U.S. patent application number 11/900566 was filed with the patent office on 2008-10-09 for methods for detection of lysosomal storage disease.
This patent application is currently assigned to Genzyme Corporation. Invention is credited to Christa Beauregard, Wei-Lien Chuang, Carole S. Elbin, Joan Keutzer, Joshua Pacheco, Sharon Pickering, Daniel Scheidegger, Xiaokui K. Zhang.
Application Number | 20080248513 11/900566 |
Document ID | / |
Family ID | 39184333 |
Filed Date | 2008-10-09 |
United States Patent
Application |
20080248513 |
Kind Code |
A1 |
Zhang; Xiaokui K. ; et
al. |
October 9, 2008 |
Methods for detection of lysosomal storage disease
Abstract
The present invention provides compositions for performing
assays of enzyme activity associated with lysosomal storage
diseases. The invention further provides methods for determining
enzyme activity, and methods for the screening for lysosomal
storage disease in an individual.
Inventors: |
Zhang; Xiaokui K.;
(Northborough, MA) ; Chuang; Wei-Lien;
(Framingham, MA) ; Elbin; Carole S.; (Framingham,
MA) ; Scheidegger; Daniel; (Liestal, CH) ;
Beauregard; Christa; (Cumberland, RI) ; Pickering;
Sharon; (Framingham, MA) ; Pacheco; Joshua;
(Holliston, MA) ; Keutzer; Joan; (Littleton,
MA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Genzyme Corporation
|
Family ID: |
39184333 |
Appl. No.: |
11/900566 |
Filed: |
September 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60923505 |
Apr 13, 2007 |
|
|
|
60844242 |
Sep 12, 2006 |
|
|
|
Current U.S.
Class: |
435/18 |
Current CPC
Class: |
A61K 31/739 20130101;
A61K 31/575 20130101; G01N 2333/924 20130101; A61K 31/164 20130101;
C12Q 1/34 20130101; A61K 31/164 20130101; A61K 2300/00 20130101;
A61K 31/575 20130101; A61K 2300/00 20130101; A61K 31/739 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
435/18 |
International
Class: |
C12Q 1/34 20060101
C12Q001/34 |
Claims
1. A method of determining acid .beta.-glucocerebrosidase activity
in a subject, the method comprising the steps of: (a) providing
acid .beta.-glucocerebrosidase in an aqueous buffer solution
comprising a detergent, wherein the acid .beta.-glucocerebrosidase
is divided into at least a first and second sample; (b) adding a
fluorogenic substrate and conduritol B epoxide to the first sample
to form a first reaction mix, and adding the fluorogenic substrate
to the second sample to form a second reaction mix; (c) determining
the fluorescence emitted from each of the first and second reaction
mixes; and (d) subtracting the level of fluorescence of the first
reaction mix from the level of fluorescence of the second reaction
mix to obtain a differential fluorescence, thereby determining the
acid .beta.-glucocerebrosidase activity in the subject.
2. The method of claim 1, wherein said step of providing acid
.beta.-glucocerebrosidase comprises extracting acid
.beta.-glucocerebrosidase from a dried blood spot obtained from the
subject in an aqueous buffer solution comprising a detergent, and
dividing the extract into a first and second sample.
3. The method of claim 1, wherein the step of determining the level
of fluorescence is preceded by a step of centrifuging the first and
second reaction mixes at 2000-3000 rpm for 30-90 minutes.
4. The method of claim 1, wherein step (d) further comprises the
step of comparing said differential fluorescence to a standard
curve to determine the acid .beta.-glucocerebrosidase activity in
the subject.
5. The method of claim 1, wherein said detergent is sodium
taurodeoxycholate.
6. The method of claim 5, wherein said sodium taurodeoxycholate is
at least 97% pure.
7. A method of identifying an individual with decreased acid
P-glucocerebrosidase activity comprising; determining acid
.beta.-glucocerebrosidase activity in the individual according to
the method of claim 1; determining acid .beta.-glucocerebrosidase
activity in a population of at least two presumptive normal
subjects according to the method of claim 1; calculating the mean
acid .beta.-glucocerebrosidase activity of the population of at
least two presumptive normal subjects; and comparing the mean acid
.beta.-glucocerebrosidase activity to the acid
.beta.-glucocerebrosidase activity of the individual, wherein if
the acid .beta.-glucocerebrosidase activity of the individual is
less than 30% of the mean acid .beta.-glucocerebrosidase activity,
the individual is identified as having decreased acid
.beta.-glucocerebrosidase activity.
Description
[0001] This application claims priority to U.S. Provisional
Application Ser. Nos. 60/844,242, filed Sep. 12, 2006 and
60/923,505, filed Apr. 13, 2007, the contents of which are
incorporated herein in their entirety.
BACKGROUND
[0002] The lysosomal storage diseases are a group of disorders that
manifest from birth to adulthood and result in damage to both
somatic organs and the central nervous system. Currently, there are
enzyme replacement therapies that have been shown effective in
treating Gaucher disease (acid .beta.-glucocerebrosidase (ABG)
deficiency), Fabry disease (acid .alpha.-galactosidase (GLA)
deficiency), and Pompe disease (lysosomal acid .alpha.-glucosidase
(GAA) deficiency). It is expected that similar therapy will be
developed for Niemann-Pick A/B disease type A and B (acid
sphingomyelinase (ASM) deficiency). In addition, it has been
suggested that presymptomatic initiation of bone marrow
transplantation may prevent the neural degeneration observed in
Krabbe disease (galactocerebroside .beta.-galactosidase (GALC)
deficiency).
[0003] For each of these diseases, early therapeutic intervention
and thus, early, presymptomatic detection of the disease will be
important to maximize treatment benefit. In particular, newborn
screening for the enzyme deficiencies associated with the lysosomal
storage diseases will provide a greater probability of effective
treatment compared to diagnosis of the disease once symptoms have
manifested.
[0004] A recent paper by Li et al. (Clinical Chemistry (2004) 50:
1785-1796) teaches assays for determining ABG, GLA, GAA, ASM, and
GALC enzyme activity from dried blood spots obtained from newborn
infants using mass spectrometry. The instant invention is based, in
part, on the independent optimization of assays for detection of
these enzymes to provide more robust, more reliable methods for
determining enzyme activity and disease diagnosis and
screening.
SUMMARY OF THE INVENTION
[0005] The present invention provides a series of assay mixtures
that can be used to determine enzyme activity in an individual. The
assay mixtures of the invention include a substrate for the enzyme
activity to be tested, an internal standard, detergent, and a
buffer. In preferred embodiment, the assay mixture can also include
one or more inhibitors of non-specific enzyme activity.
[0006] In one aspect, the invention provides a composition
comprising at least 0.6 mM C12-glucosyl ceramide, 13.33 .mu.M C14
ceramide, 16 g/L sodium taurocholate, and a buffer adjusted to a pH
of 5.1. Preferably the composition includes 0.67 mM C12-glucosyl
ceramide. Preferably, the buffer is 0.62 M phosphate/citrate.
[0007] In one aspect, the invention provides a composition
comprising 0.33 mM C6-sphingomyelin, 6.67 .mu.M C4 ceramide, 1 g/L
sodium taurocholate, 0.6 mM zinc chloride, and a buffer adjusted to
a pH of 5.7. Preferably the buffer is 0.92 M sodium acetate.
[0008] In one aspect, the invention provides a composition
comprising 1 mM C8-galactosyl ceramide, 6.67 .mu.M C10-ceramide,
9.6 g/L sodium taurocholate, 1.2 g/L oleic acid, and a buffer
adjusted to a pH of 4.4. Preferably, the buffer is 0.18 M
phosphate/citrate.
[0009] In another aspect, the invention provides a composition
comprising 0.667 mM
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl--
tetrahydro-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid
tert-butyl ester, 6.67 .mu.M
7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester, 10 g/L CHAPS, 13.3 .mu.M acarbose and a
buffer adjusted to a pH of 4.0. Preferably, the buffer is 0.3 M
phosphate/citrate.
[0010] In a further aspect, the invention provides a composition
comprising 3.33 mM
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester, 6.67 .mu.M
6-d5-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester, 3 g/L sodium taurocholate, 160 mM
N-acetylgalactosamine, and a buffer adjusted to a pH of 4.6.
Preferably, the buffer is 0.142 M sodium acetate.
[0011] In a further aspect, the above compositions can be included,
either singularly, or in combination of individual compositions in
a kit, including appropriate packaging materials. The above
compositions can also be in lyophilized form, or in a concentrated
form, such as a 0.5.times. to 50.times. formulation, including a
2.times., 10.times., 20.times., 30.times., or 40.times.
concentrated formulation.
[0012] In a further aspect any or all of the foregoing compositions
can be mixed in bulk for multiple reactions. For example, a
sufficient amount of the various components of each assay mix can
be mixed to provide a sufficient volume of assay mix to carry out 2
to 1200 or more individual enzyme reactions. Preferably a
sufficient amount of the each reaction mix will be prepared to
perform 100, 200, 300, 400, 500, 600, and up to 1200 or more enzyme
reactions.
[0013] In one aspect, the invention provides a composition
comprising C12-glucosyl ceramide, C14 ceramide, sodium
taurocholate, and a buffer adjusted to a pH of 5.1, wherein the
ratio of C12-glucosyl ceramide to C14 ceramide is 50:1, the ratio
of sodium taurocholate to C12-glucosyl ceramide is 45:1, and where
the ratio of buffer to C12-glucosyl ceramide is 925:1.
[0014] In a further aspect, the invention provides a composition
comprising C6-sphingomyelin, C4 ceramide, sodium taurocholate, zinc
chloride, and a buffer adjusted to a pH of 5.7, wherein the ratio
of C6-sphingomyelin to C4 ceramide is 50:1, the ratio of sodium
taurocholate to C6-sphingomyelin is 5.6:1, the ratio of zinc
chloride to C6-sphingomyelin is 1.82:1, and the ratio of buffer to
C-6 sphingomyelin is 2788:1.
[0015] In one aspect, the invention provides a composition
comprising C8-galactosyl ceramide, C10-ceramide, sodium
taurocholate, oleic acid, and a buffer adjusted to a pH of 4.4,
wherein the ratio of C8-galactosyl ceramide to C10-ceramide is
150:1, the ratio of sodium taurocholate to C8-galactosyl ceramide
is 17.8:1, the ratio of oleic acid to C8-galactosyl ceramide is
4.25:1, and the ratio of buffer to C8-galactosyl ceramide is
180:1
[0016] In a further aspect, the invention provides a composition
comprising
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester,
7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester, CHAPS, acarbose and a buffer adjusted to a
pH of 4.0, wherein the ratio of
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester to
7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester is 100:1, the ratio of CHAPS to
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 24.3:1, the ratio of acarbose to
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 0.02:1, and the ratio of buffer to
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 450:1.
[0017] In a still further aspect, the invention provides a
composition comprising
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester,
6-d5-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester, sodium taurocholate, N-acetylgalactosamine,
and a buffer adjusted to a pH of 4.6, wherein the ratio of
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester to
6-d5-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester is 499:1, the ratio of sodium taurocholate to
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 1.68:1, the ratio of N-acetylgalactosamine to
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 48:1, and the ratio of buffer to
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester is 43:1.
[0018] In the foregoing compositions, it will be understood by one
of skill in the art that the components may be present in the
composition at about the concentrations shown above, wherein
"about" refers to a variance of +/-2%, 5%, 7%, and up to 10% of the
stated value, wherein the concentration or amount of a given
component is determined using measurements and calculations well
known in the art.
[0019] In one aspect, the invention provides a method of
determining acid glucocerebrosidase activity in a subject, the
method comprising the steps of: extracting acid glucocerebrosidase
from a dried blood spot obtained from the subject using an aqueous
buffer solution; adding substrate and an internal standard to the
extracted acid glucocerebrosidase; reacting the substrate with the
extracted acid glucocerebrosidase; quenching the reaction by
addition of four volumes of a solution consisting essentially of
ethyl acetate/methanol (1/1); extracting the reaction product and
internal standard by adding at least 2 volumes of ethyl acetate and
at least 2 volumes of water to the quenched reaction to form a
two-phase system. In one embodiment, the assays described herein
are performed in a multiplex format (that is, where the reaction
mixes from more than one assay reaction, preferably all five
assays, are pooled following the quenching step), in which case the
pooled reaction products are extracted by adding at least a 3/5
volume of ethyl acetate and at least a 3/5 volume of water to the
quenched reaction. After extraction, the extracted product and
internal standard are harvested from the upper phase and then
purified, preferably by passage through silica gel which is then
washed with a 19:1 mixture of ethyl acetate/MeOH. The product and
the internal standard are then quantified to determine the acid
glucocerebrosidase activity.
[0020] Preferably, the substrate in the foregoing method is C12
glucosyl ceramide and the internal standard is C14 ceramide. It is
preferred that the C12 glucosyl ceramide is present at a
concentration of 0.6 mM, preferably a concentration of 0.67 mM. In
addition, it is preferred that during the step of reacting the
substrate with the extracted acid glucocerebrosidase, the
concentration of substrate is at least 0.4 mM.
[0021] The step of quantifying is preferably performed by tandem
mass spectrometry.
[0022] The invention also provides a method of identifying an
individual having decreased acid glucocrebrosidase activity by
first determining acid glucocerebrosidase activity in said
individual according to the foregoing method and also determining
acid glucocerebrosidase activity in a population of at least three
presumptive normal subjects according to the same method. A subject
is a "presumptive normal" subject when it is not known whether the
subject has a decrease level of activity for the enzyme being
assayed. The mean acid glucocerebrosidase activity is then
calculated for the population of subjects and compared to the acid
glucocerebrosidase activity of the individual. If the acid
glucocerebrosidase activity of the individual is less than 20-30%
of the mean acid glucocerebrosidase activity, preferably less than
30%, and more preferably less than 20%, then the individual is
identified as having decreased acid glucocrebrosidase activity.
[0023] In one aspect, the invention provides a method of
determining acid sphingomyelinase activity in a subject, the method
comprising the steps of: extracting acid sphingomyelinase from a
dried blood spot obtained from the subject using an aqueous buffer
solution; adding substrate and an internal standard to the
extracted acid sphingomyelinase; reacting the substrate with the
extracted acid sphingomyelinase; quenching the reaction by addition
of four volumes of a solution consisting essentially of ethyl
acetate/methanol (1/1); extracting the reaction product and
internal standard by adding at least 2 volumes of ethyl acetate and
at least 2 volumes of water to the quenched reaction to form a
two-phase system. In one embodiment, the assays described herein
are performed in a multiplex format (that is, where the reaction
mixes from more than one assay reaction, preferably all five
assays, are pooled following the quenching step), in which case the
pooled reaction products are extracted by adding at least a 3/5
volume of ethyl acetate and at least a 3/5 volume of water to the
quenched reaction. After extraction, the extracted product and
internal standard are harvested from the upper phase and then
purified, preferably by passage through silica gel which is then
washed the silica gel with a 19:1 mixture of ethyl acetate/MeOH.
The product and the internal standard are then quantified to
determine the acid sphingomyelinase activity. Preferably, the
substrate is C6 sphingomyelin and the internal standard is C4
ceramide.
[0024] The step of quantifying is performed preferably by tandem
mass spectrometry.
[0025] In one embodiment of the foregoing method, zinc chloride is
also added to the reaction mix, preferably at a concentration of
0.6 mM. In addition, it is preferred that during the step of
reacting the substrate with the extracted acid sphingomyelinase the
substrate concentration is at least 0.2 mM, and the pH is 5.7.
[0026] In one aspect, the invention provides a method of
identifying an individual having decreased acid sphingomyelinase
activity comprising, determining acid sphingomyelinase activity in
the individual according to the foregoing method and also
determining acid sphingomyelinase activity in a population of at
least three presumptive normal subjects according to the same
method. The mean acid sphingomyelinase activity is then calculated
for the population of subjects and compared to the acid
sphingomyelinase activity of the individual. If the acid
sphingomyelinase activity of the individual is less than 20% of the
mean acid sphingomyelinase activity, the individual is identified
as having decreased acid sphingomyelinase activity.
[0027] In a further aspect, the invention provides a method of
determining galactocerebroside .beta.-galactosidase activity in a
subject, the method comprising the steps of: contacting a dried
blood spot from said subject having a diameter of 3.2 mm and
comprising galactocerebroside .beta.-galactosidase with a substrate
and an internal standard; reacting the substrate with the
galactocerebroside .beta.-galactosidase; quenching the reaction by
addition of at least 2 volumes of a solution consisting essentially
of ethyl acetate/methanol (1/1); extracting the reaction product
and internal standard by adding at least 2 volumes of ethyl acetate
and at least 2 volumes of water to the quenched reaction to form a
two-phase system, and harvesting the extracted product and internal
standard from the upper phase; purifying the reaction product and
internal standard, preferably by passage of the extract through
silica gel which is then washed with a 19:1 mixture of ethyl
acetate/MeOH; and quantifying the product and the internal standard
to determine the galactocerebroside .beta.-galactosidase activity.
Preferably, the substrate is C8-galactosyl ceramide and the
internal standard is C10-ceramide. More preferably, the
C8-galactosyl ceramide is present at a concentration of 1 mM.
[0028] While preferred that the method for determining
galactocerebroside .beta.-galactosidase activity is performed using
a 3.2 mm dried blood spot, the method can also be performed by
substituting an extract of a 3.2 mm dried blood spot as described
in further detail herein below. It is also envisioned that the
method for determining galactocerebroside .beta.-galactosidase
activity can be performed using a combination of a 3.2 mm dried
blood spot and a dried blood spot extract.
[0029] In one embodiment of the foregoing method, the reaction mix
(i.e., the contacting step) includes oleic acid and sodium
taurocholate. Preferably, the oleic acid is at a concentration of
1.2 g/L. It is preferred that the substrate concentration is at
least 1 mM during the step of reacting the substrate with the
galactocerebroside .beta.-galactosidase said.
[0030] In a preferred embodiment, the enzyme activity is quantified
by tandem mass spectrometry.
[0031] The invention also provides a method of identifying an
individual having decreased galactocerebroside .beta.-galactosidase
activity comprising, determining galactocerebroside
.beta.-galactosidase activity in the individual according to the
foregoing method, and also determining galactocerebroside
.beta.-galactosidase activity in a population of at least three
presumptive normal subjects according to the same method. The mean
galactocerebroside .beta.-galactosidase activity is then calculated
for the population of subjects and compared with the
galactocerebroside .beta.-galactosidase activity of the individual.
