degradation rate of more than one peptide, especially of
two, three, four, five, six, seven, eight, nine or ten peptides
involved in the proteolytic cascade is equal to the actual
overall formation rate of said peptide(s). In still another
embodiment, in the steady state equilibrium the actual overall
degradation rate of each peptide involved in the proteolytic
cascade is equal to the actual overall formation rate of said
peptides.
Accordingly, a steady state equilibrium is reached for one
or more peptide(s) and the related enzyme(s), i.e. the enzyme(s)
forming or degrading said peptide(s). As described above, the
steady state equilibrium is reached, if the net concentration of
the at least one peptide, two or more peptides, or all peptides
involved in the proteolytic cascade, does not vary more than
%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or
1% over a time period of at least 30 minutes, 60 minutes, 90
minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240
minutes, 270 minutes, or 300 minutes. Said steady state
equilibrium is reached after incubation of the sample for 15
minutes, 20 minutes, 25 minutes, 30 minutes, 60 minutes, 90
minutes, 120 minutes, 150 minutes, 180 minutes, 210 minutes, 240
minutes, 270 minutes, or 300 minutes, or for 8, 12, 18, 24, or
48 h. The steady state equilibrium continues for at least 30
minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180
minutes, 210 minutes, 240 minutes, 270 minutes, or 300 minutes.
In an embodiment of the invention, in the steady state
equilibrium the overall maximal achievable degradation rate of
at least one peptide involved in the proteolytic cascade is
equal to or higher than its actual overall formation rate.
According to the invention, the maximal achievable degradation
rate of a peptide is the overall degradation rate which could be
achieved under the given conditions in the presence of a vast
molar excess of said peptide compared to each enzyme degrading
said peptide (or a virtually inexhaustible amount of said
peptide), i.e. by addition of external peptide. Accordingly, the
maximal achievable degradation rate of a peptide is the sum of
maximal achievable turnover rates of all enzymes involved in the
degradation of said peptide.
In one embodiment, at the start of the incubation time the
amount of all enzymes involved in the proteolytic cascade is in
excess of the amount of their respective substrate(s), except
for the feeding enzyme of the proteolytic cascade. In another
embodiment, the amount of said one or more enzymes or of all
enzymes involved in the proteolytic cascade is in excess of the
amount of its/their respective substrate(s) during the entire
time period of incubation, except for the feeding enzyme of the
proteolytic cascade.
In another embodiment, the conditions as specified above
(i.e. that the amount of enzyme is in excess of the amount of
the respective substrate, and/or that the rate of formation of a
peptide is equal to the rate of degradation in steady state
equilibrium) apply at least to. the one or more peptides to be
analysed, and/or the respective enzyme(s) which form or degrade
said peptide(s) to be analysed.
For example, if the method of the present invention is used
to examine one or more components of the RAS proteolytic
cascade, and the peptides to be analysed are, e.g. Ang 1-10 and
Ang 1-8, this means that until a steady state equilibrium is
reached, the actual rate of formation of Ang 1-10 by renin is
higher than the sum of the actual degradation rates of all
enzymes involved in Ang 1-10 degradation, including but not
limited to ACE, Aminopeptidases and ACE2; and the actual rate of
formation of Ang 1-8 by ACE is higher than the sum of the actual
degradation rates of all enzymes involved in Ang 1-8
degradation, including but not limited to ACE2, AP, and/or DAP.
Accordingly, the Ang 1-10 and Ang 1-8 concentration increases,
i.e. Ang 1-10 and Ang 1-8 accumulates, until a steady state
equilibrium is reached. When the steady state equilibrium is
reached, the actual rate of formation of Ang 1-10 is equal to
the sum of the actual turnover rates of all enzymes involved in
Ang 1-10 degradation (the actual overall degradation rate of Ang
1-10); and the actual rate of formation of Ang 1-8 is equal to
the sum of the actual turnover rates of all enzymes involved in
Ang 1-8 degradation (the actual overall degradation rate of Ang
1-8) . If, for example, all ten peptides involved in the RAS are
to be analysed (as shown, e.g. in Figures 2, 3 and 4A, 4C1 and
4C2), the above specified conditions should apply to all ten
peptides or to all enzymes involved in the RAS.
Accordingly, in an embodiment of the steady state
equilibrium of the present invention, at least one proteolytic
degradation reaction has to be active or "open" for at least
one, especially for each peptide of the proteolytic cascade to
an extent which allows that the actual overall degradation rate
is equal to the actual overall formation rate of said peptide,
i.e. is not inhibited or "closed"/ e.g. by use of one or more
protease inhibitors, to an extent that the actual overall
formation rate exceeds the actual or maximum achievable overall
degradation rate of the said peptide.
In one embodiment, chelating agents, such as e.g. EDTA,
EGTA, 8-hydroxyquinoline, phenanthroline and dimercaprol (also
called British-anti-Lewisite or BAL), are not added before
and/or during the incubation until a steady state equilibrium is
reached, especially not for the RAS cascade or other proteolytic
cascades where metalloproteases are involved, since chelating
agents have an inhibitory effect on metalloproteases through
chelating of bivalent ions.
In one embodiment, chaotropic agents, such as e.g. sodium
iodide, sodium perchlorate, lithium perchlorate, magnesium
chloride, guanidine thiocyanate (GTC), guanidinium chloride,
phenol, propanol, butanol, ethanol, sodium dodecyl sulfate,
thiourea, urea, and/or others, are not added before and/or
during the incubation until a steady state equilibrium is
reached.
In one embodiment, the steady state equilibrium is reached
under physiological conditions for said proteolytic cascade,
which means that the components of the proteolytic cascade
(enzymes and substrate or product peptides), their total and/or
relative amounts as present in the biological sample as taken
from the body, as well as the matrix in respect to composition
and/or pH of the sample are not or not substantially modified
before and/or during the incubation until a steady state
equilibrium is reached. In one embodiment, the concentrations of
the enzymes and/or peptides involved in the proteolytic cascade
present in the biological sample as taken from the body are not
modified before and/or during the incubation until a steady
state equilibrium is reached.
In another embodiment, substrates or substrate analogues of
any enzyme(s) involved in the proteolytic cascade, such as e.g.
internal standards or degradation standards, whether in their
native form or modified by labelling (e.g. isotopic and/or
fluorescent labelling, and/or amino acid modifications or
exchanges of at least one amino acid) , are not added before
and/or during the incubation until a steady state equilibrium is
reached. In one embodiment of the present invention, the
proteolytic cascade is the RAS, and neither angiotensinogen,
angiotensin I and/or angiotensin II, nor any analogues thereof,
are added before and/or during the incubation until a steady
state equilibrium is reached.
In another embodiment, further substances or reagents, such
as e.g. buffer substances (Tris, PBS, MES, HEPES, citrate,
borate, carbonate or hydrogen carbonate (or bicarbonate) and/or
other buffer substances or respective buffer solutions are not
added before and/or during the incubation until a steady state
equilibrium is reached.
In still another embodiment, substances or reagents, such as
e.g. EDTA, EGTA, PMSF, AEBSF, BSA, maleic acid/ maleic
anhydride, formic acid, and/or water (in any form, e.g.
deionised and/or distilled etc.) are not added before and/or
during the incubation until a steady state equilibrium is
reached.
However, and not withstanding the foregoing, one or more
such afore mentioned protease inhibitors, chelating agents,
chaotropic agents, substrates, standards, BSA, buffers, and/or
other substances or reagents may be added once the steady state
equilibrium is reached and optionally quenched.
Especially, one or more standards, e.g. internal standards
and/or degradation standards, may be added once a steady state
equilibrium is reached and frozen. Standards are, for example,
peptides of the proteolytic cascade, which are modified by mass
labelling and/or chemical labelling (e.g. isotopic and/or
fluorescent labelling, and/or amino acid modifications, and ore
the use of mass tags, and/or exchanges of at least one amino
acid). Accordingly, internal standards are stable isotope
labelled internal standards, e.g. disclosed in WO 03/016861 A.
In one embodiment, the biological sample is incubated as
taken from the subject (ex vivo), i.e. the matrix of the sample
and/or the concentrations of the components of the proteolytic
cascade to be analysed are not modified, but optionally further
processed (e.g. to obtain plasma or serum), either before or
after a steady state equilibrium is reached and stabilised.
Optionally, anti-coagulants, i.e. substances, which prevent
coagulation (stopping blood from clotting), may be added to the
biological sample before and/or during incubation until a steady
state equilibrium is reached. However, such anti-coagulants
should not substantially affect the proteases of the proteolytic
cascade to be analysed. A suitable anti-coagulant for use in the
method according to the present invention is heparin.
