Title: Reverse isotope dilution assay and lactose intolerance assay
Abstract: A "reverse isotope dilution assay" herein, wherein a pathway that produces a given metabolite is assayed by diluting a labelled metabolite produced by a second constitutive pathway. In one aspect, the invention relates to a method for monitoring lactose maldigestion or lactose intolerance in humans. Specifically, the method requires administering a reverse tracer of labeled glucose and unlabeled lactose to an individual and assessing labeled carbon dioxide in breath or blood. If the lactose is digested, the labeled CO2 produced by the labeled glucose is diluted by the metabolism of the lactose.
Patent Number: 6,902,719 Issued on 06/07/2005 to Wagner
| Inventors:
|
Wagner; David A. (460 Amherst St., Nashua, NH 03063)
|
| Appl. No.:
|
094309 |
| Filed:
|
March 7, 2002 |
Foreign Application Priority Data
| Jan 18, 2002[JP] | 2001-583819 |
| Current U.S. Class: |
424/1.81; 424/1.11; 424/1.65; 424/1.73; 424/9.1; 424/9.2 |
| Intern'l Class: |
A61K 051/00; A61M036/14 |
| Field of Search: |
424/111,165,169,91,181,92,941
435/183,173
|
References Cited [Referenced By]
U.S. Patent Documents
Other References
Harrison, M. and Walker-Smith, J.A., Gut, Jan. 1977, p. 48-52, v. 18-1, BMJ Publishing
Group, UK.
Davidson, G.P., et al., J. Pediatr., Oct. 1984, p. 587-90, v. 105-4, Elsevier
Science, USA.
Douwes, A. C., et al., Arch. Dis. Child. Apr. 1985, p. 333-7, v. 60-4, BMJ Publishing
Group, UK.
Arola, H. Scand. J. Gastroenterol., Apr. 1994, p. 26-35, v. 29-Suppl 202, Taylor
and Francis Ltd., Norway.
Suarez, F.L., et al., N. Engl. J. Med., Jul. 1995, p. 1-4, v. 333-1, The Massachusetts
Medical Society, USA.
Stallings, V.A., Am J. Ther. Jul. 1997, p. 259-273, v. 4-7/8, Chapman & Hall, UK.
Carrocio, A., et al., J. Am. Coll. Nutr., Dec. 1998, p. 631-36, v. 17-6, Am.
Coll. Nutr., USA.
Saltzman, J.R., et al., Am. J. Clin. Nutr., Jan. 1999, p. 140-6, v. 69-1, Am.
Soc. Clin. Nutr., USA.
Peuhkuri, K., et al., Am. J. Clin. Nutr., Feb. 2000, p. 600-1, v. 71-2, Am. Soc.
Clin. Nutr., USA.
|
Primary Examiner: Jones; Dameron L.
Parent Case Text
PRIOR RELATED APPLICATIONS
This application claims priority to prior foreign application Japan No. 2001-583819,
filed on Jan. 18, 2002, is a continuation and claims priority to prior International
patent application No. PCT/US01/15143, filed May 10, 2001, which claims priority
to U.S. provisional patent application Ser. No. 60/205,342, filed on May 18, 2000,
all of which are incorporated herein by reference in their entirety.
Claims
1. A method of assaying enzyme activity in a subject, said method comprising:
a) administering to a subject an effective amount of a reverse tracer wherein
said reverse tracer is a carbon labeled molecule that is constitutively metabolized
by the subject to produce a labeled metabolite wherein said carbon labeled molecule
is selected from the group consisting of acetate, glucose, bicarbonate, glycine,
octanoate, palmitate, formate, propionate, and urea;
b) administering to said subject an effective amount of an unlabeled substrate,
wherein said substrate is specifically metabolized by an enzyme to be assayed and
wherein said substrate is metabolized by said enzyme to produce and unlabeled metabolite
that is the same as the metabolite from step a);
c) collecting a specimen from said subject; and
d) measuring the amount of labeled metabolite in said specimen to determine the
activity of said enzyme in said subject wherein the dilution of labeled metabolite
indicates enzyme activity.