If the galactocerebroside .beta.-galactosidase activity of the
individual is less than 10% of the mean galactocerebroside
.beta.-galactosidase activity, the individual is identified as
having decreased galactocerebroside .beta.-galactosidase
activity.
[0032] In a further aspect, the invention provides a method of
determining acid glucosidase activity in a subject, the method
comprising the steps of: extracting acid glucosidase from a dried
blood spot obtained from the subject using an aqueous buffer
solution; adding substrate and an internal standard to the
extracted acid glucosidase; reacting the substrate with the
extracted acid glucosidase; quenching the reaction by addition of
four volumes of a solution consisting essentially of ethyl
acetate/methanol (1/1); extracting the reaction product and
internal standard by adding at least 2 volumes of ethyl acetate and
at least 2 volumes of water to the quenched reaction to form a
two-phase system. In one embodiment, the assays described herein
are performed in a multiplex format (that is, where the reaction
mixes from more than one assay reaction, preferably all five
assays, are pooled following the quenching step), in which case the
pooled reaction products are extracted by adding at least a 3/5
volume of ethyl acetate and at least a 3/5 volume of water to the
quenched reaction. After extraction, the extracted product and
internal standard are harvested from the upper phase and then
purified, preferably by passage through silica gel which is then
washed with a 19:1 mixture of ethyl acetate/MeOH. The product and
the internal standard are then quantified to determine the acid
glucosidase activity. It is preferred that the substrate is
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydr-
o-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester and the internal standard is
7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester.
[0033] In one embodiment, 10 g/L CHAPS is included in the reaction
mixture (i.e., the adding step). In a further preferred embodiment,
the step of adding also includes adding 13.3 .mu.M acarbose.
[0034] It is preferred that the substrate concentration is at least
0.4 mM during the step of reacting the substrate with the acid
glucosidase.
[0035] In a preferred embodiment, the enzyme activity is quantified
by tandem mass spectrometry.
[0036] In one aspect, the invention provides a method of
identifying an individual having decreased acid glucosidase
activity comprising, determining acid glucosidase activity in said
individual according to the foregoing method, and also determining
acid glucosidase activity in a population of at least three
presumed normal subjects according to the same method. The mean
acid glucosidase activity of said population of subjects is
calculated and compared to the acid glucosidase activity of the
individual. If the acid glucosidase activity of the individual is
less than 20-30% of the mean acid glucosidase activity, preferably
less than 30%, and more preferably less than 20%, the individual is
identified as having decreased acid glucosidase activity.
[0037] In another aspect, the invention provides a method of
determining acid .alpha.-galactosidase A activity in a subject, the
method comprising the steps of: extracting acid
.alpha.-galactosidase A from a dried blood spot obtained from the
subject using an aqueous buffer solution; adding substrate and an
internal standard to the extracted acid .alpha.-galactosidase A;
reacting the substrate with the extracted acid
.alpha.-galactosidase A; quenching the reaction by addition of four
volumes of a solution consisting essentially of ethyl
acetate/methanol (1/1); extracting the reaction product and
internal standard by adding at least 2 volumes of ethyl acetate and
at least 2 volumes of water to the quenched reaction to form a
two-phase system. In one embodiment, the assays described herein
are performed in a multiplex format (that is, where the reaction
mixes from more than one assay reaction, preferably all five
assays, are pooled following the quenching step), in which case the
pooled reaction products are extracted by adding at least a 3/5
volume of ethyl acetate and at least a 3/5 volume of water to the
quenched reaction. After extraction, the extracted product and
internal standard are harvested from the upper phase and then
purified, preferably by passage through silica gel which is then
washed with a 19:1 mixture of ethyl acetate/MeOH. The product and
the internal standard are then quantified to determine the acid
.alpha.-galactosidase A activity.
[0038] In a preferred embodiment, the substrate is
(6-benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester and the internal standard is
6-d5-benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester. Preferably, the substrate is at a
concentration of 3.33. mM and the internal standard is at a
concentration of 6.67 .mu.M.
[0039] In one embodiment, the step of adding also includes adding 3
g/L sodium taurocholate, and further includes adding 160 mM
N-acetylgalactosamine. In a further preferred embodiment, the step
of adding includes adding 0.142 M sodium acetate.
[0040] It is preferred that the substrate concentration is at least
2 mM. during the step of reacting the substrate with the
.alpha.-galactosidase A.
[0041] In a preferred embodiment, the step of quantifying is by
tandem mass spectrometry.
[0042] In a further aspect, the invention provides a method of
identifying an individual having decreased .alpha.-galactosidase A
activity comprising determining acid .alpha.-galactosidase A
activity in said individual according to the foregoing method, and
also determining acid .alpha.-galactosidase A activity in a
population of at least three subjects according to the same method.
The mean acid .alpha.-galactosidase A activity of the population of
subjects is calculated and compared to the acid
.alpha.-galactosidase A activity of the individual. If the acid
.alpha.-galactosidase A activity of the individual is less than
10-20% of the mean acid .alpha.-galactosidase A activity,
preferably less than 20%, and more preferably less than 10%, the
individual is identified as having decreased .alpha.-galactosidase
A activity.
[0043] In a further aspect, the invention provides a method of
determining the activity of acid glucocerebrosidase, acid
sphingomyelinase, galactocerebroside .beta.-galactosidase, acid
glucosidase, and acid .alpha.-galactosidase in a subject, the
method comprising the steps of: extracting acid glucocerebrosidase
from a first dried blood spot punch obtained from the subject using
an aqueous buffer solution; extracting acid sphingomyelinase from a
second dried blood spot punch obtained from the subject using an
aqueous buffer solution; extracting acid glucosidase from a third
dried blood spot punch obtained from the subject using an aqueous
buffer solution; extracting acid .alpha.-galactosidase A from a
fourth dried blood spot punch obtained from the subject using an
aqueous buffer solution; adding a first substrate and first
internal standard to the extracted acid glucocerebrosidase to form
a first reaction mix; adding a second substrate and second internal
standard to the extracted acid sphingomyelinase to form a second
reaction mix; adding a third substrate and third internal standard
to the extracted acid glucosidase to form a third reaction mix;
adding a fourth substrate and fourth internal standard to the
extracted acid .alpha.-galactosidase A to form a fourth reaction
mix; contacting a fifth dried blood spot punch from said subject
having a diameter of 3.2 mm and comprising galactocerebroside
.beta.-galactosidase with a fifth substrate and fifth internal
standard to form a fifth reaction mix; reacting the first, second,
third, and fourth substrate with the extracted acid
glucocerebrosidase, acid sphingomyelinase, acid glucosidase, and
acid .alpha.-galactosidase, respectively to form a first, second,
third, and fourth reaction product; reacting the fifth substrate
with the galactocerebroside .beta.-galactosidase to form a fifth
reaction product quenching each reaction by adding four volumes of
a solution consisting essentially of ethyl acetate/methanol (1/1)
to each reaction mix; combining each reaction mix in a single
container; extracting the reaction products and internal standards
by adding at least a 3/5 volume of ethyl acetate and at least a 3/5
volume of water to the quenched reactions to form a two-phase
system, and harvesting the extracted product and internal standard
from the upper phase; purifying the reaction products and internal
standards, preferably by passage of the extract through silica gel
which is then washed with a 19:1 mixture of ethyl acetate and
water; and quantifying the products and the internal standards to
determine the acid glucocerebrosidase, acid sphingomyelinase,
galactocerebroside .beta.-galactosidase, acid glucosidase, and acid
.alpha.-galactosidase activities.
[0044] In a preferred embodiment, the concentration of substrate
added to the extracted acid glucocerebrosidase is 0.67 mM. A
concentration of 0.6 mM zinc chloride can be added to the extracted
acid sphingomyelinase, and the second reaction mix is preferably at
a pH of 5.7.
[0045] A concentration of 10 g/L CHAPS, and optionally a
concentration of 13.3 .mu.M acarbose can be added to the extracted
acid glucosidase.
[0046] It is preferred that the concentration of substrate added to
prepare the fourth reaction mix is 3.33 mM, and that the
concentration of internal standard added to prepare the fourth
reaction mix is 6.67 .mu.M.
[0047] In a preferred embodiment, 3 g/L sodium taurocholate, 160 mM
N-acetylgalactosamine, and 0.142 M sodium acetate are added to
prepare the fourth reaction mix.
[0048] In a further embodiment, the concentration of substrate
added to prepare the fifth reaction mix is 1 mM. In addition, 1.2
g/L oleic acid is preferably added to prepare the fifth reaction
mix.
[0049] In each of the foregoing aspects, it is possible to omit the
step of purifying the reaction products by passage through a silica
gel. The silica gel purification step can be omitted or replaced by
an alternate procedure to clean up the reaction products and
internal standards. Such methods are well known in the art.
[0050] The invention also provides a method of determining acid
glucocerebrosidase activity in a subject, including the steps of:
(a) extracting acid glucocerebrosidase from a dried blood spot
obtained from the subject in an aqueous buffer solution that
includes a detergent, and dividing the extract into a first and
second sample; (b) adding a fluorogenic substrate and conduritol B
epoxide to the first sample to form a first reaction mix, and
adding the fluorogenic substrate to the second sample to form a
second reaction mix; (c) reacting the substrate with the extracted
glucocerebrosidase in the first and second reaction mixes; (d)
quenching the reactions; (e) centrifuging said first and second
reaction mixes at 2000-3000 rpm for 30-90 minutes; (f) determining
the fluorescence emitted from each of the first and second reaction
mixes; and (g) subtracting the level of fluorescence of the first
reaction mix from the level of fluorescence of the second reaction
mix to obtain a differential fluorescence, thereby determining the
acid glucocerebrosidase activity in the subject.
[0051] The fluorogenic substrate can be a substrate that is cleaved
by a .beta.-glucosidase enzyme. For example, fluorogenic substrates
that can be used in the method include, but are not limited to
4-methylumbelliferyl-.beta.-D-glucopyranoside (4-MU-.beta.-Glu),
4-pentafluoroethylumbelliferyl-beta-D-glucoside,
4-trifluoromethylumbelliferyl-beta-D-glucoside, and
4-heptylumbelliferyl-beta-D-glucoside. Preferably, the substrate is
4-methylumbelliferyl-.beta.-D-glucopyranoside
(4-MU-.beta.-Glu).
[0052] This method can further include the step of comparing the
differential fluorescence to a standard curve to determine the acid
glucocerebrosidase activity in the subject.
[0053] The detergent used in the foregoing method is preferably
sodium taurodeoxycholate. It is further preferred that the sodium
taurodeoxycholate by of high purity, such as for example, 90, 95,
97, 98, 99, or up to 100% pure. Preferably, the sodium
taurodeoxycholate is at least 97% pure.
[0054] The foregoing method for determining acid glucocerebrosidase
activity in a subject can be used to screen or diagnose Gaucher
disease, or can be used to identify a subject for treatment.
[0055] In the foregoing methods, it will be understood by one of
skill in the art that the components used in the method (i.e., the
assay components, such as substrate, internal standard, and the
like) may be used at about the concentrations shown above, wherein
"about" refers to a variance of +/-2%, 5%, 7%, and up to 10% of the
stated value, wherein the concentration or amount of a given
component is determined using measurements and calculations well
known in the art.
[0056] The invention still further provides a method of identifying
an individual with decreased acid glucocerebrosidase activity
including the steps of: determining acid glucocerebrosidase
activity in the individual according to the foregoing method;
determining acid glucocerebrosidase activity in a population of at
least two presumptive normal subjects according to the foregoing
method; calculating the mean acid glucocerebrosidase activity of
the population of at least two presumptive normal subjects; and
comparing the mean acid glucocerebrosidase activity to the acid
glucocerebrosidase activity of the individual, wherein if the acid
glucocerebrosidase activity of the individual is less than 30% of
the mean acid glucocerebrosidase activity, the individual is
identified as having decreased acid glucocerebrosidase
activity.
[0057] In one aspect, the invention provides a method for selecting
a treatment regimen for a patient based on the activity of the ABG,
ASM, GAA, GALC, or GLA enzymes. For example, by determining the
activity of one or more of these enzymes in an individual, a
physician or other health care professional can use that
information to make decisions as to the proper follow-up, or
treatment (e.g., enzyme replacement therapy or bone marrow
transplantation) for the individual.
[0058] The methods of the instant invention can also be used to
monitor treatment in a patient. For example, after starting a
patient on a treatment program for deficiency in one or more of the
ABG, ASM, GAA, GALC, or GLA enzymes, enzyme activity can be assayed
at relevant time points (as determined by the patient's physician)
to determine whether enzyme activity levels are higher than prior
to the commencement of treatment, and thus, monitor the efficacy of
a particular treatment.
BRIEF DESCRIPTION OF THE FIGURES
[0059] FIG. 1 (A-E) shows a schematic summary of the activity of
the target enzyme on the substrate and internal standards used in
the methods of the invention.
[0060] FIG. 2 (A-C) shows the calibration curves used to generate
the RF for the ASM, ABG, and GALC enzyme activity calculations.
[0061] FIG. 3 shows the results of a GLA activity assay.
[0062] FIG. 4 shows the results of a GAA activity assay.
[0063] FIG. 5 shows the results of a GALC activity assay.
[0064] FIG. 6 shows the results of a ABG activity assay.
[0065] FIG. 7 shows the results of a ASM activity assay.
[0066] FIG. 8 shows a 4-MU standard curve.
[0067] FIG. 9 shows an example of assay results obtained using the
second ABG assay.
DETAILED DESCRIPTION
[0068] The present invention is based, in part, on the discovery
that specific combinations of substrate, internal standard, buffer,
and enzyme inhibitors, present in predetermined ratios, and at
specific pH can be used to accurately assay for the activity of the
lysosomal enzymes implicated in the etiology of Fabry, Gaucher,
Krabbe, Niemann-Pick A/B, and Pompe diseases.
[0069] The invention is also based on the discovery that, using the
specific components and ratios of components for determining enzyme
activity, assays for activity can be multiplexed such that a single
round of screening assays can determine the enzyme activity
associated with all five lysosomal storage diseases. Alternatively,
each assay and determination of enzyme activity can be performed
separately. These assays can be performed on blood samples from
newborn infants, and provide a robust method for making an early
determination of decreased enzyme activity associated with one or
more of Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe
diseases, thus permitting early therapeutic intervention.
Assay Components
[0070] The compositions and methods described herein useful for the
determination of the activity of enzymes associated with the five
lysosomal storage diseases: Gaucher disease (acid
.beta.-glucocerebrosidase (ABG) deficiency), Fabry disease (acid
.alpha.-galactosidase (GLA) deficiency), Pompe disease (lysosomal
acid .alpha.-glucosidase (GAA) deficiency), Niemann-Pick A/B
disease (acid sphingomyelinase (ASM) deficiency), and Krabbe
disease (galactocerebroside .beta.-galactosidase (GALC)
deficiency).
[0071] Described broadly, the components utilized to determine ABG,
GLA, GAA, ASM, or GALC enzyme activity include a substrate,
internal standard, and detergent. The substrates used in the
activity assays can be the natural substrates for each of ABG, GLA,
GAA, ASM, and GALC, or can be a modified version of the natural
substrate, or a synthetic substrate. In a preferred embodiment, the
substrate for ABG, ASM, and GALC assays are synthetic sphingolipids
containing N-linked fatty acyl chains that are shorter than the
typical natural substrates (the substrates, products, and internal
standards for each enzyme assay are shown in FIG. 1). These
synthetic substrates have the advantage that the corresponding
products (i.e., the product produced by the action of ABG, ASM, and
GALC on its corresponding substrate) are non-natural ceramides and
are produced without interference from endogenous natural
ceramides. In a further preferred embodiment, the substrate for GAA
and GLA are water-soluble polysaccharide substrates, or synthetic
lipid substrates. Preferably, the GAA and GLA substrates are the
synthetic lipidated substrates shown in FIG. 1.
[0072] The internal standards used in the activity assays are
generally similar in structure to the product generated by the
action of the five enzymes on their respective substrates. The GAA
and GLA assays utilize internal standards that are chemically
identical but isotopically distinguishable from the enzymatically
generated products. The ABG, ASM, and GALC assays utilize internal
standards that are close in structure to the product but not
chemically identical. The specific internal standards used for each
of the five enzyme assays are shown in FIG. 1.
[0073] Substrates and internal standards can be obtained
commercially from vendors known to those of skill in the art (e.g.,
Avanti Polar Lipids, Alabaster, Ala.). Alternatively, substrates
and internal standards may be synthesized prior to use in the
methods of the invention. A specific description of the synthesis
of the internal standards and substrates for the GAA and GLA
substrates and internal standards is provided in Example 1.
[0074] In one embodiment, the enzyme assay reaction mixtures can
include components that inhibit the activity of other enzymes
(e.g., non-ABG, GLA, GAA, ASM, or GALC enzymes) that may interfere
with the assay. Inhibitors for interfering enzymes are known in the
art and are collectively referred to as glucose-like competitive
inhibitors. For example, in the GAA assay, blood cells contain a
second acid .alpha.-glucosidase that can interfere with the assay
for GAA. Maltose or acarbose can be used in the assay mixture to
inhibit the activity of this contaminating enzyme. In the GLA
assay, N-acetyl galactosamine can be used as an inhibitor of
alpha-galactosidase B, an interfering enzyme that can confound the
assay if not inhibited. In another embodiment, the enzyme assay
reaction mixture can include components that boost the activity of
the enzymes. For example in the ASM assay, zinc chloride provides
the Zn++ cofactor needed for optimal enzyme activity.
Synthesis of Substrates and Internal Standards
[0075] The substrates and internal standards used in the instant
invention may be obtained from commercial sources (e.g., Avanti
Polar Lipids), or may be synthesized prior to use. The following
section provides a description of the synthesis of substrates and
internal standards for each of the five assays.
[0076] ABG Substrate/Internal Standard Synthesis
[0077] The currently synthesis strategy is based on condensation of
the two key intermediates and a following deprotection step using
sodium methylate. Alternative to the synthesis shown below would be
a direct glycosylation of the C12-ceramide (the ABG assay product).