If the proteolytic cascade to be analysed is the blood
clotting cascade, the skilled person in the art can determine
suitable anti-coagulant agents to be added to the sample prior
to or during incubation until a steady state equilibrium is
reached, which would prevent blood clotting, but would still
allow the establishment of a steady state equilibrium for at
least one peptide to be analysed. For example, an anti-coagulant
could be selected that inhibits blood clotting at a step further
downstream in the proteolytic cascade, i.e. not inhibiting any
enzyme(s) involved in the degradation of the peptide(s) to be
analysed (upstream in the proteolytic cascade).
Prior to analysis, the samples may be pre-treated or further
processed, e.g. by plasma or serum separation (e.g. by
centrifugation or activation of coagulation followed by
centrifugation), and/or purification by solid phase extraction
(SPE), e.g. for matrix depletion and/or peptide enrichment.
Accordingly, the solid phase extraction may be carried out with
a reversed phase chromatography material, a hydrophobic
interaction chromatography material, an ion exchange material,
affinity chromatography material, e.g. a reversed phase
chromatography material, especially a C18, C8 or C6H5 (Phenyl)
material.
In one embodiment, the one or more analyte(s) is/are
concentrated to dryness after eluting from the solid surface and
may be reconstituted in a HPLC compatible solvent/ meaning that
the composition of the solvent does not interfere with binding
of the one or more analytes to the MS coupled HPLC column. The
reconstitution solvent is e.g. an aqueous solvent which might be
supplemented with additives including propanol, butanol, 2-
butanol, pentanol, 2-propanol, acetone, methyl ethyl ketone,
acetonitrile, methanol, ethanol, acids or bases in order to
enhance solubility of analytes and/or facilitate binding of
analytes to the HPLC column.
In another embodiment, the methods according to the
invention comprise the steps: providing a sample treated with an
anti-coagulant; optionally, further processing the sample to
obtain a plasma or serum sample; incubating the sample until a
steady state equilibrium is reached for at least one peptidic
degradation product involved in the proteolytic cascade;
conserving said steady state equilibrium; optionally, adding one
or more internal standard(s) once the steady state equilibrium
is conserved; conducting a solid phase extraction with the
sample; and analysing the sample. The plasma or serum separation
may be done either prior to or after the step of incubation
until a steady state equilibrium is reached (and optionally
stabilised), depending on whether the steady state equilibrium
is to be investigated in plasma, serum, or whole blood.
The analysis of the at least one peptidic degradation
product in steady state equilibrium concentration may be done
e.g. by mass spectrometry (MS); by liquid chromatography, such
as high pressure liquid chromatography (also called high
performance liquid chromatography, HPLC); especially by liquid
chromatography-electrospray ionisation-mass spectrometry (LC-
MS) , and/or liquid chromatography-tandem mass spectrometry (LC-
MS/MS) . For example. Gui et al. (Anal Biochem. 369 (2007), 27-
33) disclose liquid chromatography-electrospray ionisation-mass
spectrometry and liquid chromatography tandem mass spectrometry
methods for quantifying angiotensin peptides. For each peptide
and corresponding internal standards, different mass transitions
can be measured. The performance of the method may be monitored
using quality control samples.
Such quality control samples may include, for example,
biological samples with pre-defined analyte concentrations, as
well as synthetic samples comprising a mixture of pre-defined
concentrations of synthetic peptides. For example/ the quality
control sample may be a pooled blood, plasma, or serum sample or
pooled tissue homogenate sample with pre-defined concentrations
of one or more peptides. Angiotensin peptide concentrations may
be calculated by relating endogenous peptide signals to internal
standard signals provided that integrated signals achieved a
signal-to-noise ratio above 10.
Furthermore, the analysis may be done by radio immune assay
(RIA) or enzyme linked immunosorbent assay (ELISA). Optionally,
a HPLC purification step may be done prior to the RIA or ELISA
based quantification of peptidic degradation products of the
proteolytic cascade.
In one embodiment, the sample pre-treatment, sample
processing, and/or the analysis of the samples may be done in a
multiwell format, e.g. on 96 well plates.
The present invention provides a novel tool for the analysis
of the physiologic or biochemical status of a subject based on
the observation of one or more proteolytic cascades in the
subject, especially in the blood system of the subject.
Especially, the methods of the invention allow the determination
of multiple peptide levels under steady state conditions of
proteolytic cascades. Moreover, the methods of the invention
allow the evaluation of a proteolytic cascade in a compartment
specific manner, e.g. the RAS can be analysed in different
compartments, such as full blood, serum as well as plasma. The
measurement of peptidic degradation products of a proteolytic
cascade according to the invention provide a "fingerprint-like"
result of the subjecfs physiologic or biochemical status, or a
"fingerprint-like" enzyme activity profile of a subject, which
can be indicative for presence or absence of certain diseases or
for the effectiveness or ineffectiveness of a treatment of this
disease (provided, of course, that the . disease directly or
indirectly affects the proteolytic cascade) . In addition, the
methods of the invention allow a comparison of in vivo
fingerprints (samples are immediately stabilised and thus,
reflect the circulating peptide levels) , and ex vivo
fingerprints (steady state equilibrium peptide levels in plasma,
serum or full blood).
Accordingly, the methods according to the present invention
can be used to study a proteolytic cascade in general, its
physiologic or biochemical status in a patient or in patient
groups, for diagnosing a disease directly or indirectly related
to the proteolytic cascade (or the aberrance thereof), to
determine a treatment or treatment regime for such disease, to
determine fixed dose combinations, to examine the mechanism of
action, to minimise drug associated side effects or adverse
events, or to examine the pharmacology of drugs in use or of
drug candidates, e.g. to examine short term and long term
effects or toxicity.
Furthermore, the methods according to the present invention
can be used for screening and development of new drugs,
biomarkers or biomarker parameters, especially for such
screening in the physiologic matrix of said drugs or biomarkers.
In one embodiment, the methods according to the invention
are used as a biomarker assay for pathologic conditions or
diseases related to the proteolytic cascade(s) under
investigation with the methods according to the invention. For
example, not only the concentration of one peptidic degradation
product, but the entire steady state equilibrium fingerprint
(SSE-Fingerprint) comprising several or all peptides involved in
the proteolytic cascade under investigation according to the
invention, and/or mathematical functions (e.g. products, ratios,
sums, differences) of concentrations of two or more peptidic
degradation products, and/or combinations of at least two of
said mathematical functions, can be used as biomarker (or
biomarker compilation).
In another embodiment, the methods according to the
invention can be used for determining a specific patient group
for a treatment, such as e.g. for the identification of
responders and non-responders of a treatment.
The methods according to the present invention can also be
used as companion diagnostics. Companion diagnostics are assays
(tests or measurements) intended to assist physicians in making
treatment decisions for their patients. They do so by providing
information on how a drug works in the body and thus elucidating
the efficacy and/or safety of a specific drug or class of drugs
for single patients, a targeted patient group or sub-groups.
There are two main groups of companion diagnostics that include
tests that are used for drugs that are already on the market as
well as tests that are already used in the preclinical or
clinical development phase of a potential drug. The use of
companion diagnostics for drugs already in early development has
the potential to significantly alter the drug development
process and commercialisation of drug candidates by yielding
safer drugs with less side effects, with enhanced therapeutic
efficacy in a faster, more cost-effective manner.
Accordingly, the subject from which the biological sample
is taken may have been treated with one or more pharmaceutical
compositions (in vivo), e.g. a pharmaceutical composition
comprising a protease inhibitor, especially, if the method
according to the present invention is used to monitor the
effects of a pharmaceutical composition, or to determine the
optimal dose of a pharmaceutical composition, or to asses any
potential toxic interactions of one or more pharmaceutical
compositions, or to examine multiple drug interactions.
In an embodiment of the invention, the subject is a human.
In another embodiment, the subject is an animal, for example a
mammalian, a rodent, a pig, or a monkey.