2. The method according to claim 1, wherein said carbon-labeled molecule is selected
from the group consisting of a
13C labeled molecule, a
14C
labeled molecules, and mixtures thereof.
3. The method according to claim 1, wherein said carbon-labeled molecule is labeled
at the 1-position.
4. The method according to claim 1, wherein said carbon-labeled compound comprises
a plurality of labeled carbons.
5. The method according to claim 1, wherein said metabolite is carbon dioxide.
6. The method according to claim 5 wherein said labeled metabolite is
13C
carbon dioxide.
7. The method according to claim 1, further comprising comparing said amount
of labeled metabolite with a standard, whereby said comparing yields a measure
of enzyme activity, and whereby said standard is the mean amount of labelled metabolite
produced by a control population of healthy subjects.
8. A method according to claim 1 wherein said enzyme is lactase, said reverse
tracer is selected from the group consisting of
13C labeled glucose,
13C labeled glucose, and mixtures thereof said unlabeled substrate is
lactose; and said labeled metabolite is selected from the group consisting of
13C
labeled CO
2,
14C labeled CO
2, and mixtures thereof.
9. A method according to claim 1 wherein:
a) said enzyme is lactase, said reverse tracer is
13C labeled glucose,
said unlabeled substrate is lactose, and said labeled metabolite is
13CO
2;
b) said enzyme is lactase, said reverse tracer is
14C labeled glucose,
said unlabeled substrate is lactose, and said labeled metabolite is
14CO
2;
c) said enzyme is lactase, said reverse tracer is
13C labeled acetate,
said unlabeled substrate is lactose, and said labeled metabolite is
13CO
2;
d) said enzyme is lactase, said reverse tracer is
14C labeled acetate,
said unlabeled substrate is lactose, and said labeled metabolite is
14CO
2;
e) said enzyme is lactase, said reverse tracer is
13C labeled bicarbonate,
said unlabeled substrate is lactose, and said labeled metabolite is
13CO
2;
f) said enzyme is lactase, said reverse tracer is
14C labeled bicarbonate,
said unlabeled substrate is lactose, and said labeled metabolite is
14CO
2;
g) said enzyme is a fructose transporter protein, said reverse tracer is
13C
labeled glucose, said unlabeled substrate is fructose, and said labeled metabolite
is
13CO
2;
h) said enzyme is a fructose transporter protein, said reverse tracer is
14C
labeled glucose, said unlabeled substrate is fructose, and said labeled metabolite
is
14CO
2;
i) said enzyme is a fructose transporter protein, said reverse tracer is
13C
labeled acetate, said unlabeled substrate is fructose, and said labeled metabolite
is
13CO
2;
j) said enzyme is a fructose transporter protein, said reverse tracer is
14C
labeled acetate, said unlabeled substrate is fructose, and said labeled metabolite
is
14CO
2;
k) said enzyme is a fructose transporter protein, said reverse tracer is
13C
labeled bicarbonate, said unlabeled substrate is fructose, and said labeled metabolite
is
13CO
2; and
l) said enzyme is a fructose transporter protein, said reverse tracer is
14C
labeled bicarbonate, said unlabeled substrate is fructose, and said labeled metabolite
is
14CO
2.
Description
FEDERALLY SPONSORED RESEARCH STATEMENT
Not applicable.
REFERENCE TO MICROFICHE APPENDIX
Not applicable.
FIELD OF THE INVENTION
The invention relates to a novel assay for monitoring for disease or metabolic
dysfunction called a "reverse isotope dilution assay" or "RID" herein, wherein
a pathway that produces a given metabolite is assayed by diluting the metabolite
with the same metabolite produced by a second, constitutive pathway. More specifically,
the invention relates to co-administering a "reverse tracer" molecule and an unlabeled
substrate molecule to an individual. Both the reverse tracer molecule and the substrate
molecule are metabolized to an equivalent end point, for example, CO
2.