If this later route is followed, however, the resulting
.beta.-glucocerebrosidase would show a 2:1 distribution of the two
possible isomers. To avoid this, the functional group at C-3 of the
C12-ceramide is protected with a benzoyl group. Accordingly, the
following route of synthesis is preferred. The overall synthesis
scheme for the ABG substrate is shown below:
##STR00001##
[0078] As outlined in the scheme, the two components
3-O-benzoyl-ceramide C12 (Key intermediate A) and
2,3,4,6-tetra-O-acetyl-.alpha.-D-glucopyranosyl-trichloracetamidate
(Key intermediate B) are key to the synthesis of the ABG substrate.
Key intermediate A is accessible from ceramide C12 by a 2-step
protection reaction with tri-tylchloride and benzoylchloride,
followed by cleavage of the trityl-protection group using
P-toluenesulfonic acid. Key intermediate B is accessible from
.beta.-D-glucose penta-acetate by reaction with hydrazine acetate
and trichloroacetonitrile.
[0079] The sugar configuration on the substrate is preferably
beta-linked glucose. Substrates that have multiple sugar
configurations will be converted to product by additional enzymes.
Thus, the activity of a second enzyme would mask the deficiency of
ABG and result in a false negative result. The beta-linked glucose
is clearly discernible from the alpha-linked glucose using
.sup.1H-NMR analysis (H-.alpha.=5.5 ppm chemical shift,
H-.beta.=4.9 ppm chemical shift). The product must be essentially
absent from the substrate, and can be clearly discerned from the
substrate in HPLC. Separation of both substances is therefore
efficiently possible during column chromatography applied in the
process due to the significantly different polarity.
[0080] For the second ABG assay described herein, the substrate is
preferably a fluorogenic substrate such as 4-MU-.beta.-Glu.
4-MU-.beta.-Glu can be obtained from commercial sources such as
Sigma Chemical Company (Cat. no. M3633). Other substrates useful in
the second ABG assay include
4-pentafluoroethylumbelliferyl-beta-D-glucoside,
4-trifluoromethylumbelliferyl-beta-D-glucoside, and
4-heptylumbelliferyl-beta-D-glucoside. Preferably, the substrate is
4-methylumbelliferyl-.beta.-D-glucopyranoside
(4-MU-.beta.-Glu).
[0081] The internal standard for the ABG assay mix can be purchased
from a commercial supplier. The commercially available ceramide is
obtained fully synthetically, which assures a high degree of
accuracy of the structure including the resulting fatty acid chain
length. The synthesis principle for the C14 ceramide (ABG internal
standard) is shown below:
##STR00002##
[0082] ASM Substrate/Internal Standard Synthesis
[0083] The substrate for the Niemann-Pick A/B assay (ASM-S) can be
purchased from a supplier such as Avanti Polar Lipids. The
sphingomyelin is obtained fully synthetically, which assures a high
degree of accuracy of the structure including the resulting fatty
acid chain length. The synthesis principle for the C6 sphingomyelin
is shown below.
##STR00003##
The product must be essentially absent from the substrate, and can
be clearly discerned from the substrate in HPLC and a separation of
both substances is efficiently possible during the column
chromatography applied in the process due to the significantly
different polarity.
[0084] The internal standard for the ASM assay can also be
purchased from a commercial supplier. The ceramide is obtained
fully synthetically, which assures a high degree of accuracy of the
internal standard structure including the resulting fatty acid
chain length. The synthesis principle for the C4 ceramide internal
standard is shown below.
##STR00004##
[0085] GAA Substrate/Internal Standard Synthesis
[0086] The current synthesis strategy is based on condensation of
the two key intermediates as shown below. One alternate route would
be the glycosylation of the corresponding intermediate without the
sugar moiety
(7-d.sub.5-Benzoylamino-heptyl)-[2-(4-hydroxyphenyl-carbamoyl)-ethyl]-car-
bamic acid tertbutyl ester also defined as GAA-P. This alternate
route is shown as follows:
##STR00005##
If this route is used, the potential impurities in the substrate
would include the product along with the sugar (glucose for GAA-S).
Therefore the level of the impurity GAA-P could possibly adulterate
the test since it would not be possible differentiate the level of
product deriving from the synthesis compared to the level deriving
from enzyme reaction. For this reason the route of synthesis
described below is preferred route of synthesis is most appropriate
for the production of these components.
[0087] The overall synthesis of the GAA substrate is shown as
follows:
##STR00006##
[0088] As outlined in the scheme, the two components
4-Acrylaminophenyl-.alpha.-D-glucopyranoside (Key intermediate A)
and N-(7-Amino-heptyl)-benzamide (Key intermediate B) are defined
to be key intermediates. Key intermediate A is easily accessible by
reduction of the starting material
4-Nitrophenyl-.alpha.-D-glucopyranoside followed by reaction with
acryloylchloride. Key intermediate B is synthesized by coupling
1,7-diaminoheptane with benzoyl chloride to obtain an amide
bond.
[0089] The sugar configuration on the substrate must be
alpha-linked glucose. Substrates that have multiple sugar
configurations will be converted to product by additional lysosomal
enzymes; the activity of a second lysosomal enzyme will mask the
deficiency of GAA and result in a false negative result. The
Alpha-linked glucose is clearly discernible from the beta linked
glucose using .sup.1H-NMR-analysis (H-.alpha.=5.63 ppm chemical
shift, H-.beta.=5.09 ppm chemical shift). The distinction between
glucose and galactose can be done by HPLC analysis (1 minute
difference in retention time) and 1D or 2D NMR.
[0090] The following reaction scheme shows the process principles
of the GAA-internal standard synthesis
##STR00007##
[0091] As outlined in the scheme, the two components
N-(4-Hydroxy-phenyl)-acrylamide (Starting Material C) and
N-(7-Amino-heptyl)-d5-benzymide (Starting Material D) are defined
to be key intermediates from a process qualification perspective
and represent the starting point of the process qualification. Key
intermediate C is easily accessible by reduction of the starting
material 4-Nitrophenylacetate followed by reaction with
acryloylchloride. The acetyl is then saponified using sodium
methylate in methanol. Key intermediate D is synthesized by
coupling 1,7-diaminoheptane with benzoyl chloride-d5 leading to the
corresponding amide, at similar reaction conditions as for key
intermediate B.
[0092] A more detailed description of an example of GAA substrate
and internal standard synthesis is provided in Example 1.
[0093] GLA Substrate/Internal Standard Synthesis
[0094] The synthesis strategy is based on the condensation of two
key intermediates as shown below. One other potential route for
synthesis would be the glycosylation of the corresponding
intermediate without the sugar moiety
(6-d.sub.5-benzoylamino-hexyl)-[2-(4-hydroxy-phenyl-carbamoyl)-ethyl]-car-
bamic acid tertbutyl ester (i.e., the GLA assay product). This
alternate synthesis route is shown as follows:
##STR00008##
If this route would have been used, the potential impurities in the
substrate would have been the product along with the sugar
(galactose for GLA substrate). Therefore the level of the impurity
(the GLA assay product) could possibly adulterate the test since it
would not be possible to differentiate the level of product
deriving from the synthesis compared to the level deriving from the
enzyme reaction. For this reason, the following synthesis route is
preferred as the most appropriate method for the production of GLA
substrate and internal standard. The general synthesis scheme for
the GLA substrate is as follows:
##STR00009##
[0095] As outlined in the above scheme, the two components
4-acrylaminophenyl-.alpha.-D-galactopyranoside (key intermediate A)
and N-(6-amino-hexyl)-benzamide (key intermediate B) are defined to
be key intermediates. Key intermediate A is readily accessible by
reduction of the starting material
4-Nitrophenyl-.alpha.-D-galactopyranoside followed by reaction with
acryloylchloride. Intermediate B is synthesized by coupling
1,6-diaminohexane with benzoyl chloride to obtain an amide
bond.
[0096] The sugar configuration on the substrate must be
alpha-linked galactose. Substrates that have multiple sugar
configurations will be converted to product by additional lysosomal
enzymes, and the activity of a second lysosomal enzyme will mask
the deficiency in GLA activity and result in false negative
results. The alpha-linked galactose is clearly discernible from the
beta linked galactose using .sup.1H-NMR analysis (H-.alpha.=5.63
ppm chemical shift, H-.beta.=5.09 ppm chemical shift). The
distinction between glucose and galactose can be done by HPLC
analysis (1 minute difference in retention time) and 1D or 2D NMR.
In addition, if product is already present in the substrate, it may
lead to false positive results. The product is clearly discernible
from substrate in HPLC and a separation of both substances is
efficiently possible during the column chromatography applied in
the process due to the significantly different polarity.
[0097] The following reaction scheme shows the process of
production of the GLA internal standard:
##STR00010##
[0098] As outlined in the scheme, the two components
N-(4-hycrosy-phenyl)-acrylamide (key intermediate C) and
N-(6-amino-hexyl)-d5-benzamide (key intermediate D) are defined to
be key intermediates. Key intermediate C is easily accessible by
reduction of the starting material 4-nitrophenyl-acetate followed
by reaction with acryloylchloride. The acetyl group is then
saponified using sodium methylate in methanol. Key intermediate D
is synthesized by coupling 1,6-diaminohexane with benzoyl
chloride-d5 leading to the corresponding amide, at similar reaction
conditions as for key intermediate B.
[0099] A more detailed description of an example of GLA substrate
and internal standard synthesis is provided in Example 1.
[0100] GALC Substrate/Internal Standard Synthesis
[0101] The current synthesis strategy is based on condensation of
the two key intermediates and a subsequent de-protection step using
sodium-methylate. An alternate synthesis route would be the direct
galactosylation of the C8-ceramide (the GALC reaction product) as
shown below:
##STR00011##
If this synthesis method is used, however, the resulting
.beta.-galactocerebrosidase (GALC substrate) would show a 2:1
distribution of the two possible isomers. To avoid this scenario,
the functional group at C-3 of the C8-ceramide would have to be
protected with a benzoyl-group. Accordingly, the preferred
synthesis scheme is shown as follows:
##STR00012##
[0102] As outlined in the above scheme, the two components
3-O-benzoyl-ceramide C8 (key intermediate A) and
2,3,4,6-tetra-O-acetyl-.alpha.-D-galactopyranosyl-trichloracetamidate
(key intermediate B) are defined to be key intermediates. Key
intermediate A is accessible from ceramide C8 by a 2-step
protection reaction with trityl-chloride and benzoylchloride,
followed by cleavage of the trityl-protection group using
p-toluenesulfonic acid. Key intermediate B is accessible from
.beta.-D-galactose penta-acetate by reaction with hydrazine acetate
and trichloroacetonitrile.
[0103] The sugar configuration on the substrate is preferably
beta-linked galactose. Substrates that have multiple sugar
configurations will be converted to product by additional enzymes.
Thus, the activity of a second enzyme would mask the deficiency of
GALC and result in a false negative result. The beta-linked
galactose is clearly discernible from the alpha-linked galactose
using .sup.1H-NMR analysis (H-.alpha.--5.5 ppm chemical shift,
H-.beta.=4.9 ppm chemical shift). The product must be essentially
absent from the substrate, and can be clearly discerned from the
substrate in HPLC. Separation of both substances is therefore
efficiently possible during column chromatography applied in the
process due to the significantly different polarity.
[0104] The internal standard for the GALC assay can be purchased
from a commercial supplier such as Avanti Polar Lipids. The
ceramide can be obtained fully synthetically, which assures a high
degree of accuracy of the structure including the resulting fatty
acid chain length. The synthesis principle for the GALC internal
standard is as follows:
##STR00013##
[0105] The following section outlines the identity and
concentrations of the various components of the five lysosomal
enzyme activity assays of the invention.
[0106] ABG Assay Mix
[0107] In one embodiment, the invention provides a first assay to
determine the activity of ABG, a deficiency of which is the cause
of Gaucher disease. The assay is performed by combining either a
3.2 mm punch out from a dried blood spot (DBS; described in further
detail below) or DBS punch extract with an assay mix comprising
substrate, internal standard, detergent and buffer. Preferably the
assay is performed using DBS punch extract.
[0108] The substrate used to assess ABG enzyme activity is
D-Glucosyl-.beta.1-1'-N-dodecanoyl-D-erythro-sphingosine (C12
glucosyl ceramide; C.sub.36H.sub.69NO.sub.8) and is present in the
assay mix at a concentration of between 0.3 and 0.9 mM, preferably
at a concentration of 0.6 mM, and still more preferably at a
concentration of 0.67 mM. The internal standard for the ABG assay
is N-myristoyl-D-erythro-sphingosine (C14 ceramide;
C.sub.32H.sub.63NO.sub.3), and is present in the mix at a
concentration of between 6.5 and 19.5 .mu.M, preferably at a
concentration of 13 .mu.M, and still more preferably at a
concentration of 13.33 .mu.M. The detergent for the ABG assay is
sodium taurocholate, present in the assay mix at a concentration of
between 8 and 24 g/L, preferably at a concentration of 16 g/L. The
buffer for the ABG assay is a phosphate/citrate buffer at a
concentration of between about 0.3 and 0.9 M, preferably at a
concentration of 0.6 M, and still more preferably at a
concentration of 0.62 M. The ABG assay mix should be at a pH of
between 5 and 5.2, preferably pH 5.1.
[0109] To perform the first ABG assay (described in further detail
below) 10-20 .mu.l, preferably 13-17 .mu.l, and more preferably 15
.mu.l of the ABG assay mix is combined with 10 .mu.l of the DBS
punch extract. Since, as noted above, the DBS punch contains 2-3.5
.mu.l blood and is preferably extracted in 70 .mu.l buffer, each
ABG assay reaction will contain between 0.25 and 0.58 .mu.l blood.
Preferably, each assay will contain between 0.33 and 0.43 .mu.l
blood, and more preferably will contain 0.4 .mu.l blood. The DBS
punch or punch extract is reacted with the ABG assay mix according
to the steps described herein below. During the reaction, however,
the concentration of substrate in the assay is between 0.2 and 0.6
mM, preferably 0.3 and 0.5 mM, and more preferably 0.4 mM.
[0110] In addition to the absolute concentrations of assay mix
components described above, the invention contemplates that the
ratio of assay components to the amount of substrate in the mix is
an important factor for optimizing the assay reaction. Accordingly,
to assay for ABG activity the ratio of substrate to internal
standard is about 50:1, and in a preferred embodiment is 50:1. The
ratio of detergent to substrate in the ABG assay mix is about 45:1,
and in a preferred embodiment, is 45:1. The ratio of buffer to
substrate is about 925:1, and in a preferred embodiment, is
925:1.
[0111] In one embodiment, the invention provides a second assay to
determine the activity of ABG, a deficiency of which is the cause
of Gaucher disease. The assay is performed by combining either a
3.2 mm punch from a dried blood spot (DBS; described in further
detail below) or, preferably, DBS punch extract with an assay mix
comprising substrate, a glucosidase inhibitor, detergent and
buffer.
[0112] The substrate used to assess ABG enzyme activity in the
second assay is 4-Methylumbelliferyl-.beta.-D-glucopyranoside
(4-MU-.beta.-Glu), and is present in the assay mix at a
concentration in the range of 5 to 10 mM, preferably at a
concentration of about 8 mM, and more preferably at a concentration
of 8.333 mM. The detergent for the second ABG assay is preferably
sodium taurodeoxycholate, present in the reaction at a
concentration in the range of 1 to 12 mM, preferably at a
concentration of 6 mM. Importantly, the impurities in the sodium
taurodeoxycholate must be kept to a minimum, because impurities can
precipitate and affect the precision of the assay. Thus, it is
preferred that the sodium taurodeoxycholate be at least 90% pure,
preferably 95, 97, 98, 99, or up to 100% pure. Most preferably, the
sodium taurodeoxycholate used in the reaction mix is at least 97%
pure.
[0113] In addition to the foregoing, the second ABG assay utilizes
the specific ABG inhibitor, conduritol B epoxide (CBE; Calbiochem
Product No. 234599). Use of an ABG-specific inhibitor permits an
evaluation of the contribution of other .beta.-glucosidase enzymes
to the overall activity of the assay. The difference between
activities in the absence and presence of CBE is used to quantitate
ABG activity.
[0114] More specifically, the second ABG assay utilizes the
following assay components:
Substrate Stock Solution, 1 M: 774.26 mg of 4-MU-.beta.-Glu in 2.29
mL DMSO
[0115] Buffered Extractant: 0.30 M citrate phosphate with 1% sodium
taurodeoxycholate and 1% triton X-100, pH 5.2 CBE Stock Solution:
8.30 mg conduritol B epoxide in 200 .mu.L of DMSO. 4-MU Stock
Solution, 25 mM. (Used for standard curve): 5 mg of 4-MU in 1.14 mL
DMSO
Stop Buffer: 0.5 M EDTA, pH 11.3 to 12.0.
[0116] Uninhibited Working Substrate Solution: 100 .mu.l Substrate
Stock Solution plus 7.9 ml pure water
Inhibited Working Substrate Solution: 7.5 .mu.L of 0.26 M CBE+4 ml
of Uninhibited Working Substrate Solution
[0117] To perform the second ABG assay, a 3 mm DBS is first
extracted in an extraction buffer containing a detergent,
preferably sodium taurodeoxycholate (described further below). The
extracted DBS is then divided into at least two samples. The first
sample is mixed with the inhibited working substrate solution, and
the second sample is mixed with the uninhibited working substrate
solution. The reaction mixtures are then incubated for 10-30 hours,
preferably 15-25, still more preferably 20 hours at 35-39.degree.
C., preferably 37.degree. C. After the allotted time, the reactions
are quenched by the addition of stop buffer (0.5M EDTA at pH
11.3-12). The reaction mixtures are then centrifuged for 30-90
minutes, preferably 60 minutes, at 2000-3000 rpm, preferably 2500
rpm. The amount of product generated (amount of 4-MU present) in
the reaction mixtures is then measured using a fluorometer with 355
nm excitation and 460 nm emission wavelengths.
[0118] The amount of ABG enzyme activity is determined using the
second assay by subtracting the amount of product generated (amount
of 4-MU present) in the first reaction mixture (containing the
inhibited working substrate solution) from the second reaction
mixture (containing the uninhibited working substrate solution) to
produce a differential fluorescence signal. As used herein, a
"differential fluorescence signal" refers to the difference between
the amount of product generated in a reaction mix using the
inhibited working substrate solution and the amount of product
generated in a reaction mix using the uninhibited working substrate
solution, and is attributable to the level of fluorescence in the
uninhibited working substrate reaction that is contributed by the
ABG enzyme activity. The differential fluorescence signal is then
compared to a standard curve to determine the enzyme activity.