The method
according to the present invention is a standardised procedure
for the overall assessment of a proteolytic cascade in blood
samples but is also suited for proteolytic cascades in other
samples, especially tissue biopsies, tissue homogenates, tissue
slices, liquor, bile fluid, urine, etc. The present invention
can be used in principle for all proteolytic cascades (under the
control of enzymatic cleavage of proteins or peptides into
peptidic degredation products being either metabolites or
intermediates or end products of a proteolytic cascade), which
occur in the blood system. The present invention is specifically
suited for the most relevant proteolytic cascades in the human
blood system, such as the RAS, the blood-clotting cascade, the
complement system, apoptosis pathways, neuropeptide cascades,
endothelin peptide cascades, natriuretic peptide cascades. With
the present invention, a method for the measurement of steady
state equilibrium peptide concentrations, which reflect the
activity and conversion rates of their metabolising enzymes, is
provided. A steady state equilibrium concentration of a peptide
in that context means that its rate of formation is equal to its
rate of degradation leading to a peptide concentration, which
does not substantially or significantly change over time for a
certain period of time, and which is strongly dependent on
affinities of the enzymes to their substrates under the given
conditions rather than maximal enzyme conversion rates, as
further described above. Since the present invention deals with
biological systems, it is clear that the term "steady state
equilibrium" cannot be regarded as a single point to be reached,
but more as a kinetic target region of peptide concentrations,
which do not significantly change over time for a certain period
of time. More accurate time periods until such steady state
equilibrium concentrations are reached are mainly dependent on
the given proteolytic cascade, on the peptidic analytes to be
quantified, on the nature of the sample and on the incubation
parameters. This can easily be determined for each cascade. In
general, the "steady state equilibrium window" wherein the
quantification according to the present invention can be
performed is rather large, at least for some of the proteolytic
cascades, especially those in blood. Usually, the steady state
equilibrium is reached after a certain incubation time/ which is
empirically determined (e.g. 30 minutes for the RAS system) and
then stays stable for an extended period of time (e.g. 6 hours
(h) for the RAS system) . Then, the steady state equilibrium is
disturbed by effects such as degradation and inactivation of the
involved enzymes or a lack of feeding substrate in the sample.
The feeding substrate concentration based time of stability (tg)
for a given cascade can be calculated by dividing the
concentration of the feeding substrate (or feeding precursor
peptide) (Cf) reduced by an enzyme- and sample-specific constant
defining the minimal substrate concentration to achieve the
maximal turnover rate of the feeding enzyme in the sample (Cmin)/
by the turnover rate of the feeding enzyme of the cascade,
thereby defining the feed rate (Vf) of the cascade.
ts = (Cf - Cmin) /Vf
ts feeding substrate concentrationbased time of stability [h]
Cf feeding substrate concentration [mol/L]
Cmin sample specific minimal substrate concentration to achieve
the maximal turnover rate of the feeding enzyme in the
sample [mol/L]
Vf feed rate of the cascade [[mol/L]/[h]]
cmin = r ' CE
f excess factor
CE feeding enzyme concentration
For example, the application of these formulas above on the
RAS, where the feeding conversion is carried out by renin,
yields a calculated feeding substrate concentration based time
of stability of the RAS steady state equilibrium of about 60 to
200 hours based on different published values for PRA (plasma
renin activity, PRC (plasma renin concentration), see e.g.
[Nishiyama et al. 2010, and Bystrom et al., Clin. Chem.
56(2010), 1561-1569], and applying a AGT concentration of e.g.
VO^g/ml plasma and an excess factor of e.g. 1000. Of course,
said calculated feeding substrate concentration based time of
stability should serve as a rough and theoretic reference point
only, since the actual time of stability of the steady state
equilibrium may differ significantly in the samples.
The method according to the present invention is
specifically suited for the monitoring and analysis of Ang 1-10
and its degradation products, thereby observing the status of
the RAS in human blood samples. The steady state equilibrium
concentrations of Ang 1-10 and its degradation products are
highly indicative of the physiologic or biochemical status of
the subject. For example, deviations from normal distribution of
the Ang 1-10 degradation products are indicative for enzymatic
malfunction of the RAS and/or the bradykinin system, which can
lead to e.g. a pathologic increase in blood pressure or other
pathologic conditions or diseases. On the other hand, from the
distribution and relative concentrations of the Ang 1-10
degradation products in the steady state equilibrium according
to the present invention type and effectiveness of therapeutic
treatments targeting the RAS can be monitored. Surprisingly, the
steady state equilibrium concentrations according to the present
invention provide much better indications and correlations than
the determination of Ang 1-10 or other degradation products in
blood samples without allowing them to reach the steady state
equilibrium (i.e. the "classical" blood samples immediately
stabilised).
In contrast to state-of-the-art methods, where inhibitors
are used to immediately stabilise peptides produced by certain
enzymes with limited success [Bystrom et al., Clin. Chem.
56(2010), 1561-1569], according to the present invention the
sample is allowed to reach an enzyme activity defined steady
state equilibrium for at least one peptide involved in the
proteolytic cascade. This innovative approach allows a highly
reproducible overall assessment of the proteolytic cascade,
especially RA.S, in the physiological sample matrix while
integrating all enzyme activities involved in the metabolism of
the peptides of the proteolytic cascade. Another advantage over
state-of-the-art technologies is that substrate concentrations
in the assay according to the present invention generally remain
below the concentration of metabolising enzymes (except for the
feeding enzyme), taking into consideration the affinity of the
enzyme for each single substrate under the given conditions in
the sample (e.g. physiologic conditions) in contrast to in vitro
enzyme activity assays, where this important feature is
neglected for means of simplification by using excess amounts of
substrate.
Finally, the measurement of steady state equilibrium peptide
levels by the method according to the present invention allows
the identification of potential sites of de-regulation in
patient samples, paving the way for a patient specific approach
in the therapeutic manipulation of the proteolytic cascades
affected by the patient's disease (e.g. selection of the type of
RAS targeted medication in the treatment of hypertension and
prediction of drug resistances) and bearing significant
potential for the use of the method in the discovery of
biomarkers.
The present invention therefore provides an excellent ex-
vivo platform for diagnostic analyses, especially for the
diagnosis of a disease related to a proteolytic cascade,
especially the RAS. Such disease related to the RAS includes,
for example, hypertension, cardiac diseases, especially
congestive heart failure, chronic heart failure, acute heart
failure, arteriosclerosis, myocardial infarction, kidney
dieases, especially renal failure, diabetic nephropathy, lung
diseases, especially acute lung injury and/or acute respiratory
distress syndrome (ARDS), liver diseases, especially fibrosis,
inflammatory diseases, especially sepsis, arthritis,
rheumatitis, and/or cancer. The invention is also suited for
large-number routine analysis of patients, for e.g. identiflying
RAS affecting substances, assessing the effects of RAS affecting
substances, and/or monitoring patients being under RAS affecting
medication. Such RAS affecting medication may include one or
more active ingredients such as, for example, aminopeptidase
inhibitors, especially amastatin; ACE inhibitors, especially
captopril, enalapril, fosinopril, lisinopril, perindopril,
quinapril, ramipril, trandolapril, benazepril; angiotensin II
receptor antagonists, especially candesartan, eprosartan,
irbesartan, losartan, olmesartan, telmisartan, valsartan;
aldosterone receptor antagonists, especially eplerenone,
spironolactone; and/or ACE2, especially recombinant soluble
human ACE2 and renin inhibitors, especially aliskiren.
Specifically for blood samples, it is important for reaching
the steady state equilibrium for at least one peptide involved
in the proteolytic cascade, that the proteases of the
proteolytic cascade to be observed with the present method are
not inhibited by addition of protease inhibitors to the sample,
at least not to an extend which does not allow at least one
enzyme involved in the degradation of said at least one peptide
to work until the steady state is reached for said at least one
peptide, i.e. at least one degradation enzyme of said peptide(s)
has to be active to an extend to allow steady state equilibrium
for said peptide(s). Therefore, in one embodiment, protease
inhibitors are not added to the sample to an extent, that the
activities of the proteases involved in the formation and
degradation of the at least one peptide to be analysed are
significantly inhibited, before and/or during the incubation
until a steady state equilibrium is reached. According to said
embodiment, the samples are not combined with such protease
inhibitors or, if such inhibitors have already been added, such
inhibitors are inhibited (in their protease inhibiting function)
or removed before and/or during the incubation until a steady
state equilibrium is reached. Of course, inhibitors which do not
affect the proteases of the relevant proteolytic cascade which
should be studied by the method according to the present
invention, but which inhibit other proteolytic activities (e.g.
inhibitors of blood coagulation if the RA.S is studied) , can be
added to the sample, because this would not affect the ability
of the relevant proteolytic cascade (i.e. the cascade to be
analysed, e.g. the RAS) to reach a steady state equilibrium for
at least one peptide of the cascade.
Proteolytic cascades are present in a high number of
physiological processes from the simple digestion of proteins
(e.g. food protein or proteins to be eliminated from body cells
or body fluids) to highly regulated cascades, such as the RA.S or
the blood coagulation system. Proteases can either break
specific peptide bonds (limited proteolysis) / depending on the
amino acid sequence of a protein, or break down a complete
peptide to amino acids (unlimited proteolysis). The specific
cleavage of peptide bonds is a central mechanism for regulation
of complex biological processes. Especially peptide hormones are
frequently produced as inactive precursor peptides, which are
present in circulation in excessive amounts. Selective proteases
are necessary to liberate the active hormones, which are then in
turn degraded by other proteases.