However, the "reverse tracer" molecule, by definition, is metabolized via a fast
acting, constitutive pathway that differs from the pathway to be assayed. Thus,
when the substrate molecule is added, and the pathway of interest is active, the
reverse tracer metabolite will be diluted by the activity of the pathway of interest.
In contrast, if the pathway is not active, the labeled metabolite will not be diluted.
Thus, the activity of the pathway of interest can be determined from the dilution
of the reverse tracer metabolite.
In one aspect, the invention relates to a method for monitoring "lactose maldigestion"
or "lactose intolerance" in humans. Specifically, the method requires administering
a tracer amount of labeled glucose and a physiological or pharmacological dose
of unlabeled lactose to an individual and assessing labeled carbon dioxide in breath
or blood.
BACKGROUND OF THE INVENTION
Carbon dioxide is an end product of cellular metabolism. It is expired in
humans at a rate of 9 mmol/kg-hour (1) The rate of
13CO
2
production form
13C-labeled substrates has been demonstrated in cells,
tissues, perfused organs and whole animals since the 1940s (2). Moreover, this
approach has been used in biomedicine to measure liver function, malabsorption,
bacterial infection, enzyme deficiency, pancreatic insufficiency and protein metabolism.
The principle of
13CO
2 breath tests is to administer a
substrate labeled with
13C either orally or intravenously. The substrate
must possess a target bond that is attacked by a specific enzyme whose activity
is to be measured. The enzymatic cleavage of the
13C bond is the rate
limiting step. Ultimately, the
13C moiety is directly hydrolyzed or
rapidly converted to
13CO
2.
Existing CO
2 tests generally require large amounts of labeled
substrate. Tests based on radioactive labels are problematic because the patient
consumes radioactive material. Disposal and handling costs also increase with radioactive
labels. If non-radioactive labels are employed, the problems are not eliminated
because labeled substrates are very expensive, thus increasing the costs of such
tests significantly. What is needed in the art is a method that decreases the amount
and cost of label required for a metabolic test, without sacrificing the needed
sensitivity. The invention described herein, fulfills this need and, although exemplified
with respect to a lactose intolerance assay, can be used wherever CO
2 breath
tests are used. The invention can also be used for metabolites other than CO
2
and for samples other than breath samples.
Lactose maldigestion is the inability to digest significant amounts of lactose,
the predominant sugar of milk. This inability results from a shortage of the enzyme
lactase that is normally produced by the cells that line the small intestine. When
there is not enough lactase to digest the amount of lactose consumed, the results
may be very distressing and can result in dangerous dehydration among children.
Common symptoms include nausea, cramps, bloating, gas, and diarrhea, which begin
about 30 minutes to 2 hours after eating or drinking foods containing lactose.
The severity of the symptoms varies depending on the amount of lactose each individual
can tolerate.
The intestinal enzyme lactase (β-D-galactosidase) is responsible for metabolizing
lactose. At birth, humans have abundant lactase activity in the small intestine
but in most ethnic groups this activity decreases significantly during childhood
between ages 3 to 5. Under conditions of lactase deficiency the lactose passes
unmetabolized through the small intestine, drawing in copious amounts of water
by osmosis. Next, the lactose passes into the large intestine and is fermented
by colonic bacteria. Through these two processes, osmosis and fermentation, the
typical symptoms associated with lactose maldigestion such as bloating, cramping,
excessive gas and explosive diarrhea are derived.
Milk and other dairy products are a major source of nutrients in the American
diet. The most important of these nutrients is calcium. Calcium is essential for
the growth and repair of bones throughout life, but is a particular concern during
the developmental years. In the middle and later years, a shortage of calcium may
lead to thin, fragile bones that break easily; a condition known as osteoporosis.
A concern, then, for both children and adults with lactose maldigestion, is getting
enough calcium in a diet that contains little or no milk.
Studies have shown that nearly 50% of people who self-report milk intolerance
are not maldigesters (1-3). Instead, they suffer from a functional bowel disorder
such as irritable bowel syndrome (IBS), recurrent abdominal pain (RAP) in children
or some other gastrointestinal complication. In these self-reported milk intolerants,
it has been found that there is a significant, unnecessary reduction in milk consumption
and insufficient dietary calcium intake (4).