[0119] The standard curve can be generated along with the ABG assay
reactions, or can be determined separately. To prepare the standard
curve, a 12.5 .mu.M 4-Methlyumbelliferone (4-MU) solution is
prepared. This solution is then serially diluted to the following
concentrations of 4-MU: 750 pM, 375 pM, 188 pM, 93.8 pM, 46.9 pM,
23.4 pM, 11.7 pM, and 0 pM. One of skill in the art will appreciate
that a different serial dilution may be used, and the remainder of
the calculations adjusted accordingly. The individual fluorescence
readings from the standard curve samples are then plotted against
the corresponding molar quantity per sample. The equation (4-MU,
pmol)=.alpha..times.fluorescence is then fit to the data, wherein
.alpha. is the slope of the regression line. The differential
fluorescence levels measured above are then converted into pmol per
sample by linear regression using the standard curve. This result
is then converted into pmol/(punch*h) (pmol substrate converted per
3.2 mm punch per hour) by dividing the result (i.e., the total pmol
per sample found in each reaction using the standard curve) by the
incubation time in hours and multiplying by the fraction of extract
used per reaction.
[0120] ASM Assay Mix
[0121] In one embodiment, the invention provides an assay to
determine the activity of ASM, a deficiency of which is the cause
of Niemann-Pick A/B disease. The assay is performed by combining
either a DBS punch or DBS punch extract with an assay mix
comprising substrate, internal standard, detergent and buffer.
Preferably the assay is performed using DBS punch extract.
[0122] The substrate used to determine ASM enzyme activity is
N-Hexanoyl-D-erythro-sphingosylphosphorylcholine (C6-sphingomyelin;
C.sub.29H.sub.59N.sub.2O.sub.6P) and is present in the assay mix at
a concentration of between 0.15 and 0.45 mM, preferably at a
concentration of 0.3 mM, and still more preferably at a
concentration of 0.33 mM. The internal standard for the ASM assay
is N-butyroyl-D-erythro-sphingosine (C4-ceramide;
C.sub.22H.sub.43NO.sub.3), and is present in the mix at a
concentration of between 3.0 and 9.0 .mu.M, preferably at a
concentration of 6 .mu.M, and still more preferably at a
concentration of 6.67 .mu.M. The detergent for the ASM assay is
sodium taurocholate, present in the assay mix at a concentration of
between 0.5 and 1.5 g/L, preferably at a concentration of 1 g/L.
The buffer for the ASM assay is a sodium acetate buffer at a
concentration of between about 0.45 and 1.3 M, preferably at a
concentration of 0.9 M, and still more preferably at a
concentration of 0.92 M. In addition, the ASM assay mix includes
the enzyme co-factor zinc chloride at a concentration of between
0.5 and 1.5 mM, preferably at a concentration of 0.6 mM. The ASM
assay mix should be at a pH of between 5.5 and 5.9, preferably pH
5.7.
[0123] To perform the ASM assay (described in further detail below)
10-20 .mu.l, preferably 13-17 .mu.l, and more preferably 15 .mu.l
of the ASM assay mix is combined with 10 .mu.l of the DBS punch
extract. Since, as noted above, the DBS punch contains 2-3.5 .mu.l
blood and is preferably extracted in 70 .mu.l buffer, each ASM
assay reaction will contain between 0.25 and 0.58 .mu.l blood.
Preferably, each assay will contain between 0.33 and 0.43 .mu.l
blood, and more preferably will contain 0.4 .mu.l blood. The DBS
punch or punch extract is reacted with the ASM assay mix according
to the steps described herein below. During the reaction, however,
the concentration of substrate in the assay is between 0.1 and 0.3
mM, preferably 0.15 and 0.25 mM, and more preferably 0.2 mM.
[0124] In addition to the absolute concentrations of assay mix
components described above, the invention contemplates that the
ratio of assay components to the amount of substrate in the mix is
an important factor for optimizing the assay reaction. Accordingly,
to assay for ASM activity the ratio of substrate to internal
standard is about 50:1, and in a preferred embodiment is 50:1. The
ratio of detergent to substrate in the ASM assay mix is about
5.6:1, and in a preferred embodiment, is 5.6:1. The ratio of buffer
to substrate is about 2788:1, and in a preferred embodiment, is
2788:1. The ratio of zinc chloride to substrate is about 1.82:1,
and in a preferred embodiment, is 1.82:1.
[0125] GAA Assay Mix
[0126] In one embodiment, the invention provides an assay to
determine the activity of GAA, a deficiency of which is the cause
of Pompe disease. The assay is performed by combining either a DBS
punch or DBS punch extract with an assay mix comprising substrate,
internal standard, detergent and buffer. Preferably the assay is
performed using DBS punch extract.
[0127] The substrate used to determine GAA enzyme activity is
(7-benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxyethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester (C.sub.34H.sub.49N.sub.2O.sub.10) and is present in the assay
mix at a concentration of between 0.3 and 0.9 mM, preferably at a
concentration of 0.6 mM, and still more preferably at a
concentration of 0.667 mM. The internal standard for the GAA assay
is
7-d5-benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester (C.sub.28H.sub.34N.sub.3O.sub.5D.sub.5), and
is present in the mix at a concentration of between 3.3 and 9.9
.mu.M, preferably at a concentration of 6 .mu.M, and still more
preferably at a concentration of 6.67 .mu.M. The detergent for the
GAA assay is CHAPS, present in the assay mix at a concentration of
between 5 and 15 g/L, preferably at a concentration of 10 g/L. The
buffer for the GAA assay is a phosphate citrate buffer at a
concentration of between about 0.15 and 0.45 M, preferably at a
concentration of 0.3 M. In addition, the GAA assay mix includes the
non-specific enzyme inhibitor acarbose at a concentration of
between about 6.5 and 19.5 .mu.M, preferably at a concentration of
13.3 .mu.M. The GAA assay mix should be at a pH of between 3.8 and
4.2, preferably pH 4.0.
[0128] To perform the GAA assay (described in further detail below)
10-20 .mu.l, preferably 13-17 .mu.l, and more preferably 15 .mu.l
of the GAA assay mix is combined with 10 .mu.l of the DBS punch
extract. Since, as noted above, the DBS punch contains 2-3.5 .mu.l
blood and is preferably extracted in 70 .mu.l buffer, each GAA
assay reaction will contain between 0.25 and 0.58 .mu.l blood.
Preferably, each assay will contain between 0.33 and 0.43 .mu.l
blood, and more preferably will contain 0.4 .mu.l blood. The DBS
punch or punch extract is reacted with the GAA assay mix according
to the steps described herein below. During the reaction, however,
the concentration of substrate in the assay is between 0.2 and 0.6
mM, preferably 0.3 and 0.5 mM, and more preferably 0.4 mM.
[0129] In addition to the absolute concentrations of assay mix
components described above, the invention contemplates that the
ratio of assay components to the amount of substrate in the mix is
an important factor for optimizing the assay reaction. Accordingly,
to assay for GAA activity the ratio of substrate to internal
standard is about 100:1, and in a preferred embodiment is 100:1.
The ratio of detergent to substrate in the GAA assay mix is about
24.3:1, and in a preferred embodiment, is 24.3:1. The ratio of
buffer to substrate is about 450:1, and in a preferred embodiment,
is 450:1. The ratio of inhibitor (acarbose) to substrate is about
0.02:1, and in a preferred embodiment, is 0.02:1.
[0130] GLA Assay Mix
[0131] In one embodiment, the invention provides an assay to
determine the activity of GLA, a deficiency of which is the cause
of Fabry disease. The assay is performed by combining either a DBS
punch or DBS punch extract with an assay mix comprising substrate,
internal standard, detergent and buffer. Preferably the assay is
performed using DBS punch extract.
[0132] The substrate used to determine GLA enzyme activity is
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-
-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid tert-butyl
ester (C.sub.33H.sub.47N.sub.3O.sub.10) and is present in the assay
mix at a concentration of between 1.5 and 4.5 mM, preferably at a
concentration of 3.0 mM, and still more preferably at a
concentration of 3.33 mM. The internal standard for the GLA assay
is
6-d5-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbamic
acid tert-butyl ester (C.sub.27H.sub.32N.sub.3O.sub.5D.sub.5), and
is present in the mix at a concentration of between 3.3 and 9.9
.mu.M, preferably at a concentration of 6 .mu.M, and still more
preferably at a concentration of 6.67 .mu.M. The detergent for the
GLA assay is sodium taurocholate present in the assay mix at a
concentration of between 2 and 4 g/L, preferably at a concentration
of 3 g/L. The buffer for the GLA assay is a sodium acetate buffer
at a concentration of between about 0.07 and 0.21 M, preferably at
a concentration of 0.142 M. In addition, the GLA assay mix includes
the non-specific enzyme inhibitor N-acetylgalactosamine at a
concentration of between about 80 and 240 .mu.M, preferably at a
concentration of 160 mM. The GLA assay mix should be at a pH of
between 4.4 and 4.8, preferably pH 4.6.
[0133] To perform the GLA assay (described in further detail below)
10-20 .mu.l, preferably 13-17 .mu.l, and more preferably 15 .mu.l
of the GLA assay mix is combined with 10 .mu.l of the DBS punch
extract. Since, as noted above, the DBS punch contains 2-3.5 .mu.l
blood and is preferably extracted in 70 .mu.l buffer, each GLA
assay reaction will contain between 0.25 and 0.58 .mu.l blood.
Preferably, each assay will contain between 0.33 and 0.43 .mu.l
blood, and more preferably will contain 0.4 .mu.l blood. The DBS
punch or punch extract is reacted with the GLA assay mix according
to the steps described herein below. During the reaction, however,
the concentration of substrate in the assay is between 1.0 and 1.5
mM, and preferably at a concentration of 2.0 mM.
[0134] In addition to the absolute concentrations of assay mix
components described above, the invention contemplates that the
ratio of assay components to the amount of substrate in the mix is
an important factor for optimizing the assay reaction. Accordingly,
to assay for GLA activity the ratio of substrate to internal
standard is about 499:1, and in a preferred embodiment is 499:1.
The ratio of detergent to substrate in the GLA assay mix is about
1.68:1, and in a preferred embodiment, is 1.68:1. The ratio of
buffer to substrate is about 43:1, and in a preferred embodiment,
is 43:1. The ratio of inhibitor (N-acetylgalactosamine) to
substrate is about 48:1, and in a preferred embodiment, is
48:1.
[0135] GALC Assay Mix
[0136] In one embodiment, the invention provides an assay to
determine the activity of GALC, a deficiency of which is the cause
of Krabbe disease. The assay is performed by combining either a DBS
punch or DBS punch extract with an assay mix comprising substrate,
internal standard, detergent and buffer. Preferably the assay is
performed using a DBS punch, that is, there is no blood extraction
step, and the 3.2 mm DBS punch is placed in direct contact with the
assay mix described below.
[0137] The substrate used to determine GALC enzyme activity is
D-galactosyl-.beta.-1-1'-octanoyl-D-erythro-sphingosine (C8
galactosyl ceramide; C.sub.32H.sub.61NO.sub.8) and is present in
the assay mix at a concentration of between 0.5 and 1.5 mM,
preferably at a concentration of 1 mM. The internal standard for
the GALC assay is N-decanoyl-D-erythro-sphingosine (C10 ceramide;
C.sub.28H.sub.55NO.sub.3), and is present in the mix at a
concentration of between 3.3 and 9.9 .mu.M, preferably at a
concentration of 6 .mu.M, and still more preferably at a
concentration of 6.67 .mu.M. The detergent for the GALC assay is
sodium taurocholate, present in the assay mix at a concentration of
between 4.5 and 18 g/L, preferably at a concentration of 9.6 g/L.
The detergent for the GALC assay also includes oleic acid at a
concentration of between 0.6 and 1.8 g/L, preferably at a
concentration of about 1 g/L, and more preferably at a
concentration of 1.2 g/L. The buffer for the GALC assay is a
phosphate/citrate buffer at a concentration of between about 0.09
and 0.27 M, preferably at a concentration of 0.18 M. The GALC assay
mix should be at a pH of between 4.2 and 4.6, preferably pH 4.4
[0138] To perform the GALC assay (described in further detail
below) 20-40 .mu.l, preferably 25-35 .mu.l, and more preferably 30
.mu.l of the GALC assay mix is combined with a single 3.2 mm DBS
punch. Thus, each GALC reaction will contain between about 2-3.5
.mu.l blood (i.e., the amount of blood in a single 3.2 mm punch).
Preferably, each assay will include about 3 .mu.l blood, and more
preferably, will contain 2.8 .mu.l blood. The DBS punch is reacted
with the GALC assay mix according to the steps described herein
below. During the reaction, however, the concentration of substrate
in the assay is between 0.5 and 1.5 mM, preferably 0.8 and 1.2 mM,
and more preferably 1.0 mM.
[0139] In addition to the absolute concentrations of assay mix
components described above, the invention contemplates that the
ratio of assay components to the amount of substrate in the mix is
an important factor for optimizing the assay reaction. Accordingly,
to assay for GALC activity the ratio of substrate to internal
standard is about 150:1, and in a preferred embodiment is 150:1.
The ratio of sodium taurocholate to substrate in the GALC assay mix
is about 17.8:1, and in a preferred embodiment, is 17.8:1. The
ratio of oleic acid to substrate is about 4.25:1, and in a
preferred embodiment, is 4.25:1. The ratio of buffer to substrate
is about 180:1, and in a preferred embodiment, is 180:1.
[0140] The specific components and amounts used for each of the
five assay mixtures are shown in Table 1.
TABLE-US-00001 TABLE 1 ABG (Gaucher) Assay Mix ASM (Niemann-Pick)
Assay Mix GAA (Pompe) Assay Mix Substrate (mmol/L) 0.67 Substrate
(mmol/L) 0.33 Substrate (mmol/L) 0.667 Internal standard 13.33
Internal standard 6.67 Internal standard 6.67 (.mu.mol/L)
(.mu.mol/L) (.mu.mol/L) Detergent (g/L Sodium 16 Detergent (g/L 1
Detergent (g/L 10 Taurocholate) Sodium CHAPS) Taurocholate)
Inhibitor (mmol/L 0.6 Inhibitor (umol/L 13.3 Zinc Chloride)
Acarbose) Buffer (mol/L 0.62 Buffer (mol/L 0.92 Buffer (mol/L 0.3
Phosphate/Citrate) Sodium Acetate) Phosphate/Citrate) pH 5.1 pH 5.7
pH 4.0 Incubation solution 15 .mu.L reagent + Incubation solution
15 .mu.L reagent + Incubation solution 15 .mu.L reagent + 10 .mu.L
dbs extract 10 .mu.L dbs extract 10 .mu.L dbs extract Concentration
during incubation Substrate (mmol/L) 0.4 Substrate (mmol/L) 0.2
Substrate 0.4 (mmol/L) Internal standard 8.0 Internal standard 4.0
Internal standard 4.0 (.mu.mol/L) (.mu.mol/L) (.mu.mol/L) Detergent
(g/L Sodium 9.6 Detergent (g/L 0.6 Detergent (g/L 6 (Chaps)
Taurocholate) Sodium Chaps) Taurocholate) Buffer (mol/L 0.4
Inhibitor (mmol/L 0.4 Inhibitor (umol/L 8 Phosphate/Citrate) Zinc
Chloride) Acarbose) pH 5.1 Buffer (mol/L 0.6 Buffer (mol/L 0.18
Sodium Acetate) Phosphate/Citrate) pH 5.7 pH 4.0 Incubation volume
(ul) 25 Incubation volume 25 Incubation volume 25 (ul) (ul) Ratio
S/IS 50 S/IS 50 S/IS 100 Detergent/S (g/mmol) 45 Detergent/S 5.6
Detergent/S 24.3 (g/mmol) (g/mmol) Buffer/S 925 Inhibitor/S 1.82
Inhibitor/S 0.02 Buffer/S 2788 Buffer/S 450 GLA (Fabry) Assay Mix
GALC (Krabbe) Assay Mix Substrate 3.33 Substrate 1 (mmol/L)
(mmol/L) Internal Standard 6.67 Internal Standard 6.67 (.mu.mol/L)
(.mu.mol/L) Detergent (g/L 3 Detergent (g/L 9.6 Sodium Sodium
Taurocholate) Taurocholate) Inhibitor (mmol/L 160 Detergent (g/L
1.2 N-Acetyl- Oleic Acid) galactosamine) Buffer (mol/L 0.142 Buffer
(mol/L 0.18 Sodium Acetate) Phosphate/ Citrate) pH 4.6 pH 4.4
Incubation 15 .mu.L reagent + Incubation 30 .mu.L cktl + 1 solution
10 .mu.L dbs extract solution dbs (3 mm) Concentration during
incubation Substrate 2.0 Substrate 1 (mmol/L) (mmol/L) Internal
standard 4.0 Internal standard 6.67 (.mu.mol/L) (.mu.mol/L)
Detergent (g/L 1.8 Detergent (g/L 9.6 Sodium Sodium Taurocholate)
Taurocholate) Inhibitor (mmol/L 96 Detergent 1.2 N- (mmol/L Oleic
Acetylgalactosamine) Acid) Buffer (mol/L 0.085 Buffer (mol/L 0.18
Sodium Acetate) Phosphate/Citrate) pH 4.6 pH 4.4 Incubation volume
25 Incubation 30 volume (ul) Ratio S/IS 499 S/IS 150 Detergent/S
1.68 Sodium 17.8 (g/mmol) Taurocholate/S (g/mmol) Inhibitor/S 48
Oleic Acid/S 4.25 (g/mmol) Buffer/S 43 Buffer/S 180
[0141] In one embodiment, any or all of the above enzyme assay
mixtures can be prepared in a concentrated form that is then
diluted prior to use. For example, the assay mixtures can be
prepared as 0.5.times., 2.times., 5.times., 10.times., or 20.times.
or more. The concentrated assay mix can then be diluted to an
appropriate concentration with the DBS extract. For example, a
smaller amount (or larger amount in the case of a 0.5.times.
concentration) of the concentrated assay mixture can be used, as
would be determined by one of skill in the art, to achieve the
concentrations and/or ratios of components described above for each
assay mixture. Alternatively, the concentrated assay mix could be
diluted with a non-reactive buffer (e.g., water) to the correct
concentration prior to use in the enzyme activity assays described
herein. In a further embodiment, the assay mix could be lyophilized
for long term storage, and then re-hydrated prior to use. Methods
for lyophilizing are known in the art. In addition, the assay mixes
described herein can be prepared in bulk form. That is, the
components can be mixed in to achieve the concentrations and/or
ratios described above, but in a volume sufficient for multiple
reactions. The assay mixtures can be prepared in volumes
appropriate for 2, 10, 50, 100, 600, or 1200 or more individual
reactions. In addition, subsets of the components for each assay
mix can be premixed prior to use in the assay. For example, the
substrate and internal standard may be premixed at an appropriate
ratio (see Table 1). The other assay components can then be added
to the premixed substrate/internal standard. Any and all
subcombinations of the components of the assay mix can be premixed
prior to initiation of the assay and are contemplated by the
invention (e.g., substrate/internal standard/buffer or
substrate/internal standard/detergent or
buffer/detergent/inhibitor).