In one embodiment, the type of sample, which can be analysed
according to the present invention, is a blood sample. "Blood
sample" according to the present invention can be any sample
containing blood or one or more of its fractions (containing the
proteases of the relevant proteolytic cascade). Specifically,
all blood samples, which are routinely provided from human
donors can be used according to the present invention, i.e. full
blood, plasma or serum, especially fresh or frozen anti-
coagulated full blood or fresh or frozen anti-coagulated plasma.
"Anti-coagulated" blood or plasma contains anti-coagulants, i.e.
substances, which prevent coagulation (stopping blood from
clotting), of course, without affecting the proteases of the
proteolytic cascade to be analysed in their ability to reach a
steady state equilibrium. A suitable anti-coagulant is heparin;
heparinised blood samples are therefore a suitable source
material for the method according to the present invention.
Heparinised blood samples may be further processed before the
method according to the present invention is applied. For
example, heparinised plasma or serum may be derived from the
blood sample. Alternatively, the method according to the present
invention may also be performed on full blood, i.e. with all
blood cells still being present (as well as the proteases on or
in such cells) . More generally acting protease inhibitors as
anti-coagulants, such as citrate or EDTA are less suitable; or,
in the alternative (if the sample e.g. already contains such
anti-coagulants) may require the addition of neutralising
substances for such protease inhibitors in order to allow
incubation of the sample until a steady state equilibrium is
reached.
With the method according to the present invention, not only
one specific peptide or peptidic degradation product as a marker
for the status of a proteolytic cascade can be identified. It is
possible to analyse more than one of the peptide members of the
cascade, thereby allowing an even further fine-tuned analysis of
the physiologic or biochemical status of this cascade in the
subject. It has turned out in the course of the present
invention that the relative amounts of different peptide members
of a proteolytic cascade in the steady state equilibrium are
highly indicative of the physiologic or biochemical status of
this cascade in the subject. For example, the ratio of molar
amounts or concentrations of two or more proteolytic degradation
products in steady state equilibrium can be indicative of an
enzyme activity, a related physiologic condition or pathology.
An example provided with the present invention is the molar
ratio of Ang 1-8 to Ang 1-10 (or relative ratios of other Ang 1-
degradation products) in steady state equilibrium, which is
indicative of the RAS status of the subject and/or the RA.S
influencing treatment regime applied to the subject (e.g.
treatment with substances like Lisinopril, Amastatin, ACE, ACE2,
NEP, etc.).
Therefore, one embodiment of the present invention comprises
the quantification of at least two, at least three, especially
at least four peptidic degradation products of the proteolytic
cascade in the sample in steady state equilibrium concentration.
With the methods of the present invention, it is possible to
quantify as many degradation products in the proteolytic cascade
in steady state equilibrium as possible (or: as known) to get a
"fingerprint-like" assessment of the status of the proteolytic
cascade.
According to the present invention it is necessary to
quantify the one or more proteolytic degradation products in the
steady state equilibrium. This is essentially different from
prior art analyses, which usually apply quantification of
analytes in a status of the proteolytic cascade immediately
stabilised after the samples (i.e. blood samples) are taken from
the subjects, i.e. not in a steady state equilibrium. Usually/
such prior art samples have been treated with protease
inhibitors immediately after taking of the samples in order to
inhibit unwanted enzyme dependent changes in the cascade. The
present invention, however, uses such enzyme dependent changes
in analysing the physiologic or biochemical status of the
subject concerning the proteolytic cascade by specifically
allowing the proteases of said cascade under investigation to
perform their proteolytic activity until a steady state
equilibrium is reached. This will usually lead to a change in
the amount and composition of the peptidic degradation products
in the proteolytic cascade under investigation compared to the
sample immediately stabilised after the taking of the sample
from the subject. According to the invention, the sample
specific proteolytic activity leads to a steady state
equilibrium which is much more indicative of the biochemical
status of the subject concerning this cascade than the
immediately stabilised sample (without the incubation step until
a steady state equilibrium is reached according to the present
invention).
As already indicated above, the steady state equilibrium
according to the present invention is not a single,
quantitatively exactly determined and isolated point, but a
status where changes in the relative ratios have been
substantially reduced in the sample. Usually, such a steady
state equilibrium can be reached by applying usual incubation
conditions for the given samples and the cascade under
investigation. As specified above, the sample may be incubated
for up to 15 minutes, 20 minutes, 25 minutes, 30 minutes, 60
minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, 210
minutes, 240 minutes, 270 minutes, or 300 minutes. For the RAS
and/or the bradykinin system, the samles may be incubated for at
least 30 min to up to 300 min, or for at least 30 min to up to
180 min, or for at least 30 min to up to 120 min, or for at
least 30 min to up to 90 min, or for at least 30 min to up to 60
min. Suitable incubation temperatures are those present in the
physiologic system or those, wherein the proteases of the
proteolytic cascade under investigation have their optimal
temperature of action, e.g. at a temperature of 30 to 50"C, 35
to 40 °C, or especially of about 37 °C (specifically for human
blood samples).
As already stated, the present invention is further
exemplified in the example section, especially on the renin-
angiotensin-system (RAS) and the bradykinin system. The one or
more peptidic degradation products to be analysed can be
selected from angiotensin peptides, such as angiotensin I (Ang
1-10), angiotensin 2-10 (Ang 2-10), angiotensin II (angiotensin
1-8 or Ang 1-8), angiotensin III (angiotensin 2-8 or Ang 2-8),
angiotensin IV (angiotensin 3-8 or Ang 3-8), angiotensin 1-9
(Ang 1-9), angiotensin 1-7 (Ang 1-7), angiotensin 2-7 (Ang 2-7),
angiotensin 3-7 (Ang 3-7) and angiotensin 1-5 (Ang 1-5) ; and/or
from kinin peptides, such as kallidin (KD or Lys-bradykinin) and
its biological degradation products, such as bradykinin 1-9 (BK
1-9), bradykinin 2-9 (BK 2-9), bradykinin 1-8 (BK 1-8),
bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5).
In an embodiment, the peptidic degradation product is
selected from angiotensin I (1-10), angiotensin II (angiotensin
1-8 or Ang 1-8), angiotensin 1-7 (Ang 1-7), and angiotensin 1-5
(Ang 1-5). In another embodiment, the peptidic degradation
product is selected from angiotensin II (angiotensin 1-8 or Ang
1-8), angiotensin 1-7 (Ang 1-7), and angiotensin 1-5 (Ang 1-5).
In still another embodiment/ the peptidic degradation product is
selected from bradykinin 1-9 (BK 1-9), bradykinin 1-8 (BK 1-8),
bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5).
Since the method according to the present invention applies
the proteolytic activities contained in the sample, the one or
more samples, especially blood samples, should be free of added
protease inhibitors for the proteolytic cascade before the
steady state equilibrium is reached.
Such protease inhibitors may be added after the incubation
until a steady state equilibrium is reached and stabilised. This
safeguards that the peptide concentrations reflecting the steady
state equilibrium are still present during the quantification
step (although the steady state equilibrium is usually stable
over a certain period of time, this provides additional quality
assurance for the method according to the present invention).
With the methods according to the present inventions it is
possible to monitor the status of the RAS and/or the bradykinin
system. In one embodiment, at least two peptidic degradation
products are quantified and a ratio is calculated of the steady
state equilibrium quantifications of the at least two peptidic
degradation products. Accordingly, one embodiment of the present
invention employs the quantification of at least two of
angiotensin I (Ang 1-10), angiotensin 2-10 (Ang 2-10),
angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin III
(angiotensin 2-8 or Ang 2-8), angiotensin IV (angiotensin 3-8 or
Ang 3-8), angiotensin 1-9 (Ang 1-9), angiotensin 1-7 (Ang 1-7),
angiotensin 2-7 (Ang 2-7), angiotensin 3-7 (Ang 3-7) and
angiotensin 1-5 (Ang 1-5) , kallidin (KD or Lys-bradykinin) and
its biological degradation products, such as bradykinin 1-9 (BK
1-9), bradykinin 2-9 (BK 2-9), bradykinin 1-8 (BK 1-8),
bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5) . Then, a
ratio or product of those at least two peptidic degradation
products may be calculated to provide an especially indicative
parameter for the status of the proteolytic cascade, e.g. the
RAS, in the sample. For example, the steady state equilibrium
concentration ratio between Ang 1-8 and Ang 1-10 is indicative
of the physiologic or biochemical status, function, and/or
activity of ACE. Accordingly, the ratio between a peptidic
degradation product and its precursor peptide (i.e. the
substrate of the enzyme forming said peptidic degradation
product) in steady state equilibrium is indicative of the
physiologic or biochemical status, function, and/or activity of
the enzyme which cleaves said substrate into said peptidic
degradation product. In an embodiment, more than one ratio
between a peptidic degradation product and its precursor peptide
are calculated. Said at least two ratios may be again related to
each other, e.g. by calculating the ratio or product of the at
least two ratios, or by addition or subtraction of ratios, or
both (calculating the ratio (s) between sums and/or differences
or subtractions). In another embodiment, the ratio between at
least one in vivo peptide level (immediately stabilised) and the
same peptide level in steady state equilibrium is calculated.
The method according to the present invention is dependent
on an exact and accurate quantification of the peptidic
degradation products. Since many samples, especially blood
samples contain proteins, salts, acids, bases, lipids,
phospholipids or other components, which can disturb peptide
quantification; methods for pre-treatment of the samples before
quantification may be applied.
The method according to the invention may be conducted by
using a kit.
Accordingly, in another aspect, the present invention
concerns a kit for the measurement of peptidic degradation
products of a proteolytic cascade in a biological sample in
steady state equilibrium concentration. The kit may comprise an
instruction to incubate the sample for a certain time period
until a steady state equilibrium is reached for at least one
peptidic degradation product of the proteolytic cascade. The kit
may further comprise one or more instructions for conducting the
method according to the present invention, as specified above.
Accordingly, in one embodiment, the kit for the measurement
of peptidic degradation products of a proteolytic cascade in a
biological sample in steady state equilibrium concentration
(i.e. a kit for conducting the method according to the
invention) comprises an instruction to incubate the one or more
samples for a certain time period until a steady state
equilibrium is reached for at least one peptidic degradation
product of the catalytic cascade. Optionally, the kit further
comprises one or more chemical, biochemical and/or
biotechnological reagents selected from peptides, enzymes,
enzyme inhibitors, buffers, solvents, chaotropic agents,
detergents, and combinations thereof. Optionally, the kit
further comprises one or more chemical, biochemical and/or
biotechnological laboratory items selected from solid phase
extraction materials or other purification materials,
containers, and combinations thereof. Optionally, the kit
further comprises one or more biological samples selected from
blood samples, serum samples, plasma samples, tissue samples,
and combinations thereof. For example, said biological samples
can be blood, serum, plasma and/or tissue samples and may be
used e.g. as a standard and/or quality control. Said containers
can be tubes or sample collection vials, or any other standard
container suitable to comprise chemical, clinical, biochemical
and/or biotechnological material. In an embodiment, the
container is a blood or tissue container. Said container
optionally comprises one or more anti-coagulants, such as e.g.
heparin, EDTA, and/or other anti-coagulants. Said container may
further comprise one or more enzyme inhibitors or a protease
inhibitor cocktail, such as further specified above or in the
example section.
In an embodiment, the kit further comprises one or more
protease inhibitors or inhibitor cocktails, one or more feeding
substrates, one or more blood collection tubes, optionally
coated with heparin, one or more standards, one or more quality
control samples, one or more solid phase extraction materials,
and/or one or more solvents/ or combinations thereof.
The components of the kit and the instructions comprised by
the kit are further defined above with regard to the methods of
the present invention. The instruction to incubate the samples
for a certain time period until a steady state equilibrium is
reached for at least one peptidic degradation product or a
catalytic cascade may, for example, include information about
the methods, reagents, and/or conditions as specified above with
regard to the methods of the present invention, for example,
information as to the duration and conditions for incubation,
sample stabilisation, and/or analysis. Accordingly, the
instruction may include further instructions, such as e.g. not
to add any protease inhibitors prior to and/or during incubation
until a steady state equilibrium is reached, and/or to
inactivate or remove any protease inhibitor which may have been
added. Specific applications like the measurement of single
protease activities based on steady state peptide measurements
or the influence of certain protease inhibitors (or respective
pharmaceutical compositions) on the proteolytic cascade and
peptide levels in steady state equilibrium might require the
addition of certain protease inhibitors before the incubation to
reach steady state equilibrium. Thus, the instructons may
include instructructions to add certain protease inhibitors.
However, as specified above, at least one proteolytic
degradation reaction has to be active for at least one peptide
of the proteolytic cascade to an extent which allows that the
actual overall degradation rate is equal to the actual overall
formation rate of said peptide.
Such instruction may be provided, for example, in a paper
format (e.g. as a leaflet or manual) or electronically. Such
instruction may directly or indirectly accompany the kit, e.g.
it is provided by the manufacturer and/or supplier of the kit
and comprised by the kit package or provided otherwise together
with the kit (e.g. by email from the manufacturer and/or
supplier of the kit or by download from a web page of the
manufacturer and/or supplier of the kit) .
In another aspect, the present invention relates to the use
of the kit for the measurement of peptidic degradation products
of a proteolytic cascade in a biological sample in steady state
equilibrium concentration. Such use of the kit is further
specified above with regard to the methods according to the
invention. In an embodiment, the use of the kit comprises the
step of incubating the sample until a steady state equilibrium
is reached for at least one peptidic degradation product
involved in said proteolytic cascade. In another embodiment, the
use of the kit comprises the step of quantifying said at least
one peptidic degradation product in a steady state equilibrium
concentration in the sample.
According to another aspect, the present invention concerns
a physical or electronic representation of the result of
quantification of the method according to the present invention
or of the use of the kit according to the present invention,
wherein at least two peptidic degradation products have been
quantified on a physical or electronic carrier and wherein the
quantified amounts of the at least two degradation products are
provided in the sequence of the proteolytic cascade. In other
embodiments, at least three or at least four peptidic
degradation products have been quantified on a physical or
electronic carrier and the quantified amounts of the at least
three or four degradation products are provided in the sequence
of the proteolytic cascade.
In one embodiment of the physical or electronic
representation according to the present invention, the results
of quantification of at least two or at least three of
angiotensin I (Ang 1-10), angiotensin 2-10 (Ang 2-10),
angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin III
(angiotensin 2-8 or Ang 2-8), angiotensin IV (angiotensin 3-8 or
Ang 3-8), angiotensin 1-9 (Ang 1-9), angiotensin 1-7 (Ang 1-7),
angiotensin 2-7 (Ang 2-1), angiotensin 3-7 (Ang 3-7) and
angiotensin 1-5 (Ang 1-5) , kallidin (KD or Lys-bradykinin) and
its biological degradation products, such as bradykinin 1-9 (BK
1-9), bradykinin 2-9 (BK 2-9), bradykinin 1-8 (BK 1-8),
bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5) are included
and provided in dependence of their relative quantities.
Accordingly, this physical or electronic representation may
be provided in a form, wherein the results of quantification of
angiotensin I (Ang 1-10), angiotensin 2-10 (Ang 2-10),
angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin III
(angiotensin 2-8 or Ang 2-8), angiotensin IV (angiotensin 3-8 or
Ang 3-8), angiotensin 1-9 (Ang 1-9), angiotensin 1-7 (Ang 1-7),
angiotensin 2-7 (Ang 2-7), angiotensin 3-7 (Ang 3-7) and
angiotensin 1-5 (Ang 1-5) are included and provided in
dependence of their relative quantities. According to another
embodiment, this physical or electronic representation may be
provided in a form, wherein the results of quantification of
kallidin (KD or Lys-bradykinin) and its biological degradation
products, such as bradykinin 1-9 (BK 1-9), bradykinin 2-9 (BK 2-
9), bradykinin 1-8 (BK 1-8), bradykinin 1-7 (BK 1-7) and
bradykinin 1-5 (BK 1-5) are included and provided in dependence
of their relative quantities.
In one embodiment, this physical or electronic
representation may be provided in a form, wherein the results of
quantification of angiotensin I (1-10), angiotensin II
(angiotensin 1-8 or Ang 1-8), angiotensin 1-7 (Ang 1-7), and
angiotensin 1-5 (Ang 1-5) are included and provided in
dependence of their relative quantities.
In another embodiment, this physical or electronic
representation may be provided in a form, wherein the results of
quantification of angiotensin II (angiotensin 1-8 or Ang 1-8),
angiotensin 1-7 (Ang 1-7) , and angiotensin 1-5 (Ang 1-5) are
included and provided in dependence of their relative
quantities.
In still another embodiment, this physical or electronic
representation may be provided in a form, wherein the results of
quantification of bradykinin 1-9 (BK 1-9), bradykinin 1-8 (BK 1-
8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5) are
included and provided in dependence of their relative
quantities.