Methods for Determining Enzyme Activity in DBS
[0142] The instant invention provides a method to determine the
activity of one or more lysosomal enzymes, specifically, ABG, ASM,
GAA, GLA, and GALC. The assay for enzyme activity is performed by
contacting a 3.2 mm DBS (that is, dried blood carried on an inert
surface such as filter paper) punch or an extract prepared from a
3.2 mm DBS punch with the assay mix described above that is
specific for the enzyme sought to be assayed.
[0143] The assays are designed to be used in combination with blood
samples that are routinely taken from newborn infants after birth.
The blood samples are typically prepared as a blood drop or smear
on a piece of filter paper or other suitable substrate. These drops
are generally referred to as newborn screening cards, but for
purposes of the invention are referred to as a dried blood spot
(DBS). While the invention is likely to be used to screen for
enzyme activity from newborn DBS, it is understood that enzyme
activity can be screened in DBS obtained from individuals of any
age, such as children, adolescents, and/or adults, including
populations of individuals at high risk for lysosomal storage
disease.
[0144] The invention utilizes 3.2 mm diameter "punches" taken from
the DBS; that is, a 3.2 mm circular piece of the paper containing
the dried blood is cut out of the DBS using a hole-punch. The 3.2
mm piece could also be hand-cut from the DBS, or separated from the
DBS by other means sufficient to produce a 3.2 mm sample from the
DBS. While typically circular in shape, the 3.2 mm sample could
take any shape, provided that the amount of dried blood in the
sample is equivalent to that in a 3.2 mm circular punch. Typically,
a 3.2 mm circular punch from a newborn DBS will contain between
about 2 and 3.5 .mu.l of blood, preferably between about 2.5 and
3.2 .mu.l of blood, more preferably about 2.8-3 .mu.l of blood, and
still more preferably, 2.8 .mu.l of blood.
[0145] It is generally possible to take between 3-7 3.2 mm punches
from a single DBS. The punches are preferably taken from the
perimeter of the DBS, rather than the center, because the amount of
blood in the perimeter of the DBS is more consistent than that in
the center of the DBS (where the amount of blood is usually higher
relative to the perimeter).
[0146] The 3.2 mm punch can be used directly in an assay for enzyme
activity, or the blood from a 3.2 mm punch can be extracted from
the substrate on which it is dried. The DBS punch can be extracted
by incubating the 3.2 mm DBS punch with suitable buffer. For
example a single 3.2 mm punch can be extracted by incubating it
with 60-80 .mu.l of sodium phosphate buffer (pH 7.1) at
20-45.degree. C. for between 10 minutes and 5 hours. Preferably the
extraction is performed in 70 .mu.l sodium phosphate buffer (pH
7.1) at 37.degree. C. for 1 hour. The DBS punch extract can then be
used in the assays for specific enzyme activity described below.
Based on the amount of blood in a single 3.2 mm DBS punch, the
amount of blood in the DBS extract will be between 0.25 and 0.33
.mu.l per 10 .mu.l DBS extract at the low end of the range
(extraction of a 3.2 mm DBS containing 2 .mu.l blood in a volume of
between 60 and 80 .mu.l extraction buffer) and between 0.438 and
0.583 .mu.l per 10 .mu.l DBS extract at the high end of the range
(extraction of a 3.2 mm DBS containing 3.5 .mu.l blood in a volume
of between 60 and 80 .mu.l extraction buffer). In a preferred
embodiment, the amount of blood in a 3.2 mm DBS is about 2.8 .mu.l
blood and the amount of blood in 10 .mu.l of DBS extract is about
0.4 .mu.l (based on an extraction buffer volume of 70 .mu.l).
[0147] For the second ABG assay described herein, the DBS is
preferably extracted in an aqueous buffer solution that includes
detergent. The detergent is preferably sodium taurodeoxycholate
with a minimum purity of 97% TLC (Sigma T0557). Preferably, the DBS
extraction buffer for the second ABG assay includes 0.30 M citrate
phosphate with 1% sodium taurodeoxycholate and 1% triton X-100, pH
5.2. To extract the DBS, 200 .mu.l of the extract buffer is added
to each 3.2 mm DBS punch, and incubated for 30-90 minutes,
preferably 60 minutes, at room temperature. The samples are then
centrifuged at 10,000-18,000 rpm, preferably 14,000 rpm for 15-45
minutes, preferably 30 minutes. This centrifugation step is
necessary to augment assay precision and accuracy. While it is
preferred that the second ABG assay is performed using a DBS
extract as described, it is contemplated that the assay could also
be performed using a DBS punch, provided that the sodium
taurodeoxycholate detergent is added to the reaction mixture.
Reaction of DBS and DBS Extract with Assay Mix
[0148] The assay mixtures are added to appropriate containers to be
reacted with the DBS punches or DBS punch extract (or both). Any
type of container (e.g., a microfuge tube, or multiwell plate) can
be used, however, it is preferred that the reactions are carried
out in a multiwell plate, such as a 96 well polypropylene plate.
Other containers known to those of skill in the art may be used
according to the methods of the invention.
[0149] The assay mixtures described above are each added to
separate wells of a multiwell plate (or separate containers if
other types of containers are being used). For ASM, ABG (first
assay), GAA, and GLA assays 10-20 .mu.l of assay mix (specific for
each enzyme), preferably 13-17 .mu.l, and more preferably 15 .mu.l
is added to each well followed by 5-20 .mu.l of DBS extract,
preferably 10 .mu.l DBS extract. For the GALC assay, one 3.2 mm DBS
punch is added to a well, followed by 20-40 .mu.l, preferably 25-35
.mu.l, and more preferably 30 .mu.l GALC assay mix. A sixth
container or well may also be used as a blank and contains the same
amount of assay mix used to assay for enzyme activity (i.e., 10-20
.mu.l for ASM, ABG, GAA, GLA; 20-40 .mu.l for GALC) combined with
either DBS extraction buffer or a 3.2 mm punch taken from the same
substrate as the DBS, minus the dried blood.
[0150] In one embodiment, one or more assay wells or containers
contain both a 3.2 mm DBS punch and DBS extract. Likewise, while it
is preferred that the ASM, ABG, GAA, and GLA assays are performed
using DBS extract, and the GALC assay is performed using a 3.2 mm
DBS punch, each assay may be performed using either DBS extract or
a 3.2 mm DBS punch.
[0151] The present invention can be used to determine the activity
of a single enzyme in a single DBS sample or may be used in a
multiplex format to assay multiple enzyme activities in a given DBS
sample, and/or multiple enzyme activities from multiple
individuals. In the multiplex format, DBS samples from one or a
plurality of individuals are incubated under appropriate conditions
("reacted") with the assay mix corresponding to the enzyme
activities to be assayed. For example, the methods of the invention
may be used to assay DBS samples from "n" individuals for each of
the five enzyme activities described herein (ASM, ABG, GAA, GLA,
and GALC). When expanded to include at least one blank reaction,
the multiplex format will require (5.times.n+the number of blanks)
separate reactions, all of which can be performed simultaneously in
a multiwell plate (or a plurality of multiwell plates). Once each
individual enzyme reaction is performed using extract or a whole
DBS punch from a given individual, the quenched reactions can be
pooled prior to mass spectrometry. Thus, from a single sample
analyzed by mass spectrometry, under the multiplex format, the
activities of all five enzymes can be determined.
[0152] Once the assay mix is combined with the DBS extract and/or
DBS punch for each enzyme activity to be assayed (including at
least one control reaction), the combination, referred to as a
reaction mix is incubated at between 36 and 38.degree. C. for 1 to
48 hours. Preferably the reaction mix is incubated at 37.degree. C.
for 20-30 hours, and more preferably for 20-24 hours.
[0153] After incubation, the enzyme activity of each reaction is
stopped or "quenched" by the addition of a mixture of 1:1 ethyl
acetate:MeOH to each well. For the reaction volumes described
herein, a volume of 100-200 .mu.l is added to each reaction. In a
preferred embodiment, where a reaction volume of between 25 and 30
.mu.l is used, 100 .mu.l ethyl acetate:MeOH is used to quench each
reaction. Provided that the ratio of ethyl acetate to MeOH remains
1:1, the concentration of ethyl acetate and MeOH can vary. For
example, 20-100% ethyl acetate can be used and 20-100% MeOH can be
used, provided that the ratio of ethyl acetate to MeOH is 1:1.
Preferably the concentration of each is 100%.
[0154] Once the reactions are quenched, in a multiplexed assay
format, each assay reaction (excluding the control) for a given DBS
sample can be combined in a single container (i.e., in a single
well of a multiwell plate). If a non-multiplex format is being
used, then each reaction is processed individually through the
following steps. The combined reaction mix is then extracted by
adding ethyl acetate followed by an equal amount of water. As used
herein, the term "extraction" or "extract" refers to the separation
of the assay sample into an organic phase and inorganic phase.
Between 1 and 16 volumes of ethyl acetate and water can be used,
however it is preferred that in a non-multiplex format, at least 2
volumes of each of ethyl acetate and water are added to each well.
In contrast, in a multiplex format, that is, where the enzyme assay
reactions have been pooled, at least a 3/5 volume of each of ethyl
acetate and water are added to each well. The ethyl acetate and
water is then mixed with the combined reaction assays and then
centrifuged. This results in a separation of the organic phase from
the inorganic phase (i.e., extraction). Substantially all of the
top phase (organic) is then removed and placed in a clean container
or well. The extracted assay reactions are then dried under a
stream of nitrogen. In addition, the container or well comprising
the extracted organic phase can be warmed to any temperature
between room temperature and about 25.degree. C. to assist in the
drying process.
[0155] The samples are then reconstituted in a mixture of ethyl
acetate and MeOH at a ratio of 19:1 ethyl acetate:MeOH, and then
purified to remove buffer components by passing them over silica
gel under vacuum. As used herein, the term "purify" or "purifying"
refers to a step of removing the buffer components of the assay
mixture. The ethyl acetate:MeOH mixture can contain between about
90-99% ethyl acetate and 1-10% MeOH. Prior to adding the samples to
the silica gel, the gel is washed with a 19:1 mixture of ethyl
acetate:MeOH. Preferably the silica gel used in the purification
step has a particle size of between 43 and 60 .mu.m and a pore size
of between 50-70 .ANG., preferably 60 .ANG.. Although silica gel is
the preferred mode of purification, other commercially available
methods may be used by one of skill in the art to purify the enzyme
assays including, but not limited to, online solid phase
extraction, bead solid phase extraction, or high turbo flow liquid
chromatography (HTLC; available from Cohesive). After the samples
are purified through the silica gel, the gel is washed with a 19:1
mixture of ethyl acetate:MeOH. The clean-up steps that include both
liquid-liquid or solid phase extraction can be omitted or can be
replaced with other methods of separation provided that the other
methods can separate lipid-like compounds from detergent, buffer,
or other compounds eluted from the blood. A more specific protocol
for silica gel purification of the assay samples is described in
the Examples.
[0156] The resulting purified assay sample is then dried under
nitrogen, and can be optionally heated to between room temperature
and about 25.degree. C. to speed the drying process. The amount of
product (produced by the activity of the enzyme to be assayed) and
internal standard present in the assay samples is then determined
by mass spectrometry analysis. Prior to analysis, the samples are
reconstituted in a mixture of acetonitrile and water with the
addition of formic acid. Preferably the reconstitution buffer
comprises 80% acetonitrile, 20% water, and 0.2% formic acid.
Alternate organic solvent compositions could also be used to
reconstitute the samples, such as, but not limited to, 5 mmol/L
ammonium formate in acetonitrile-water (4:1 volume). The
reconstituted samples are then analyzed by mass spectrometry,
preferably tandem mass spectrometry, to determine the amount of
each product and internal standard present in the each sample,
which is then used to calculate the activity of each enzyme.
[0157] The second ABG DBS assay measures the ABG-catalyzed cleavage
of the fluorogenic substrate 4-MU-.beta.-Glu by detecting the
product 4-MU in a fluorometer. Enzymes are eluted from dried blood
spots with a buffered detergent solution. The eluate is incubated
with the substrate at acidic pH. After 20 hours the reaction is
stopped by the addition of alkaline EDTA solution. Conduritol B
epoxide (CBE), a specific and irreversible inhibitor of ABG, is
used to assess the contribution of other .beta.-glucosidase
isoenzymes to the activity. The difference between activities in
the absence presence of CBE is used to quantitate ABG activity. A
bile salt, sodium taurodeoxycholate, acts as a detergent and as an
activator of ABG, however, it is contemplated that other detergents
could be used such as n-dodecymaltoside, ASB14, C7B20, zwettergent
3-14, or sodium taurocholoate. The purity of sodium
taurodeoxycholate is important to the success of the assay.
Impurities in the sodium taurodeoxycholate can precipitate and
affect the precision of the assay. To minimize the impact of
precipitation, it is preferred that the second ABG assay utilizes
sodium taurodeoxycholate of at least 97% purity.
[0158] More specifically, to each 3.2 mm DBS is added 100-300
.mu.l, preferably 200 .mu.l Buffered Extractant (defined above),
and the DBS is allowed to incubate at room temperature for 30-90
minutes, preferably 60 minutes. The filter paper or other substrate
that the DBS was contained on is then removed and the sample is
centrifuged at 10,000 to 20,000 rpm for 15-45 minutes, preferably
at 14,000 rpm for 30 minutes.
[0159] Each test sample is assayed in duplicate, preferably for
both uninhibited and inhibited working substrate solutions. For
example, 60-100 .mu.l, preferably 80 .mu.l, of uninhibited working
substrate solution is added to a well (or other container) per
sample, and 60-100 .mu.l, preferably 80 .mu.l, of inhibited working
substrate solution is added to a well (or other container) per
sample (optionally uninhibited and inhibited working substrate
solution are separately added to one additional well per sample).
To each test well is then added 20-60 .mu.l, preferably 40 .mu.l of
the DBS extract. The reaction is then allowed to incubate at
35-39.degree. C., preferably 37.degree. C. for 15-25 hours,
preferably 20 hours. The reactions are then stopped by adding an
appropriate amount of stop buffer (80-120 .mu.l, preferably 100
.mu.l). The assay reactions are then centrifuged for 30-90 minutes,
preferably 60 minutes, at 2000-3000 rpm, preferably 2500 rpm.
[0160] The reactions can then be assessed by fluorometry at 355 nm
excitation and 460 nm emission to determine the amount of product
(fluorescence) produced by the enzymatically processed substrate.
The amount of product (fluorescence) in the reaction mixtures
without CBE is determined, and the amount of product (fluorescence)
of the reaction mixtures with CBE is determined. The amount of
product in the reaction mix with CBE is then subtracted from the
amount of product in the reaction mix without CBE. The result is
the differential fluorescence and it is then compared to a standard
curve. The equation (4-MU, pmol)=.alpha..times.fluorescence is then
fit to the data, wherein a is the slope of the regression line. The
differential fluorescence levels measured above are then converted
into pmol per sample by linear regression using the standard curve.
This result is then converted into pmol/(DBS punch*h) by dividing
the result by the incubation time in hours, and multiplying by the
fraction of extract used per well.
[0161] The standard curve used in the second. ABG assay can be
generated in parallel with the sample assays, or can be generated
separately and kept as a reference for future tests. The standard
curve may degrade over time and should be made fresh is there is a
noticeable change in the slope. To prepare a standard curve, a 12.5
.mu.M 4-MU standard working solution is first prepared by diluting
5 .mu.l of 25 mM 4-MU stock solution into 10 ml water. Serial
dilutions of the 4-MU standard are then prepared. For example, a
serial dilution, in one embodiment, has 4-MU concentrations per
well of 750, 375, 188, 93.8, 46.9, 23.4, 11.7, and 0 pmol per well.
100 .mu.l of stop buffer is then added to each well, and the
standard curve reactions are centrifuged for 30-90 minutes,
preferably 60 minutes, at 2000-3000 rpm, preferably 2500 rpm. The
fluorescence of the standard curve wells is determined using a
fluorometer with 355 nm excitation and 460 nm emission
wavelengths.
Mass Spectrometry Analysis
[0162] The methods of the invention, in part, use mass spectrometry
for determining the amount or presence of the product or products
of the enzyme assays (i.e., the products generated by the action of
the enzyme in a DBS punch on the substrate), and the internal
standard. It should be noted, that for the second ABG assay, enzyme
activity is measured using a fluorescence-based assay. A variety of
configurations of mass spectrometers can be used in a method of the
invention. Several types of mass spectrometers are available or can
be produced with various configurations. In general, a mass
spectrometer has the following major components: a sample inlet, an
ion source, a mass analyzer, a detector, a vacuum system, and
instrument-control system, and a data system.
[0163] Difference in the sample inlet, ion source, and mass
analyzer generally define the type of instrument and its
capabilities. For example, an inlet can be a capillary-column
liquid chromatography source or can be a direct probe or stage such
as used in matrix-assisted laser desorption. Common ion sources
are, for example, electrospray, including nanospray and microspray
or matrix-assisted laser desorption. Common mass analyzers include
a quadruple mass filter, ion trap mass analyzer and time-of-flight
mass analyzer.
[0164] The ion formation process is a starting point for mass
spectrum analysis. Several ionization methods are available and the
choice of ionization method depends on the sample to be analyzed.
For example, for the analysis of amino acids a relatively gentle
ionization procedure such as electrospray ionization (ESI) can be
desirable. For ESI, a solution containing the sample is passed
through a fine needle at high potential which creates a strong
electrical field resulting in a fine spray of highly charged
droplets that is directed into the mass spectrometer. Other
ionization procedures include, for example, fast-atom bombardment
(FAB) which uses a high-energy beam of neutral atoms to strike a
solid sample causing desorption and ionization. Matrix-assisted
laser desorption ionization (MALDI) is a method in which a laser
pulse is used to strike a sample that has been crystallized in an
UV-absorbing compound matrix.