In still another embodiment, this physical or electronic
representation may be provided in a form, wherein the results of
quantification of bradykinin 1-8 (BK 1-8), bradykinin 1-7 (BK 1-
7) and bradykinin 1-5 (BK 1-5) are included and provided in
dependence of their relative quantities.
This physical or electronic representation of the peptidic
degradation products according to the present invention is a
valuable diagnostic tool for any physician allowing a
significant assistance in the diagnosis of a disorder or disease
(e.g. a disorder or disease connected to the RAS) . It allows
comparison between healthy and diseased status (or the
comparison of different stages of the disease or different
stages in the treatment) in a "fingerprint-like" manner, i.e. by
a multi-parameter representation of the quantification results
obtained (as shown in the graphic representations in the figures
according to the present invention ("RAS-Fingerprints") ) . These
results can be provided electronically. In the electronic
format, comparison and development of these patterns over time
either from the same patient, or over a population or patient
group, can easily be analysed and monitored. In graphic
representations, the proteolytic cascade can be depicted so that
the results of the present method are not only quantification
results, but also complex and interdependent "fingerprint-like"
results being of high value in the diagnosis of a disease and/or
treatment depicting the biochemical relations underlying the
peptide concentrations. This tool according to the present
invention therefore allows an improved use of the results
obtained with the method according to the present invention.
The invention is further described by the following examples
and the figures, of course without being limited thereto:
Figure 1 shows the RAS steady state equilibrium (RSSE). (A)
Heparin blood was collected from a healthy donor and incubated
at 37 C. After indicated time periods protease inhibitor
cocktail was added to an aliquot of the heparinised blood sample
to conserve the steady state equilibrium. Concentrations of
angiotensin 1-10 (blue) and angiotensin 1-8 (green) are shown
and compared with an aliquot of the blood sample collected from
the same donor, which was stabilised immediately during
collection (ICE, t=0) . (B) Steady state equilibrium angiotensin
concentrations in heparinised blood were determined in the
absence (red; control) and presence of indicated RAS inhibitors
(blue) . The steady state equilibrium was frozen by adding a
protease inhibitor cocktail to the samples immediately (Oh) or
after 2h and 4h of incubation at 37 C. (C) Angiotensin
concentrations in heparinised blood were determined in the
absence (red; control) and presence of EDTA and AEBSF (blue) .
The incubations were frozen by adding a protease inhibitor
cocktail to the samples immediately (Oh) or after 2h and 4h of
incubation at 37°C. Concentrations of angiotensin 1-10 and
angiotensin 1-8 are shown.
Figure 2 shows the pharmacologic manipulation of the RAS
steady state equilibrium. Indicated agents were added prior to
an incubation period of 2h followed by LC-MS/MS based RSSE-
Fingerprinting. Results are shown in fingerprint illustrations
displaying angiotensin peptide concentrations as differently
sized spheres and metabolising enzymes represented by blue
arrows and letters. Annotations beneath the spheres are
constituted by peptide name and peptide concentration in pg/ml
blood. RSSE-Fingerprints are shown for low-molecular-weight RAS
inhibitors (A), exogenously added RAS enzymes (B) and
combinations of both (C).
Figure 3 shows matrix dependence of RSSE-Fingerprinting.
Heparinised blood, plasma and plasma subjected to one
freeze/thaw cycle were prepared from the same donor and
subjected to RSSE-Fingerprinting performing a 2h-37 C incubation
followed by conserving the steady state equilibrium adding a
protease inhibitor cocktail to the samples and LC-MS/MS
analysis. Fingerprint graphs of control samples without
pharmacologic manipulation (A) are given and compared with
samples where Amastatin (B) or ACE2 in combination with
Lisinopril (C) where added before the incubation period.
Figure 4 shows RAS- as well as RSSE-Fingerprinting in
healthy volunteers. Blood samples were collected from healthy
volunteers in the presence of protease inhibitor cocktail to
conserve angiotensin peptide levels as present in vivo, i.e.
prior to any equilibration (RAS-Fingerprint or in vivo RAS-
Fingerprint) and compared to heparinised blood and plasma
subjected to incubation until a steady state equilibrium is
reached (RSSE-Fingerprint or ex vivo Fingerprint) in parallel.
The mean RAS-Fingerprint (conserved in vivo peptide levels) and
the mean RSSE-Fingerprints (conserved ex vivo generated steady
state equilibrium peptide levels) for heparin blood and plasma
for 12 healthy volunteers are shown (A). The molar SSE-Ratio for
ACE [fmol/ml Ang 1-8]/[fmol/ml Ang 1-10] was calculated and
presented in the together with corresponding angiotensin 1-10
and angiotensin 1-8 concentrations in pg/ml and fmol/ml for each
subject donor (B). The tables show the values for peptide levels
constituting the RAS-Fingerprints (Fig. 4B1; Table 1), RSSE-
Fingerprints from Blood (Fig. 4B2; Table 2) and RSSE-
Fingerprints from plasma (Fig. 4B3; Table 3) for all donors as
well as calculated MEAN and SEM. The RAS- and RSSE-Fingerprints
of 2 out of the 12 healthy donors are shown together with
corresponding values for molar SSE-Ratios below (C; 4C1: Donor
3, 4C2: Donor 6).
Figure 5 shows an overview of the main peptide degradation
steps of the bradykinin system, i.e. the main bradykinin
peptides and the respective enzymes forming and/or degrading
those peptides.
Figure 6 shows the tables with the peptide levels measured
for the RAS (A) and the bradykinin system (B) in the same plasma
samples, stabilised with GTC after incubation for the indicated
time periods.
Figure 7 shows the respective bar graphs.
EXAMPLES:
Materials:
C18 Cartridges: Sep-Pak Vac 3cc (500mg), Waters
Mass Spectrometer: Q TRAP4000 - Applied Biosystems
HPLC System: 1100 Series, Agilent
C18 RP-HPLC column: Luna 3u Cl8(2) 100A, 100x2.00mm,
(Phenomenex, Cat.no. OODBO)
Reagents:
Ethanol, abs. (Merck, Cat.no. 100983)
Methanol, (Fluka/ Cat.no. 14262)
Water, LiChrosolv (Merck, Cat.no. 115333)
Acetonitril/ LiChrosolv (Merck, Cat.no. 114291)
Formic acid, >98%, (Fluka, Cat.no. 06440)
Z-Arg, as Renin inhibitor, (Bachem, C-3195)
Pepstatin A (Bachem (N-1125)
p-Hydroxymercuribenzoic acid, sodium salt (Fluka, 55540)
1,10-Phenanthroline monohydrate (Sigma, P9375)
Lisinopril (Sigma, L6394)
Captopril (Sigma, C4043)
Amastatin • HC1, (Bachem, N-1410)
ACE, NEP and APN were purchased from R&D Systems.
rhACE2 (recombinant soluble human ACE2) was produced by Apeiron
Biologies.
EDTA (Sigma)
GTC (Sigma, Cat.no. G9277)
Trifluoroacetic acid (TFA) (Sigma-Aldrich, Cat.no. 302031)
Internal standards:
The internal standards used for absolute quantification of
peptides in biological samples were synthetic peptides, their
sequence was identical to the peptides analytes and was tagged
with a mass label allowing the discrimination between endogenous
peptides and standard peptides in LC-MS/MS analysis. The
identical physicochemical properties of these synthetic peptides
make them ideal internal standards for low abundance peptide
quantification showing identical behaviour and recovery during
sample processing compared to their corresponding peptide
analyte. The internal standards were subjected to the sample
during or directly after blood collection, taking into
consideration all manipulation induced variations. The use of
peptide specific internal standards is recoimnendable, as peptide
recoveries may differ between different peptides and individual
samples.
Furthermore, the MS/MS-fragmentation characteristics of
endogenous and standard peptides are identical allowing high
accuracy and precision in determining absolute peptide levels.
Example I: Analysis of proteolytic cascade degradation products
in blood samples according to the present invention (RSSE
fingerprint)
RSSE-Fingerprinting.
Blood samples were collected and anti-coagulated with
standardised heparin tubes (BD). As indicated in figure 3,
plasma separation has been done for the respective samples
before the incubation to reach steady state equilibrium. After
incubation of the blood or plasma samples for the time periods
as indicated in figure 1, or incubation for 2 h for figures 2, 3
and 4, in a 37 °C water bath, samples were cooled on ice followed
by immediate addition of the steady state equilibrium conserving
protease inhibitor cocktail containing Pepstatin A, 1,10-
Phenanthroline, EDTA, p-Hydroxymercuribenzoic acid, and Z-Arg,
as well as the internal standards.