[0165] Other ionization procedures known in the art include, for
example, plasma and glow discharge, plasma desorption ionization,
resonance ionization, and secondary ionization.
[0166] Electrospray ionization (ESI) has several properties that
are useful for the invention described herein. For example, ESI can
be used for biological molecules such as lipid or glycophingolipids
that are difficult to ionize or vaporize. In addition, the
efficiency of ESI can be very high which provides the basis for
highly sensitive measurements. Furthermore, ESI produces charged
molecules from solution, which is convenient for analyzing
enzymatic products and internal standards that are in solution. In
contrast, ionization procedures such as MALDI require
crystallization of the sample prior to ionization.
[0167] Since ESI can produce charged molecules directly from
solution, it is compatible with samples from liquid chromatography
systems. For example, a mass spectrometer can have an inlet for a
liquid chromatography system, such as an HPLC, so that fractions
flow from the chromatography column into the mass spectrometer.
[0168] This in-line arrangement of a liquid chromatography system
and mass spectrometer is sometimes referred to as LC-MS. A LC-MS
system can be used, for example, to separate enzymatic products and
internal standards from complex mixtures before mass spectrometry
analysis. In addition, chromatography can be used to remove salts
or other buffer components from the sample before mass spectrometry
analysis (i.e., in addition to, or in place of silica gel
purification). For example, desalting of a sample using a
reversed-phase HPLC column, in-line or off-line, can be used to
increase the efficiency of the ionization process and thus improve
sensitivity of detection by mass spectrometry.
[0169] A variety of mass analyzers are available that can be paired
with different ion sources. Different mass analyzers have different
advantages as known to one skilled in the art and as described
herein. The mass spectrometer and methods chosen for detection
depends on the particular assay, for example, a more sensitive mass
analyzer can be used when a small amount of ions are generated for
detection.
[0170] Several types of mass analyzers and mass spectrometry
methods are described below.
[0171] Quadruple mass spectrometry utilizes a quadruple mass filter
or analyzer. This type of mass analyzer is composed of four rods
arranged as two sets of two electrically connected rods. A
combination of rf and dc voltages are applied to each pair of rods
which produces fields that cause an oscillating movement of the
ions as they move from the beginning of the mass filter to the end.
The result of these fields is the production of a high-pass mass
filter in one pair of rods and a low-pass filter in the other pair
of rods. Overlap between the high-pass and low-pass filter leaves a
defined m/z that can pass both filters and traverse the length of
the quadrupole. This m/z is selected and remains stable in the
quadruple mass filter while all other m/z have unstable
trajectories and do not remain in the mass filter. A mass spectrum
results by ramping the applied fields such that an increasing m/z
is selected to pass through the mass filter and reach the detector.
In addition, quadruples can also be set up to contain and transmit
ions of all m/z by applying a rf-only field. This allows
quadrupoles to function as a lens or focusing system in regions of
the mass spectrometer where ion transmission is needed without mass
filtering. This will be of use in tandem mass spectrometry as
described further below.
[0172] A quadruple mass analyzer, as well as the other mass
analyzers described herein, can be programmed to analyze a defined
m/z or mass range. This property of mass spectrometers is useful
for the invention described herein. Since the mass range of
enzymatic products and/or internal standards will be known prior to
an assay, a mass spectrometer can be programmed to transmit ions of
the projected correct mass range while excluding ions of a higher
or lower mass range.
[0173] The ability to select a mass range can decrease the
background noise in the assay and thus increase the signal-to-noise
ratio as well as increasing the specificity of the assay.
Therefore, the mass spectrometer can accomplish an inherent
separation step as well as detection and identification of
enzymatic products and internal standards.
[0174] Ion trap mass spectrometry utilizes an ion trap mass
analyzer. In these mass analyzers, fields are applied so that ions
of all m/z are initially trapped and oscillate in the mass
analyzer. Ions enter the ion trap from the ion source through a
focusing device such as an octapole lens system. Ion trapping takes
place in the trapping region before excitation and ejection through
an electrode to the detector. Mass analysis is accomplished by
sequentially applying voltages that increase the amplitude of the
oscillations in a way that ejects ions of increasing m/z out of the
trap and into the detector. In contrast to quadruple mass
spectrometry, all ions are retained in the fields of the mass
analyzer except those with the selected m/z. One advantage to ion
traps is that they have very high sensitivity, as long as one is
careful to limit the number of ions being tapped at one time.
Control of the number of ions can be accomplished by varying the
time over which ions are injected into the trap. The mass
resolution of ion traps is similar to that of quadruple mass
filters, although ion traps do have low m/z limitations.
[0175] Time-of-flight mass spectrometry utilizes a time-of-flight
mass analyzer. For this method of m/z analysis, an ion is first
given a fixed amount of kinetic energy by acceleration in an
electric field (generated by high voltage). Following acceleration,
the ion enters a field-free or "drift" region where it travels at a
velocity that is inversely proportional to its m/z. Therefore, ions
with low m/z travel more rapidly than ions with high m/z. The time
required for ions to travel the length of the field-free region is
measured and used to calculate the m/z of the ion.
[0176] One consideration in this type of mass analysis is that the
set of ions being studied be introduced into the analyzer at the
same time. For example, this type of mass analysis is well suited
to ionization techniques like MALDI which produces ions in short
well-defined pulses. Another consideration is to control velocity
spread produced by ions that have variations in their amounts of
kinetic energy. The use of longer flight tubes, ion reflectors, or
higher accelerating voltages can help minimize the effects of
velocity spread. Time-of-flight mass analyzers have a high level of
sensitivity and a wider m/z range than quadruple or ion trap mass
analyzers. Also data can be acquired quickly with this type of mass
analyzer because no scanning of the mass analyzer is necessary.
[0177] Tandem mass spectrometry can utilize combinations of the
mass analyzers described above. Tandem mass spectrometers can use a
first mass analyzer to separate ions according to their m/z in
order to isolate an ion of interest for further analysis. The
isolated ion of interest is then broken into fragment ions (called
collisionally activated dissociation or collisionally induced
dissociation) and the fragment ions are analyzed by the second mass
analyzer. These types of tandem mass spectrometer systems are
called tandem in space systems because the two mass analyzers are
separated in space, usually by a collision cell. Tandem mass
spectrometer systems also include tandem in time systems where one
mass analyzer is used, however the mass analyzer is used
sequentially to isolate an ion, induce fragmentation, and then
perform mass analysis.
[0178] Mass spectrometers in the tandem in space category have more
than one mass analyzer. For example, a tandem quadruple mass
spectrometer system can have a first quadruple mass filter,
followed by a collision cell, followed by a second quadruple mass
filter and then the detector. Another arrangement is to use a
quadruple mass filter for the first mass analyzer and a
time-of-flight mass analyzer for the second mass analyzer with a
collision cell separating the two mass analyzers.
[0179] Other tandem systems are known in the art including
reflectron-time-of-flight, tandem sector and sector-quadrupole mass
spectrometry.
[0180] Mass spectrometers in the tandem in time category have one
mass analyzer that performs different functions at different times.
For example, an ion trap mass spectrometer can be used to trap ions
of all m/z. A series of rf scan functions are applied which ejects
ions of all m/z from the trap except them/z of ions of
interest.
[0181] After the m/z of interest has been isolated, an rf pulse is
applied to produce collisions with gas molecules in the trap to
induce fragmentation of the ions. Then the m/z values of the
fragmented ions are measured by the mass analyzer. Ion cyclotron
resonance instruments, also known as Fourier transform mass
spectrometers, are an example of tandem-in-time systems.
[0182] Several types of tandem mass spectrometry experiments can be
performed by controlling the ions that are selected in each stage
of the experiment. The different types of experiments utilize
different modes of operation, sometimes called "scans," of the mass
analyzers. In a first example, called a mass spectrum scan, the
first mass analyzer and the collision cell transmit all ions for
mass analysis into the second mass analyzer. In a second example,
called a product ion scan, the ions of interest are mass-selected
in the first mass analyzer and then fragmented in the collision
cell. The ions formed are then mass analyzed by scanning the second
mass analyzer. In a third example, called a precursor or parent ion
scan, the first mass analyzer allows the transmission of all sample
ions, while the second mass analyzer is set to monitor specific
fragment ions, which are generated by bombardment of the sample
ions with the collision gas in the collision cell.
[0183] The second mass analyzer mass-selects the product ion of
interest for transmission to the detector. Therefore, the detector
signal is the result of all precursor ions that can be fragmented
into a common product ion. Other experimental formats include
neutral loss scans where a constant mass difference is accounted
for in the mass scans. The use of these different tandem mass
spectrometry scan procedures can be advantageous when large sets of
analytes are measured in a single experiment as with multiplex
experiments. An additional scan mode useful in the present
invention is the selected or multiple reaction monitoring mode in
which both of the analyzers are static, as user-selected specific
ions are transmitted through the first analyzer and user-selected
specific fragments arising from these ions are measured by the
second analyzer. The compound under scrutiny must be known and have
been well characterized prior to using this type of scan mode. This
type of scan mode can be used to confirm unambiguously the presence
of a compound in a matrix (e.g., blood or urine). In a preferred
embodiment of the invention, the product and internal standards are
assayed using the multiple reaction monitoring mode.
[0184] In view of the above, those skilled in the art recognize
that different mass spectrometry methods, for example, quadruple
mass spectrometry, ion trap mass spectrometry, time-of-flight mass
spectrometry and tandem mass spectrometry, can use various
combinations of ion sources and mass analyzers which allows for
flexibility in designing customized detection protocols. In
addition, mass spectrometers can be programmed to transmit all ions
from the ion source into the mass spectrometer either sequentially
or at the same time. Furthermore, a mass spectrometer can be
programmed to select ions of a particular mass for transmission
into the mass spectrometer while blocking other ions. The ability
to precisely control the movement of ions in a mass spectrometer
allows for greater options in detection protocols which can be
advantageous when a large number of analytes, for example, from a
multiplex experiment, are being analyzed.
[0185] Different mass spectrometers have different levels of
resolution, that is, the ability to resolve peaks between ions
closely related in mass. The resolution is defined as R=m/delta m,
where m is the ion mass and delta m is the difference in mass
between two peaks in a mass spectrum. For example, a mass
spectrometer with a resolution of 1000 can resolve an ion with a
m/z of 100.0 from an ion with a m/z of 100.1. Those skilled in the
art will therefore select a mass spectrometer having a resolution
appropriate for the analyte (s) to be detected.
[0186] Mass spectrometers can resolve ions with small mass
differences and measure the mass of ions with a high degree of
accuracy. Therefore, analytes of similar masses can be used
together in the same experiment since the mass spectrometer can
differentiate the mass of even closely related molecules. The high
degree of resolution and mass accuracy achieved using mass
spectrometry methods allows the use of large sets of analytes
because they can be distinguished from each other.
[0187] Additional mass spectrometry methods are well known in the
art (see Burlingame et al. Anal. Chem. 70: 647R-716R (1998); Kinter
and Sherman, New York (2000)). Exemplary descriptions of mass
spectrometry methods for detecting metabolic analytes include Chace
D H, Hillman S L, Van Hove J L K, Naylor E W. Clin Chem 1997 ; 43:
210613; Rashed M S, Bucknall M P, Little D, et al. Clin Chem 1997 ;
43: 112941; Matern D, Strauss A W, Hillman S L, Mayatepek E,
Millington D S, Trefz F K. Pediatr Res 1999 : 46: 459, and
Millington D S, Kodo N, Terada N, Roe D, Chace D H. International
Journal of Mass Spectrometry and ion Processes 1991; 111:
21128.
[0188] From the mass spectrometry analysis, one of skill in the art
is able to determine the amount of product and internal standard in
each of the enzyme assays. As noted above, each enzyme product and
internal standard may be assayed individually or, in a preferred
embodiment, following the initial assay reaction, the products and
internal standards for the five enzyme reactions are combined and
assayed simultaneously by mass spectrometry. This highlights one of
the advantages of the present invention. That is, the invention
provides a method for the determination of multiple enzyme
activities utilizing a single read out assay (i.e., tandem mass
spectrometry). Thus, the invention is well adapted to be used to
perform high throughput screening of multiple DBS samples for the
five enzyme activities described herein, and is therefore useful
for performing large scale newborn screening for the lysosomal
storage diseases associated with ASM, ABG, GAA, GLA, and GALC
enzyme activity.
[0189] Once the amount of product and internal standard has been
determined by mass spectrometry, these values can be used to
calculate enzyme activity. Enzyme activity can be determined by
applying the product and internal standard values to the following
equation:
Enzyme activity (.mu.mol/hr/L)=(P/IS)*IS/RF/T/V
[0190] Wherein (P/IS) is the ratio of the amount of product to the
amount of internal standard as determined by mass spectrometry; RF
is the response factor ratio; IS is the amount of internal standard
in the enzyme assay mix; T is the incubation time; and V is the
volume of blood used in the assay reaction. If the internal
standard is a stable isotope analog of the molecule of interest
(i.e., analog of the enzyme product), then there is no need to
calculate the response factor ratio. Accordingly, it is not
necessary to calculate the RF for the GLA and GAA enzyme assays.
Thus, for calculating the activities of GLA and GAA, the RF value
is equal to 1. For the ASM, GALC, and ABG assays however, the
internal standards used are structural analogs, having similar, but
not identical structures to the products. As a result they may have
a slightly different ionization efficiency relative to the product.
The RF is determined by constructing a calibration curve to reflect
the linear relationship between spiked-in product concentrations in
the solution and the area ratio of product:internal standard
measured on the mass spectrometer. The slope of the resulting curve
represents the RF of product to internal standard. As shown in FIG.
2A-C the RF for the ASM assay is 0.9651, the RF for the ABG assay
is 1.2341, and the RF for the GALC assay is 1.1579.
Screening and Therapeutic Applications
[0191] The present invention provides methods for screening for one
or more of Gaucher, Niemann-Pick A/B, Pompe, Fabry, and Krabbe
diseases by determining the activity of the ABG, ASM, GAA, GLA, and
GALC enzymes, respectively. It will be understood by one of skill
in the art that the absolute level of activity for any of the above
enzymes will vary from individual to individual. Accordingly, to
facilitate the screening for decreased enzyme activity in an
individual, individual samples must be compared to a population of
presumptively normal samples assayed under the same conditions. One
aspect of the invention provides a method for performing newborn
screening assays. In this context, the activity of one or more of
the lysosomal enzymes described herein is compared with the daily
mean enzyme activities for that enzyme from all the newborn samples
assayed on a given day. For example, in a typical setting (e.g., a
newborn screening lab), newborn screening assays are performed on
between 200 and 1000 individual samples in a given day. The level
of activity of each enzyme assayed is compared to the mean of that
enzyme activity from all the other samples (which are presumptively
normal). Therefore, if 200 DBS samples from 200 individuals are
tested for the five enzyme activities described herein the enzyme
activity from one is compared against the mean of the 199 other
presumptively normal samples. Decreased enzyme activity is
identified if the enzyme activity for a particular enzyme is less
than 30% of the daily mean for that enzyme, preferably less than
25%, preferably less than 20%, preferably less than 15%, preferably
less than 10%, and more preferably less than 8% of the daily mean.
In particular, for Fabry disease, a patient is identified as having
decreased GLA enzyme activity (and potentially as having Fabry
disease) if the GLA enzyme activity measured in that patient is
less than 10-20% of the mean GLA activity in all the other patient
samples assayed on the same day. For the four other enzyme
activities described herein, decreased enzyme activity is
identified if the enzyme activity is at least less than 30% of the
mean activity of the same enzyme from all the other samples assayed
on the same day. This method can accordingly be used as a first
step in the diagnosis of any one or all of the five lysosomal
storage diseases described herein. This identification of enzyme
deficiency is useful to identify patients that may benefit from
enzyme replacement therapies or similar treatments.
[0192] The methods of the invention, in addition to being used for
newborn screening, can also be used to screen other populations,
such as adolescent or adult individuals identified as high risk for
developing lysosomal storage disease.
[0193] In one embodiment, following the identification of a sample
that has an enzyme activity at least less than 30% of the daily
mean, as described above, the individual from which the DBS sample
was obtained may be screened further to confirm disease diagnosis.
For example, The DBS from which the 3.2 mm punch was taken can be
re-punched to obtain additional 3.2 mm punches, that are then
re-analyzed in the same enzyme activity assays described above. In
addition, or alternatively, the a new DBS can be prepared from the
individual from which the first DBS was obtained. For example, for
a newborn infant that shows low activity of one or more of the
enzymes described herein, blood can be re-drawn to prepare a second
DBS that is then used according to the methods of the invention to
re-assess enzyme activity.
[0194] In addition, or alternatively, after a sample is identified
as having an enzyme activity at least less than 30% of the daily
mean, additional clinical diagnostic methods, such as genotyping,
or enzyme assay in another sample type (e.g., GAA assay in
lymphocytes for Pompe disease) can be used to confirm a diagnosis
of one or more of the lysosomal storage diseases described herein.
Confirmatory clinical assays are known in the art and can be found,
for example, on the world wide web at genetests.org.
[0195] Alternatively, decreased enzyme activity can be determined
by making a comparison between the enzyme activity in a test
patient and the enzyme activity in a patient known to be free of
the disease. In this example, decreased enzyme activity is
identified if the enzyme activity in the test patient is less than
the control enzyme activity by a statistically significant amount
as determined by statistical analysis known to those of skill in
the art (e.g., student's t-test where P<0.05 is
significant).
[0196] A further aspect of the invention provides a method for
selecting a treatment regimen and monitoring treatment of one or
more of the lysosomal storage diseases described herein. In one
aspect, the invention provides a method for selecting a treatment
regimen for a patient based on the activity of the ABG, ASM, GAA,
GALC, or GLA enzymes. For example, by determining the activity of
one or more of these enzymes in an individual, a physician or other
health care professional can use that information to make decisions
as to the proper treatment (e.g., enzyme replacement therapy or
bone marrow transplantation) for the individual in accordance with
the current standard of care for the particular disease. Methods
for treatment of several of the lysosomal storage diseases are
known in the art (see, e.g., Grabowski and Hopkin 2003, Annu. Rev.
Genomics, 4:403-36; Kaye 2001 Curr. Treat. Options Neurol., 3:
249-56; and Schiffmann and Brady 2002 Drugs, 62: 733-42).