LC-MS/MS sample preparation and analysis:
Following plasma separation by centrifugation at 3000 rcf
for 10 minutes at 4"C, 0.2 - 2 ml of plasma was applied onto an
activated and equilibrated Sep-Pak C18 cartridge. Sample matrix
components were removed by washing three times with 1 ml water.
Bound analytes were then eluted with 1 ml of methanol. Eluates
were evaporated to dryness and reconstituted in 10%
acetonitril/90% water supplemented with 0.1% formic acid
followed by subjection to LC-MS/MS analysis.
Solid phase extraction:
A vacuum manifold has been used for sample processing.
1. Activation EtOH abs.
2. Equilibration 2 x 1ml R^O
3. Loading: 0.2 - 1ml stabilised plasma
4. Washing: 3 x 1 ml H20
. Elution: 1ml MeOH
Quantification and signal integration.
MRM chromatograms were integrated using Analyst 1.5.1
software provided by Applied Biosystems. The threshold for the
quantification limit was set at a signal-to-noise ratio of 10.
Integration signals not reaching this ratio were set to zero.
Analyte signals were related to internal standard signals and
concentration was calculated from initially spiked amounts of
internal standards.
RESULTS
The evaluation of the RAS in respect to angiotensin peptide
concentrations is critically dependent on the conditions used
for sample collection and sample conservation. An analytical
system was developed which is able to effectively conserve in
vivo as well as ex vivo steady state equilibrium angiotensin
peptide levels in blood followed by high sensitivity LC-MS/MS-
Analysis and absolute quantification.
In general, the RA.S is a peptide hormone system constantly
producing new peptides from the pro-hormone AGT, whereas the
rate of production is primarily dependent on renin activity. The
peptide levels, which are present in circulation, are dependent
on soluble proteases, blood cell bound proteases and also
endothelium associated proteases which can be spatially
different due to organ specific expression patterns. The inner
surface of blood vessels is covered with numerous different
angiotensin receptors and therefore takes over a central part in
the establishment of angiotensin peptide concentrations in
circulation. As a consequence of organ specific expression of
angiotensin metabolising proteases like ACE or ACE2, it becomes
obvious that blood peptide levels can be spatially different
throughout the body. Nevertheless, there are plenty of enzymatic
components of the RAS present in blood either in a freely
soluble or in a blood cell associated form, which significantly
affect circulating peptide levels. Taking together previous
considerations, the RAS constitutes a system with a temporary
constant throughput of peptide hormone molecules with local
differences regarding peptide concentrations in different
tissues and organs.
In the present invention a method is described which takes
into account all blood associated factors affecting peptide
hormone systems like the RAS by incubating a blood or plasma
sample until a steady state equilibrium is reached for one or
more peptide levels, followed by quantification of peptides. As
shown in Figure 1A, the RAS-Fingerprint which represents the in
vivo circulating angiotensin peptide concentrations in blood
samples collected by arm venous puncture and immediate sample
stabilisation by addition of a protease inhibitor cocktail, is
significantly different from the ex vivo RAS-Fingerprint (or
RSSE-Fingerprint) observed when blood is incubated at 37 C
without the addition of protease inhibitors. Interestingly, the
angiotensin peptide concentrations achieved under these
conditions reach levels, which were found to be constant over a
remarkable period of time. This indicated a state of equilibrium
reached by the sample, which is characterised by equal rates of
formation and degradation for individual peptides, namely the
steady state equilibrium. The steady state equilibrium peptide
levels were reached within 30 minutes and remained stable for at
least 6h from the start of incubation (Fig. 1A) . The stability
period ended with a strong increase of Ang 1-10 concentration in
the sample indicating a change of enzyme activities within the
sample. Finally, after 24h of incubation, the concentration of
Ang 1-10 steeply dropped down. Beside Ang 1-10 and Ang 1-8,
there were no significant amounts of other angiotensin
metabolites detected in the samples during the time course. The
so-called RSSE-Fingerprint (RAS steady state equilibrium
Fingerprint) was found to be significantly affected by
pharmacologic agents interfering with the RAS (Fig. 1B, Fig.
2A) . It was further tested if the addition of RAS affecting
agents can shift the steady state equilibrium of the sample to a
different but stable steady state equilibrium condition. The ACE
inhibitor (Captopril) and the aminopeptidase inhibitor
(Amastatin) or a combination of both were added to blood prior
to incubation for 2 or 4 hours at 37 C. All treatments reached
steady state equilibrium characteristic for the inhibitor(s) as
indicated by comparable levels of Ang 1-10 and Ang 1-8 after 2
and 4 hours of incubation (Fig. 1B). The ACE inhibitors
Lisinopril or Captopril increased the Ang 1-10 and decreased the
Ang 1-8 steady state equilibrium levels when compared to control
levels. Renin inhibitors, which block the initial step of
angiotensin production, were found to completely shut down the
RAS. Amastatin, which is an inhibitor of certain
aminopeptidases, was found to massively increase Ang 1-8 levels
pointing to the important role of aminopeptidases in the
regulation of Ang 1-8 levels in vivo.
Fig. 1C compares the peptide concentrations reached after 2h and
4h incubation at 37 C in the absence (red bars) and presence of
EDTA and AEBSF (blue bars) followed by the conservation of
peptide concentrations by addition of an inhibitor cocktail at
indicated time points. The combination of EDTA and AEBSF is used
in state-of-the-art methods for the measurement of plasma renin
activity (PRA) (Bystrom et al.; Clin. Chem. 56(2010), 1561-
1569) . The concentrations of angiotensin 1-10 (upper panel) and
angiotensin 1-8 (lower panel) in control samples reach steady
state equilibrium as indicated by minor changes between 2h and
4h of incubation at 37 C (compare figure 1B) . In contrast, in
the presence of EDTA and AEBSF, there are significant changes in
the levels of either Ang 1-10 and Ang 1-8 between 2h and 4h of
incubation at 37°C. The strong accumulation of Ang 1-10 in the
EDTA/AEBSF treated sample over time clearly shows the absence of
a steady state equilibrium in these samples.
Furthermore, the effects of recombinant RAS enzymes on RSSE-
Fingerprints were tested by adding 5^g/ml ACE, ACE2, NEP, or APN
to the samples prior to the incubation periods. The RSSE-
Fingerprints shifted as expected in these samples (Fig. 2B) and
also in samples where combinations of enzymes with different
pharmacologic inhibitors were added (Fig. 2C) . Of note, the
comparison of samples treated with the ACE inhibitor Lisinopril
with the combination of ACE2 and Lisinopril revealed, that Ang
1-10 is a substrate for ACE2 at physiologic concentrations in
the original sample matrix, efficiently producing Ang 1-9 (Fig.
2A1, 2C1) . Although the steady state levels of Ang 1-10 after
addition of ACE2 remain high, there is a remarkable peptide flow
in the direction Ang 1-9, which might be an important mechanism
of action of ACE inhibitors in clinical use. Furthermore, this
ACE2 mediated Ang 1-9 production is impressively shown in the
presence of Amastatin, which further increases steady state
equilibrium Ang 1-10 levels by inhibiting its N-terminal
proteolytic degradation (Fig. 2C3). ACE inhibition was found to
be a prerequisite to detect significant steady state equilibrium
Ang 1-9 levels which points to a significant higher affinity of
Ang 1-10 to ACE than to ACE2. This higher affinity of Ang 1-10
to ACE than to ACE2 could also be confirmed by the observation
of lower steady state equilibrium Ang 1-10 concentrations
comparing addition of ACE to ACE2 (Fig. 2B1).
The results obtained from these experiments clearly
demonstrated a direct association of the RSSE-Fingerprint with
the integrated RAS enzyme activities contained in the sample.
Based on these findings, the effect of using fresh or frozen
plasma instead of blood were explored as there would be easier
handling regarding large scale analysis, knowing that blood cell
associated RAS components would be lost under these conditions.
The RSSE-Fingerprints were compared for blood, plasma and
frozen/thawed plasma from the same donor for control samples
(Fig. 3A), Amastatin spiked samples (Fig. 3B) and samples spiked
with ACE2 and Lisinopril (Fig. 3C) . The inhibitors and
combinations were selected in order to achieve a clearly visible
shift of the steady state equilibrium while using either enzymes
or low molecular weight inhibitors to proof the method's
suitability for clinical analytic questions. Freezing and
thawing of plasma was found to cause minimal variations in the
RSSE-Fingerprint whereas using blood instead of plasma resulted
in significant differences especially regarding Ang 1-10, Ang 1-
8 and Ang 1-7 which point to the presence of blood cell
associated NEP (CD10) and ACE (CD143).
Finally the variability of the RAS-Fingerprint and the RSSE-
Fingerprint was investigated among 12 healthy volunteers and
analysed in immediately stabilised blood, equilibrated blood and
equilibrated plasma from each donor. The mean of the measured
angiotensin concentrations is given as a fingerprint graph in
Fig. 4A. Donor specific data regarding Ang 1-10 and Ang 1-8,
which are the predominant peptide present in healthy volunteers,
are given for the RAS-Fingerprints (4Bl-Table 1), the RSSE-
Fingerprint in blood (4B2-Table 2) as well as the RSSE-
Fingerprint in plasma (4B3-Table 3) . Beside concentrations of
the peptides in pg/ml, the concentrations were calculated in
fmol/ml and used to constitute a molar steady state activity
ratio for ACE by dividing the Ang 1-8 concentration through the
Ang 1-10 concentration. Compared to the RAS-Fingerprints
measured, the RSSE-Fingerprints showed greater variances among
donors, which reflect potential diversities in the constitution
of the soluble RAS components among different donors. Two
representative donors are shown in Figure 4C. Donor 3 (Fig. 4C1)
had a molar SSE-Ratio for ACE in blood (1-8/1-10) of 4.5 while
Donor 6 (Fig. 4C2) displayed an ACE-SSE-Ratio of 20.5 pointing
to a more prominent role of ACE in the production of Ang 1-8 in
this donor. There is also a difference in the RAS-ratio for ACE
(0.35 vs. 0.79), however, the difference based on the RSSE-
Fingerprint are much more distinctive.
As a conclusion, a powerful method for the evaluation of the
RAS or components thereof in biological samples is provided with
the present invention. The combination of the present highly
sensitive LC-MS/MS based angiotensin peptide quantification
method with the innovative steady state equilibration of the
sample prior to stabilisation represents a highly reproducible
tool for evaluation of soluble and blood cell associated RAS
enzyme activities. The use of this new technology has a great
potential for the discovery of biomarkers as the RAS is involved
in a variety of pathologic conditions. Furthermore, soluble and
blood cell associated RA.S enzyme activities represent a major
site of pharmacologic activity of several anti-hypertensive
drugs. The understanding of the system'1 s individuality might
pave the way for patient specific approaches in the treatment of
RAS associated diseases. The technology according to the present
invention will push this development forward by providing a deep
and comprehensive insight into the renin-angiotensin system in
biological samples.
Example II: Analysis of proteolytic cascade degradation products
of the renin-angiotensin system as well as the bradykinin system
in blood samples according to the present invention
All methods were done as described in Example I, except that the
plasma samples were stabilised by the addition of 4M GTC/1% TFA,
either immediately or after incubation for 1 or 3 hours in a
37°C water bath.
RESULTS
Figures 6 and 7 show that the method according to the present
invention can be applied not only to the RAS, but also to other
proteolytic cascades, such as the bradykinin system.
Furthermore, these figures show that the steady state
equilibrium can efficiently be stabilised not only by the
addition of a protease inhibitor cocktail, but also by a
chaotropic agent such as GTC. After an incubation period of 1 h,
a steady state equilibrium has been reached for the RAS and the
bradykinin system and remained stable, as can be seen from the
comparison of peptide levels between 1 h and 3 h of incubation.
Claims (18)
1. Method for measurement of peptidic degradation products of a proteolytic cascade comprising at least two consecutive proteolytic reactions in a biological sample, wherein the sample is incubated until the actual overall degradation rate of at least one peptidic degradation product is equal to the actual overall formation rate of said at least one peptidic degradation product and a steady state equilibrium is reached for said at least one peptidic degradation product involved in said proteolytic cascade and wherein said at least one peptidic degradation product in a steady state equilibrium concentration is quantified in the sample.
2. Method according to claim 1 wherein said sample is a blood sample, such as full blood, plasma or serum, especially fresh or frozen anti-coagulated full blood or fresh or frozen anti- coagulated plasma.
3. Method according to claim 1 or 2, wherein at least two, at least three, or at least four peptidic degradation products of the proteolytic cascade are quantified in the sample in steady state equilibrium concentration.
4. Method according to claim 3, wherein at least two peptidic degradation products are quantified and wherein a ratio is calculated of the steady state equilibrium quantifications of the at least two peptidic degradation products.
5. Method according to any one of claims 1 to 4, wherein the sample is incubated for up to 15 minutes, 20 minutes, 25 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes/ 150 minutes, 180 minutes, 210 minutes, 240 minutes, 270 minutes, or up to 300 minutes.
6. Method according to any one of claims 1 to 5, wherein the sample is incubated at a temperature of 30 to 50 C, of 35 to 40°C, or of substantially 37°C.
7. Method according to any one of claims 1 to 6, wherein the proteolytic cascade is the renin-angiotensin system (RAS) or the bradykinin system, or both.
8. Method according to any one of claims 1 to 7, wherein the peptidic degradation product is selected from angiotensinogen, angiotensin I (Ang 1-10), angiotensin 2-10 (Ang 2-10), angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin III (angiotensin 2-8 or Ang 2-8), angiotensin IV (angiotensin 3-8 or Ang 3-8), angiotensin 1-9 (Ang 1-9), angiotensin 1-7 (Ang 1-7), angiotensin 2-7 (Ang 2-7), angiotensin 3-7 (Ang 3-7), angiotensin 1-5 (Ang 1-5), kallidin (KD or Lys-bradykinin), bradykinin 1-9 (BK 1-9), bradykinin 2-9 (BK 2-9), bradykinin 1-8 (BK 1-8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5).
9. Method according to any one of claims 1 to 8, wherein the peptidic degradation products that are quantified are angiotensin I (1-10), angiotensin II (angiotensin 1-8 or Ang 1- 8), angiotensin 1-7 (Ang 1-7), and angiotensin 1-5 (Ang 1-5).
10. Method according to any one of claims 1 to 8, wherein the peptidic degradation products that are quantified are angiotensin II (angiotensin 1-8 or Ang 1-8), angiotensin 1-7 (Ang 1-7), and angiotensin 1-5 (Ang 1-5).
11. Method according to any one of claims 1 to 8, wherein the peptidic degradation products that are quantified are bradykinin 1-9 (BK 1-9), bradykinin 1-8 (BK 1-8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1-5) .
12. Method according to any one of claims 1 to 8, wherein the peptidic degradation products that are quantified are bradykinin 1-8 (BK 1-8), bradykinin 1-7 (BK 1-7) and bradykinin 1-5 (BK 1- 5) .
13. Method according to any one of claims 1 to 12, wherein one or more protease inhibitors are added after the incubation until a steady state equilibrium is reached for at least one peptidic degradation product.
14. Kit when used for the measurement of peptidic degradation products of a proteolytic cascade comprising at least two consecutive proteolytic reactions in a biological sample in steady state equilibrium concentration, wherein the actual overall degradation rate of at least one peptidic degradation product is equal to the actual overall formation rate of said at least one peptidic degradation product and wherein said at least one peptidic degradation product in a steady state equilibrium concentration is quantified, wherein said kit comprises one or more chemical, biochemical and/or biotechnological reagents selected from peptides, enzymes, enzyme inhibitors, buffers, solvents, chaotropic agents, detergents and combinations thereof, and instructions for conducting the method according to any one of claims 1 to 13.
15. Kit according to claim 14 comprising an instruction to incubate the sample for a certain time period until a steady state equilibrium is reached for at least one peptidic degradation product of the proteolytic cascade.
16. Kit according to claim 14 or 15, further comprising one or more chemical, biochemical and/or biotechnological laboratory items selected from solid phase extraction materials or other purification materials, containers, and combinations thereof.
17. Kit according to any one of claims 14 to 16, further comprising one or more biological samples selected from blood samples, serum samples, plasma samples, tissue samples, and combinations thereof.
18. Kit according to any one of claims 14 to 17, further comprising one or more protease inhibitors or inhibitor cocktails, feeding substrates, one or more blood collection tubes, optionally comprising one or more anti-coagulants, one or more standards, one or more quality controls, one or more solid phase extraction materials, or one or more solvents, or combinations thereof. 180 ^ lAng 1-10 160 ^ 140 -{ iAng 1-8
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/EP2012/060678 WO2013182237A1 (en) | 2012-06-06 | 2012-06-06 | Method for measurement of peptidic degradation products of a proteolytic cascade in blood samples |
Publications (2)
Publication Number | Publication Date |
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NZ630182A NZ630182A (en) | 2016-07-29 |
NZ630182B2 true NZ630182B2 (en) | 2016-11-01 |
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