[0197] The methods of the instant invention can also be used to
monitor treatment in a patient. For example, after starting a
patient on a treatment program for deficiency in one or more of the
ABG, ASM, GAA, GALC, or GLA enzymes, enzyme activity can be assayed
at relevant time points, as determined by the patient's physician,
to determine whether enzyme activity levels are higher than prior
to the commencement of treatment, and thus, monitor the efficacy of
a particular treatment. Based on the level of enzyme activity
measured during the treatment regimen, the patient's physician can
make adjustment to the treatment (e.g., by varying the dosage of
medications, or modifying therapy).
[0198] All journal articles, references and patent citations
provided above and parentheses or otherwise, whether previously
stated or not are incorporated herein by reference.
[0199] It is understood that modifications that do not
substantially affect the function of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly the following examples are
intended to illustrate but not limit the present invention.
EXAMPLES
Example 1
Synthesis of GLA and GAA Substrate and Internal Standards
[0200] The methods used to synthesize the internal standards and
substrates for the GLA and GAA assays are essentially the same as
those described by Li et al. (2004, Clinical Chemistry 50:1785-96).
The methodology is briefly summarized as follows:
[0201] General Methods. Thin layer chromatography (TLC) is carried
out on silica plates (Merck, 60F.sub.254), and flash column
chromatography is carried out with silica gel (Merck, 230-400
Mesh). Preparative HPLC is carried out can be monitored with a UV
detector (.quadrature.=254 nm). Dry CH.sub.2Cl.sub.2 can be
obtained by distillation from CaH.sub.2 under Ar, and other dry
solvents are obtained from Aldrich (Sure-Seal). As noted below,
reactions are carried out in a round bottom flask (RBF) or in a
vial with a Teflon septum-lined screw cap. .sub.1H-NMR spectra are
obtained on a Bruker DPX200 spectrometer (200 MHz) unless otherwise
noted.
[0202] Acetic acid 4-nitro-phenyl ester (1): Acetic anhydride (50
ml) is added to a solution of 4-nitrophenol (5.56 g, 40 mmol) in
dry pyridine (50 ml). The solution is stirred at ambient
temperature for 2 hr and then at 70.degree. C. overnight with a
reflux condenser under Ar. The mixture is poured onto ice, and a
white precipitate is formed after standing for several hours. Water
(400 ml) is added, and the white solid is collected by vacuum
filtration and dried in vacuo to yield a white solid (5.1 g, 70%).
ESI-MS (M+H).sup.+: 182.2. .sub.1H-NMR (CDCl.sub.3) .delta. 8.30
(2H, d, J=9.0 Hz, NO.sub.2CCH), 7.31 (2H, d, J=9.0 Hz, OCCH), 2.25
(3H, s, CH.sub.3).
[0203] Acetic acid 4-acryloylamino-phenyl ester (2): H.sub.2 is
bubbled through a solution of 1 (280 mg, 1.54 mmol) and 10 mg of
10% Pd on carbon in 20 ml of MeOH for 1 hr. The catalyst is removed
by filtration. Triethylamine (410 .mu.l, 3.08 mmol) is added to the
filtrate which was chilled on ice, then acryloyl chloride (250
.mu.l, 3.08 mmol, Aldrich) in 10 ml of dry CH.sub.2Cl.sub.2 is
added dropwise with stirring over 0.5 hr under Ar. The reaction is
then allowed to return to ambient temperature, followed by 2 hr of
stirring. Anion exchange resin (Bio-Rad, AG-MP1, OH.) (4
equivalents based on acryloyl chloride) is added, the mixture is
filtered, and the filtrate is treated with sufficient cation
exchange resin (Dowex, 50 W.times.8, H.sub.+) to bring the mixture
to neutrality (moist pH paper). The resin is removed by filtration,
and the solvent is removed by rotary evaporation to yield an
off-white solid (268 mg, 85%). ESI-MS (M+H).sup.+: 206.1.
.sub.1H-NMR (acetone-d.sub.6) .delta. 9.15 (1H, br, NH), 7.78 (2H,
d, J=9.0 Hz, NHCCH), 7.08 (2H, d, J=9.0 Hz, NHCCHCH),
6.55.about.6.37 (2H, m, COCHCHH (anti to each other)), 5.75 (1H,
dd, J=9.8 and 2.2 Hz, COCHCHH (syn to COCH)), 2.25 (3H, s,
CH.sub.3).
[0204] N-(4-Hydroxy-phenyl)-acrylamide (3). To 2 (200 mg, 0.98
mmol) in 1.5 ml of MeOH in a 5 ml screw-capped vial is added 1.0 ml
of 0.5 M of sodium methoxide in MeOH. The mixture is stirred at
ambient temperature, and the reaction is complete in 10 min. The
mixture is neutralized by addition of cation exchange resin (Dowex,
50 W.times.8, H.sub.+) (moist pH paper). The resin is removed by
filtration and washed with MeOH. The combined filtrate and wash is
concentrated by rotary evaporation to yield an off-white solid (152
mg, 95%), ESI-MS (M+H).sup.+: 164.2. .sub.1H-NMR (acetone-d.sub.6),
.delta. 9.15 (1H, br, NH), 7.59 (2H, d, J=9.0 Hz, NHCCH), 6.82 (2H,
d, J=9.0 Hz, NHCCHCH), 6.52.about.6.35 (2H, m, COCHCHH (anti to
each other)), 5.70 (1H, dd, J=9.8 and 2.2 Hz, COCHCHH (syn to
COCH)).
[0205] 4-Acrylaminophenyl .alpha.-D-galactopyranoside (4): The
compound is prepared as described for 2 using 1 g of 4-nitrophenyl
.alpha.-D-galactopyranoside (Sigma) to obtain 0.94 g (87%) of 4;
ESI-MS (M+H).sup.+: 326.3. .sub.1H-NMR (D.sub.2O) .delta. 7.43 (2H,
d, J=9.0 Hz, NHCCH), 7.16 (2H, d, J=9.0 Hz, NHCCHCH),
6.47.about.6.24 (2H, m, COCHCHH (anti to each other)), 5.81 (1H,
dd, J=9.8 and 2.2 Hz, COCHCHH (syn to COCH)), 5.52 (1H, d, J=3.4
Hz, H-1), 4.01.about.3.86 (4H, m, H-2,3,4,5), 3.63.about.3.60 (2H,
d, J=6.2 Hz, H-6, 6').
[0206] 4-Acrylaminophenyl .alpha.-D-glucopyranoside (5): The
compound is prepared as described for 4, using 1 g of 4-nitrophenyl
.alpha.-D-glucopyranoside (Sigma) to obtain 0.97 g (90%) of 5;
ESI-MS (M+H).sup.+: 326.3. .sub.1H-NMR (D.sub.2O) .delta. 7.43 (2H,
d, J=9.0 Hz, NHCCH), 7.16 (2H, d, J=9.0 Hz, NHCCHCH),
6.47.about.6.24 (2H, m, COCHCHH (anti to each other)), 5.81 (1H,
dd, J=9.8 and 2.2 Hz, COCHCHH (syn to COCH)), 5.60 (1H, d, J=3.6
Hz, 1-H), 3.94.about.3.66 (5H, m, H-2,3,5,6,6'), 3.48 (1H, t, J=9.2
Hz, H-4). N-(6-Amino-hexyl)-benzamide (6): To a stirred solution of
1,6-diaminohexane (10.0 g, 86.3 mmol, Aldrich) in 30 ml dry
CH.sub.2Cl.sub.2 is added benzoyl chloride (1 ml, 8.6 mmol) in 300
ml dry CH.sub.2Cl.sub.2 dropwise at ambient temperature under Ar. A
white precipitate formed as the reaction proceeded, and the mixture
is stirred at ambient temperature for 5 hr after the addition is
completed. Aqueous NaOH (3 ml of 4 N) is added to dissolve the
precipitate. The reaction mixture is washed with water (3.times.60
ml), dried over Na.sub.2SO.sub.4 and solvent is removed by rotary
evaporation. The oil was purified by flash chromatograph on silica
eluting (Merck 230-400 Mesh) with 30:1 acetone/concentrated
ammonium hydroxide to yield product as a yellowish oil (0.75 g,
32%). R.sub.f=0.43 (TLC, same solvent). ESI-MS (M+H).sup.+: 221.3.
.sub.1H-NMR (acetone-d.sub.6) .delta. 7.82.about.7.75 (2H, m,
COCCH), 7.55.about.7.35 (3H, m, COCHCHCH), 6.35 (1H, br, NH), 3.42
(2H, dt, J=5.8 and 6.8 Hz, CONHCH.sub.2), 3.20 (1H, t, J=6.8 Hz,
NH.sub.2CH.sub.2), 1.90-1.32 (8H, m,
NHCH.sub.2(CH.sub.2).sub.4).
[0207] N-(6-Amino-hexyl)-d.sub.5-benzamide (7): The compound is
prepared as for 6 using d.sub.5-benzoyl chloride (Cambridge Isotope
Inc.). ESI-MS (M+H).sup.+: 226.3. .sub.1H-NMR (acetone-d.sub.6)
.delta. 6.35 (1H, br, NH), 3.42 (2H, dt, J=5.8 and 6.8 Hz,
CONHCH.sub.2), 3.20 (1H, t, J=6.8 Hz, NH.sub.2CH.sub.2), 1.90-1.32
(8H, m, NHCH.sub.2(CH.sub.2).sub.4).
[0208] N-(7-Amino-heptyl)-benzamide (8): The compound is prepared
as for 6 using 1,7-diaminoheptane (Aldrich). ESI-MS (M+H).sup.+:
235.3. .sub.1H-NMR (acetone-d.sub.6) .delta. 7.82.about.7.75 (2H,
m, COCCH), 7.55.about.7.35 (3H, m, COCHCHCH), 6.35 (1H, br, NH),
3.42 (2H, dt, J=5.8 and 6.8 Hz, CONHCH.sub.2), 3.20 (1H, t, J=6.8
Hz, NH.sub.2CH.sub.2), 1.90-1.32 (10H, m,
NHCH.sub.2(CH.sub.2).sub.5).
[0209] N-(7-Amino-heptyl)-d.sub.5-benzamide (9): The compound is
prepared as for 6 using d.sub.5-benzoyl chloride. ESI-MS
(M+H).sup.+: 240.3. .sub.1H-NMR (acetone-d.sub.6) .delta.6.35 (1H,
br, NH), 3.42 (2H, dt, J=5.8 and 6.8 Hz, CONHCH.sub.2), 3.20 (1H,
t, J=6.8 Hz, NH.sub.2CH.sub.2), 1.90-1.32 (10H, m,
NHCH.sub.2(CH.sub.2).sub.5).
[0210]
(6-Benzoylamino-hexyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tetr-
ahydro-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid
tert-butyl ester (GLA-S): Compound 4 (0.88 g, 2.7 mmol) and 6 (0.71
g, 3.2 mmol) in a solution of isopropanol (30 ml) and H.sub.2O (4
ml) is stirred at 65.degree. C. (oil bath) in a capped 100 ml RBF
for 48 hrs. TLC on silica shows that at least 85% of 4 is converted
to the Michael addition product (R.sub.f=0, 30:1
acetone-concentrated ammonium hydroxide). The reaction is allowed
to cool to ambient temperature, followed by the addition of
powdered K.sub.2CO.sub.3 (0.44 g, 3.2 mmol) and
di-tert-butylcarbonate (0.84 mg, 3.8 mmol, Aldrich). The mixture is
stirred at ambient temperature for 3 hr. TLC should show at least
80% of Michael addition product was converted to the desired
product (R.sub.f=0.17, 10:1 acetone-concentrated ammonium
hydroxide). The solid is collected by vacuum filtration and is
washed with 30 ml of MeOH. The filtrates are combined, and solvent
is removed by rotary evaporation to give an oily residue. MeOH (6.5
ml) is added to dissolve the residue, and the pH is adjusted to
.about.3-4 (moist pH paper) by addition of trifluoroacetic acid
with chilling on ice. The desired product is purified by 10 runs of
preparative HPLC: 50% MeOH in H.sub.2O, at a flow rate of 6 ml/min;
t.sub.R=27 min. Product fractions are pooled, and most of the
solvent is removed by rotary evaporation at ambient temperature.
The remaining solvent is removed by lyophilization, and the
resulting residue is dissolved in 20 ml of MeOH. Solvent is removed
by rotary evaporation, and the oily residue is dried in vacuum to
give a white solid (1.1 g, 63%). ESI-MS (M+H).sup.+: 646.6;
.sub.1H-NMR (1:2.5 D.sub.2O/acetone-d.sub.6) .delta.
7.80.about.7.75 (2H, m, COCCH), 7.55.about.7.35 (5H, m, COCHCHCH
and NHCCH), 7.05 (2H, d, J=9.0 Hz, NHCCHCH), 5.39 (1H, d, J=3.4 Hz,
H-1), 4.00.about.3.57 (6H, m, H-2,3,4,5,6,6'), 3.51 (2H, t, J=6.8
Hz, COCH.sub.2CH.sub.2), 3.30 (2H, t, J=7.0 Hz, CONHCH.sub.2), 3.16
(2H, t, J=7.0 Hz, CONH(CH.sub.2).sub.5CH.sub.2), 2.55 (2H, t, J=6.4
Hz, COCH.sub.2), 1.70-1.20 (17H, m, O-tert-C4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.4).
[0211]
(7-Benzoylamino-heptyl)-{2-[4-(3,4,5-trihydroxy-6-hydroxymethyl-tet-
rahydro-pyran-2-yloxy)-phenylcarbamoyl]-ethyl}-carbamic acid
tert-butyl ester (GAA-S): The compound is prepared as for GLA-S
starting from 0.63 g of 5 and 0.55 g of 8. HPLC t.sub.R=40 min.
Yield 60%. ESI-MS (M+H).sup.+: 660.6. .sub.1H-NMR (1:2.5
D.sub.2O/acetone-d.sub.6) .delta. 7.80.about.7.75 (2H, m, COCCH),
7.55.about.7.35 (5H, m, COCHCHCH and NHCCH), 7.05 (2H, d, J=9.0 Hz,
NHCCHCH), 5.39 (1H, d, J=3.6 Hz, H-1), 3.90.about.3.57 (5H, m,
H-2,3,5,6,6'), 3.51 (2H, t, J=6.8 Hz, COCH.sub.2CH.sub.2), 3.45
(1H, t, j=9.6 Hz, H-4), 3.30 (2H, t, J=7.0 Hz, CONHCH.sub.2), 3.20
(2H, t, J=7.0 Hz, CONH(CH.sub.2).sub.5CH.sub.2), 2.65 (2H, t, J=6.4
Hz, COCH.sub.2), 1.70-1.20 (19H, m, O-tert-C.sub.4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.5).
[0212]
(6-d.sub.5-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl-
]-carbamic acid tertbutyl ester (GLA-IS): Compound 3, 10 mg, 0.06
mmol) and 7 (21 mg, 0.09 mmol) are dissolved in 1.5 ml of
isopropanol in a screw capped vial. The mixture is stirred at
65.degree. C. overnight. TLC should show that more than 85% of 3
has been converted into the Michael addition product (R.sub.f=0.22,
30:1 acetone/concentrated ammonium hydroxide solution). After the
reaction is cooled to ambient temperature, K.sub.2CO.sub.3 (10 mg,
0.07 mmol) and di-tert-butylcarbonate (16 mg, 0.07 mmol) are added,
and the mixture is stirred for 2 hr at the same temperature. TLC
should show that all the Michael addition product has been
converted into the desired product (R.sub.f=0.93, 30:1
acetone/concentrated ammonium hydroxide solution). The final
product is purified by HPLC (solvent A, H.sub.2O; solvent B, MeOH;
Gradient 0-30 min, 30-60% B; 30-70 min, 60-85%; flow rate 6 ml/min;
t.sub.R=45.4 min) to yield 22 mg of desired product (yield 75%).
ESI-MS (M+H).sup.+: 489.5. .sub.1H-NMR (CDCl.sub.3) .delta. 8.78
and 8.48 (2H, br, NH), 7.35 (2H, d, J=9.0 Hz, NHCCH), 6.91 (1H, br,
OH), 6.77 (2H, d, J=9.0 Hz, HOCCH), 3.47 (2H, t, J=6.2 Hz,
COCH.sub.2CH.sub.2), 3.34 (2H, dt, J=5.8, 6.8 Hz, CONHCH.sub.2),
3.09 (2H, t, J=6.8 Hz, CONH(CH.sub.2).sub.5CH.sub.2), 2.55 (2H, t,
J=6.2 Hz, COCH.sub.2), 1.70-1.10 (17H, m, O-tert-C.sub.4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.4).
[0213]
(6-Benzoylamino-hexyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carbam-
ic acid tertbutyl ester (GLA-P): The compound is prepared as for
GLA-IS using 10.4 mg of 6. HPLC t.sub.R=45.3 min. Yield 72.1%.
ESI-MS (M+H).sup.+: 484.5. .sub.1H-NMR (CDCl.sub.3) .delta. 8.78
and 8.48 (2H, br, NH), 7.84.about.7.79 (2H, m, COCCH),
7.55.about.7.35 (5H, m, NHCCH, COCHCHCH), 6.91 (1H, br, OH), 6.82
(2H, d, J=9.0 Hz, HOCCH), 3.57 (2H, t, J=6.2 Hz,
COCH.sub.2CH.sub.2), 3.42 (2H, dt, J=5.8, 6.8 Hz, CONHCH.sub.2),
3.20 (2H, t, J=6.8 Hz, CONH(CH.sub.2).sub.5CH.sub.2), 2.64 (2H, t,
J=6.2 Hz, COCH.sub.2), 1.70-1.10 (17H, m, O-tert-C4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.4).
[0214]
(7-d.sub.5-Benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethy-
l]-carbamic acid tertbutyl ester (GAA-IS): The compound is prepared
as for GLA-IS using 22 mg of 9. HPLC t.sub.R=47.0 min. Yield 70.5%.
ESI-MS (M+H).sup.+: 503.5. .sub.1H-NMR (CDCl.sub.3) .delta. 8.78
and 8.48 (2H, br, NH), 7.35 (2H, d, J=9.0 Hz, NHCCH), 6.91 (1H, br,
OH), 6.77 (2H, d, J=9.0 Hz, HOCCH), 3.47 (2H, t, J=6.2 Hz,
COCH.sub.2CH.sub.2), 3.34 (2H, dt, J=5.8, 6.8 Hz, CONHCH.sub.2),
3.09 (2H, t, J=6.8 Hz, CONH(CH.sub.2).sub.6CH.sub.2), 2.55 (2H, t,
J=6.2 Hz, COCH.sub.2), 1.70-1.20 (19H, m, O-tert-C.sub.4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.5).
[0215]
(7-Benzoylamino-heptyl)-[2-(4-hydroxy-phenylcarbamoyl)-ethyl]-carba-
mic acid tertbutyl ester (GAA-P): The compound is prepared as for
GAA-IS using 11 mg of 8. HPLC t.sub.R=46.8 min. Yield 75.5%. ESI-MS
(M+H).sup.+: 498.5. .sub.1H-NMR (CDCl.sub.3) .delta. 8.78 and 8.48
(2H, br, NH), 7.84.about.7.79 (2H, m, COCCH), 7.55.about.7.35 (5H,
m, NHCCH, COCHCHCH), 6.91 (1H, br, OH), 6.82 (2H, d, J=9.0 Hz,
HOCCH), 3.57 (2H, t, J=6.2 Hz, COCH.sub.2CH.sub.2), 3.42 (2H, dt,
J=5.8, 6.8 Hz, CONHCH.sub.2), 3.20 (2H, t, J=6.8 Hz,
CONH(CH.sub.2).sub.5CH.sub.2), 2.64 (2H, t, J=6.2 Hz, COCH.sub.2),
1.70-1.10 (19H, m, O-tert-C.sub.4H.sub.9 and
NHCH.sub.2(CH.sub.2).sub.5).
Example 2
Enzyme Screening Assays
[0216] The following example describes the specific protocol used
to perform the enzyme assays (ASM, first ABG, GAA, GLA, and GALC
assay) of the present invention.
[0217] Prior to beginning the DBS extraction, the assay mixtures
should be warmed to room temperature and vortexed briefly. If
needed, the ABG and GALC assay mixtures can be warmed in hot
(40-45.degree. C.) water for 5 minutes if solutions are not clear
(the GALC cocktail may remain slightly cloudy).
DBS Extraction Method
[0218] DBS were obtained from adult, adolescent, and newborn
patients that had been previously diagnosed as having one of the
lysosomal storage diseases described herein based on other
diagnostic tests (referred to generally as "test samples"). Test
samples were obtained from patients confirmed as having one of ASM,
ABG, GAA, GLA, GALC, or MPS1 deficiencies. A 3 mm hole punch was
used to punch smaller samples from the DBS. It is possible to punch
6 or 7 times from one DBS, however, punches should be taken
preferably from the perimeter of the DBS, not the center. The hole
punch was rinsed with 70% isopropanol (or 70% ethanol) and dried
prior to use to prevent contamination. Each sample to be tested
used two 3 mm punches (one punch in one well of a 96 well plate for
the 1 hour incubation with the sodium phosphate buffer (i.e., the
punch to be extracted) and one punch for the overnight incubation
with the Krabbe (GALC) cocktail). In between samples, the punch was
used to punch blank paper 3 to 4 times to reduce sample carryover.
Control, or "normal" DBS were prepared from blood samples obtained
from a commercial source (e.g., ProMedDX, Norton, Mass.).
[0219] One 3 mm punch of dried blood (for each sample to be
assayed) was added to a well of a 96 well polypropylene plate. 70
.mu.L of 20 mM Sodium phosphate buffer, pH 7.1 as then added to
each well containing the DBS punch. The plate was then covered and
sealed with aluminum plate sealing film, and the plate was examined
to ensure that all of the DBS punches were in contact with the
extraction buffer (If necessary, the plates were centrifuged for 1
minute at 4000 rpm; all centrifugation steps of this method are
performed at 25.degree. C.). The plates were incubated for 1 hour
at 37.degree. C. with orbital shaking.
Assay Preparation
[0220] Following incubation the plates were removed from the
shaker. The assay mixes were added to the appropriate wells of a
fresh 96 well plate, followed by the DBS extract in the following
patterns.
To a 96 well polypropylene plate was added: [0221] 1) 15 .mu.L ASM
assay mix+10 .mu.L DBS extract [0222] 2) 15 .mu.L ABG assay mix+10
.mu.L DBS extract [0223] 3) 15 .mu.L GAA assay mix+10 .mu.L DBS
extract [0224] 4) 15 .mu.L GLA assay mix+10 .mu.L DBS extract
[0225] 5) one punch of 3 mm dried blood spot per well (for each
sample to be assayed)+30 .mu.L GALC assay mix.
[0226] The plates were sealed with aluminum plate sealing film and
incubated at 37.degree. C. with orbital shaking (approximately 225
rpm) for 20-24 hours.
[0227] Following incubation, the plates were centrifuged at 4000
rpm for 1 minute. Each assay reaction was quenched by adding a 100
.mu.L mixture of 1:1 Ethyl Acetate (EA):MeOH to each well. The
assay mix was then aspirated and dispensed several times to ensure
even mixture. The 5 extract/assay mix mixtures associated with one
sample were then combined-into a single well of a fresh deep well
plate. Into each well of the deep well plate containing extracts,
400 .mu.L EA and 400 .mu.L H.sub.2O were added, in that order. The
samples were aspirated and dispensed vigorously several times in
order to mix. The deep well plate was then sealed with aluminum
plate sealing tape and centrifuged for 5 minutes at 4000 rpm at
25.degree. C. to create a phase separation.
[0228] Approximately 300 .mu.L (the majority of the top phase) was
then removed from the top layer (the organic phase) and transferred
to a new deepwell microtiter plate and dried under a stream of
nitrogen (25 PSI) in a 96 well drying apparatus. Where needed, the
plate was heated to 25.degree. C. to speed drying.
[0229] A silica filter plate was prepared by adding 100 mg of
silica per well to a 96 well filter plate (Innovative Microplate
Catalog #F2005). The vacuum source was protected with the presence
of a vacuum line filter. The vacuum was set for a maximum of 5 inch
Hg. The dispense speed of a pipetor was set to gentle and the
silica was carefully washed by adding a 250 .mu.L mixture of EA and
MeOH, mixed at a ratio of 19:1. Vacuum pressure was applied and the
wash was collected as waste. (Pipetting too vigorously or having
the vacuum rate set too high can cause channeling in the silica,
which should be avoided.) The collection plate was then replaced
with clean deep well plate.
[0230] The assay samples were reconstituted by adding a 100 .mu.L
mixture of EA and MeOH, mixed at a ratio of 19:1, to each well. The
plates were covered with aluminum plate sealing film and shaken on
a microtiter plate shaker (speed 7=.about.200 rpm) for 5 minutes to
resuspend. With the pipette set to lowest dispense speed, the
samples were added to the corresponding wells of the filter plate
in the vacuum manifold (with the clean, empty deep well plate
underneath). Vacuum was applied and the plates were visually
inspected to insure that all liquid has passed through. The vacuum
was turned off and a 400 .mu.L mixture of EA and MeOH, mixed at a
ratio of 19:1, was added, and vacuum was applied to collect the
eluant. An additional 400 .mu.L of a 19:1 EA:MeOH was added, vacuum
was applied, and the eluted sample was collected in the same well.
The EA:MeOH wash was repeated 2 more times for a total wash volume
of 1600 .mu.L.
[0231] The resulting eluted samples were dried under a stream of
N.sub.2 (drying manifold set to 25.degree. C.). In the event that
the samples could not be analyzed immediately by mass spectrometry,
the plates were sealed with aluminum plate sealing film and stored
at -20.degree. C.
[0232] Prior to analysis by mass spectrometry, the plates were
warmed to room temperature and each well was reconstituted with 200
.mu.L of reconstitution buffer (a mixture of acetonitrile and water
with addition of formic acid: 80% Acetonitrile with 20% water and
0.2% Formic Acid). The plate was sealed with aluminum plate sealing
film and shaken on a microtiter plate shaker (speed 7=.about.200
rpm) for 5 minutes to resuspend. After resuspension, the plate
sealing film was removed and replaced with aluminum foil.
[0233] The samples were then analyzed by tandem mass spectrometry
using methods known in to those of skill in the art, and summarized
in brief below.
Mass Spectrometry Analysis
[0234] Data were obtained on an API 4000 triple quadrupole mass
spectrometer interfaced with PAL autosampler and Agilent 1100 HPLC
system. The electrospray source was operated in positive mode, and
the ions were detected in multi-reaction monitoring (MRM) mode. In
the MRM mode, a selected product ion was passed through the last
(Q3) mass analyzer, whereas the first mass analyzer (Q1) was fixed
to transmit the parent ion given rise to the selected product ion.
Data were acquired and analyzed by Analyst 3.5. The instrument was
adjusted to give an optimized response for all analytes (detailed
settings for a hypothetical experiment are given in Appendix 1).
Samples were introduced by autosampler and pumped by an Agilent
1100 HPLC system. The mobile phase was composed of 80/20
acetonitrile/Water with 0.1% formic acid. The flow rate was set at
200 .mu.l/min. The injector port was flushed once with 100 .mu.l
methanol/0.1% formic acid and once with 100 .mu.l 50/50
methanol/isopropanol before each injection. The amount of product
was calculated from the ion abundance ratio of the product to the
internal standard for a sample minus that of a blank, multiplied by
the amount of added internal standard and divided by the response
factor ratio of product to internal standard. The enzyme activity
in units of .mu.mol/h/.mu.L blood was calculated assuming that 10
.mu.L of DBS extraction solution contained one seventh of the total
blood contained in a 3.2 mm DBS (2.8 .mu.L of blood), i.e., 10
.mu.L DBS extract contained 0.4 .mu.L of blood.
Results
[0235] The results of the foregoing experiments are shown in FIGS.
3-7. As can be seen from the figures, the methods of the invention
are able to discriminate between normal enzyme activity vs.
abnormal enzyme activity in patients having one of the five
lysosomal storage diseases described herein. FIGS. 3-7 also
demonstrate that the enzyme assays are specific for the target
enzyme. The "Other LSD" data column represents the level of enzyme
activity for a given assay as measured in all the other test
samples, presumed to be normal for that particular enzyme activity.
The target enzyme activity was shown to be substantially lower in
patients with the target disease than control samples and samples
from patients diagnosed as having an LSD other than the target
disease. Both control samples and "other LSD" patient samples
revealed enzyme activity levels that are in the normal range.
Example 3
Second ABG Assay
[0236] The second ABG DBS assay measures the ABG-catalyzed cleavage
of the fluorogenic substrate 4-MU-.beta.-Glu by detecting the
product 4-MU in a fluorometer.
Reagent Preparation
[0237] The following preparations are adequate for 20 plates. If
possible, reagents should be made in batches large enough to cover
an entire study. This is particularly important for the Buffered
Extractant.
Substrate Stock Solution, 1 M
[0238] 2.29 mL DMSO was added to 774.26 mg of 4-MU-.beta.-Glu in a
15 mL screw cap tube. The mixture was thawed at RT until dissolved
completely. The tube may be thawed briefly in a 37.degree. C. if
necessary. The solution was well vortexed and then 110 .mu.L
aliquots were placed in 1.5 mL microtubes. Aliquots of this
solution can be thawed and refrozen several times, but should not
be left thawed for more than two hours, and should be protected
from light and moisture.
Buffered Extractant: 0.30 M Citrate Phosphate with 1% Sodium
Taurodeoxycholate and 1% Triton X-100, pH 5.2
[0239] A solution of 0.15 M citric acid was prepared by dissolving
3.78 g of citric acid monohydrate in 120 mL of pure water.
[0240] A solution of 0.30 M sodium phosphate was prepared by
dissolving 5.12 g of anhydrous dibasic sodium phosphate in 120 mL
of water.
[0241] A 0.30 M citrate phosphate buffer was prepared by combining
93 mL of 0.15 M citric acid and 107 mL of 0.3 M sodium
phosphate.
[0242] A 10% triton X-100 solution was prepared by measuring 10 mL
of Triton X-100 and adding 0.30 M citrate phosphate buffer to a
final volume of 100 mL.
[0243] The buffered extractant was prepared by adding 10 mL of 10%
Triton X-100 to 1.00 g sodium taurodeoxycholate. 0.30 M citrate
phosphate buffer was then added to a total volume of 100 mL. The pH
was adjusted to pH 5.2 with sodium hydroxide or hydrochloric acid
as necessary, and the solution was filter sterilized using a 0.22
.mu.m filter unit.
CBE Stock Solution
[0244] To prepare the 0.26 M CBE stock solution, 8.30 mg conduritol
B epoxide was added to 200 .mu.L of DMSO.
4-MU Stock Solution, 25 mM. Used for Standard Curve (Adequate for
Over 200 Plates)
[0245] To prepare the 4-MU stock solution, 5 mg of 4-MU was added
into a microcentrifuge or 15 mL conical tube, and dissolved in 1.14
mL DMSO. Aliquots of this solution can be thawed and refrozen
several times, but should not be left thawed for more than two
hours, and should be protected from light and moisture.
Stop Buffer: 0.5 M EDTA, pH 11.3 to 12.0
[0246] To prepare the stop buffer, 5.20 g EDTA was dissolved in
approximately 20 mL of water. The pH was adjusted to be in the
range of 11.3 and 12.0. The final volume was adjusted to 25 mL with
water.
[0247] Assay Procedure
Preparation of Test Samples:
[0248] DBS were prepared from 153 control subjects and 43 Gaucher
disease patients using standard procedures (see, Appendix II). DBS
were stored at -20.degree. C. or below in tightly sealed plastic
bags, and protected from moisture and condensation at all
times.
[0249] To obtain DBS punches, a paper punch was first cleaned with
water and then with 100% ethanol. The punch was wiped with a tissue
and used to punch a clean blank Guthrie card several times to
ensure that all residual ethanol is removed from the tool. One 3.2
mm disk was punched from a DBS onto a nonabsorbent surface, and
then placed in a sample tube (one DBS punch per sample tube).
Sufficient clearance from the edge of the spot and previous punch
holes was left to ensure that a complete 3.2 mm circle saturated
with blood is obtained. A clean, blank Guthrie card was punched
several times between each additional test sample.
[0250] 200 .mu.L of Buffered Extractant was added to each sample
tube. The DBS were mixed gently for 1 hour at RT on a rocking
platform, and checked to make sure that paper discs were in
constant contact with the moving liquid. After incubation, the
filter paper punches were removed from the sample tubes, and the
tubes were centrifuged at 14000 RPM for 30 minutes.
[0251] An Uninhibited Working Substrate Solution (4-MU-.beta.-Glu)
was prepared by combining 100 .mu.l substrate stock solution with
7.9 ml water. An Inhibited Working Substrate Solution was prepared
by combining 4 ml of the Uninhibited Working Substrate Solution
with 7.5 .mu.l CBE Stock Solution. Each of the Uninhibited and
Inhibited Working Substrate Solutions were used within four
hours.
Starting the Reaction:
[0252] Each test sample was assayed in duplicate for both
Uninhibited and Inhibited Working Substrate solutions and, thus,
requires four wells per sample. 80 .mu.L of Uninhibited Working
Substrate Solution was added to two wells per sample, and 80 .mu.L
of Inhibited Working Substrate Solution was added to two wells per
sample. 16 wells were reserved for a standard curve. The enzyme
reactions were initiated by adding 40 .mu.L each of a test sample
(extracted DBS) to all assay wells, being careful not disturb the
pellet in the DBS sample tubes when removing 40 .mu.L. The contents
were mixed in well by pipetting up and down. The plate was sealed
with an adhesive sealer or capmat, making sure that all wells were
individually sealed. The plate was incubated in a 37.degree. C.
water bath for 20 hours.
Terminating the Reaction:
[0253] Each plate was centrifuged at approximately 2500 RPM for
5-10 minutes to remove condensate from sealing film, which was then
carefully removed. 100 .mu.L of Stop Buffer was added to all assay
wells, but was not yet added to the wells for the standard curve.
The assay wells were then covered with a plate sealer to protect
them from contamination while making the standard curve.
Fluorescence Measurement:
[0254] A 12.5 .mu.M 4-MU standard was prepared by diluting 5 .mu.L
of 25 mM 4-MU Stock Solution into 10 mL water. 120 .mu.L of water
was added to all standard curve wells, following by the addition of
120 .mu.L of 12.5 .mu.M 4-MU standard to the first two standard
curve wells, which were mixed by pipetting up and down.
[0255] The standard was diluted serially by transferring 120 .mu.L
from the first duplicate standard curve wells to the consecutive
pairs of wells. The dilution was ended after 6 transfer steps by
discarding 120 .mu.L from the 7th pair of wells, leaving the last
two wells with water only. This serial dilution yielded the
following quantities of standard in pmol per well: 750, 375, 188,
93.8, 46.9, 23.4, 11.7 and 0.
[0256] 100 .mu.L Stop Buffer was then added to all standard curve
wells. The plate was sealed and centrifuged for one hour at 2500
RPM. The sealant was removed and the plate was immediately read in
a fluorometer with 355 nm excitation and 460 nm emission
wavelengths.
Data Processing
[0257] The individual fluorescence readings from the standard curve
wells were plotted against the corresponding molar quantities per
well. The equation (4-MU, pmol)=.alpha.*fluorescence was fit to the
data with a being the slope of the regression line (Note: If the
correlation coefficient r of the standard curve is less than 0.98
the standard curve is invalid and should be repeated).
[0258] The average fluorescences of the duplicate pairs containing
reaction mixture without CBE was then calculated. The average
fluorescences of the duplicate pairs containing reaction mixture
with CBE were also calculated, and subtracted from the average
fluorescence of the reactions without CBE. The fluorescences of the
standard wells was used to calculate a linear standard curve. The
fluorescence differentials were converted into pmol per well by
linear regression using the standard curve, and were then converted
into pmol/(punch*h) by dividing the result by the incubation time
in hours (normally 20 hours), and then multiplying by the fraction
of each extract used per well (in this assay, the factor is
200:40=5).
[0259] A test for bad duplicates was also performed in which the
difference of fluorescence within each pair of duplicate wells was
calculated, wherein f the absolute difference was greater then 25%
of the average fluorescence of the corresponding pair the result
was invalid and the assay was repeated for the corresponding
sample.
[0260] FIG. 8 shows the standard curve that was generated according
to the foregoing description. FIG. 9 shows a comparison of the 153
control and 43 Gaucher disease patient samples. The solid lines
indicate the mean for control and Gaucher disease samples at 10.87
and 1.58 pmol/(punch*h), respectively. These results demonstrate
the ability of the second ABG assay to determine levels of ABG
enzyme activity. and to discriminate between diseased and
non-diseased patient samples.
* * * * *