Lactose maldigestion is relatively easy to treat. No treatment exists to
improve the body's ability to produce lactase, but the symptoms can be controlled.
Many foods are now available that are lactose-reduced or even lactose-free. Moreover,
chewable tablets of lactase are available without prescription that, when taken
just prior to a lactose-containing meal, can alleviate many symptoms.
However, all of these proposed therapies and remedies are only advisable
in the person who is truly a lactose maldigesters (truly deficient in the enzyme
lactase). For the person who suffers, for example, from irritable bowel syndrome
(IBS) but is misdiagnosed as lactase-deficient, the addition of lactase in the
form of tablets or the change to lactose-free dairy products will not alleviate
symptoms. Moreover, those self-treaters who avoid dairy under the mistaken impression
that they are maldigesters, put themselves at risk for poor bone growth and repair,
osteoporosis and other conditions that results from the unnecessary removal of
dairy products from their diet.
The diagnosis of lactose maldigestion has relied on the interview process coupled
with removing milk (and milk products) from the diet, laboratory tests and jejunal
biopsy. We briefly describe the state of each measure.
The interview process during which a patient is quizzed as to the history of
their gastrointestinal symptoms and its relation to milk intake is easy to perform
and inexpensive. It is also overly simplistic and quite imprecise. First, nearly
50% of people who self-report milk intolerance are normal digesters of lactose
and secondly, 70% of the people with lactase-deficiency (although symptomatic)
fail to correlate the broad gastrointestinal symptoms of this disease to the intake
of lactose or "milk sugar" (7).
A number of laboratory tests are available for the assessment of lactose maldigestion.
The most often cited tests are the hydrogen breath test, lactose tolerance test
and the stool acidity test. The hydrogen breath test measures the amount of hydrogen
in the breath. Normally, very little hydrogen is detectable in the breath. However,
in the case of the lactose maldigesters, the lactose passes into the colon unmetabolized
where bacteria ferment it and various gases, including hydrogen are produced. The
hydrogen is absorbed from the intestines, carried through the blood stream to the
lungs and exhaled. In this test, the patient drinks a lactose-loaded beverage,
and the breath is analyzed at regular intervals over several hours. Raised levels
of hydrogen in the breath indicates that the lactose is not being properly digested.
The interpretation of the hydrogen breath test results can be confounded by a
number of factors. First, 5-20% of maldigesters do not produce hydrogen, resulting
in a lowered sensitivity for the test (8). A comparable percentage of non-producers
has been found in children (9). This is due to either not having the flora capable
of producing hydrogen or utilization of the hydrogen to produce methane. Secondly,
careful patient preparation, including no teeth brushing on the morning of the
exam, no smoking, sleeping or strenuous activity during the exam is absolutely
mandatory in order to produce a reliable test (10). Also, for one month prior to
the test, there should be no mechanical bowel cleansing or antibiotic use since
both influence the type and quantity of colonic bacteria (10). Finally, a low carbohydrate,
low fiber dinner the night before the test is advised. Any deviations from these
recommendations will compromise the test.
In the lactose tolerance test, a fasted individual (>10 hours without eating)
is given a liquid that contains a large lactose load (typically 2g/kg to a maximum
of 50 g which is equivalent to the lactose content of one liter of milk). Several
blood samples are taken over a period of two hours to measure the subject's blood
glucose level. This result is used as an indication of how well that patient digests lactose.
Again, there are several drawbacks to this test method. This test uses a supraphysiological
dose of lactose, which makes its generalization to normal milk or dairy ingestion
questionable. It requires a minimum of four (4) needle sticks over 2 hours to measure
glucose concentration and strict patient compliance to a fasted state. Moreover,
it suffers from decreased specificity (13% false positive rates have been reported)
since a flattened response requires differentiation from defective glucose absorption
resulting from small bowel disease (11). It has been suggested in the medical literature
that due to both false negative and false positive results "that routine estimation
of blood glucose after lactose load is not a useful measurement in children and
adults and should be abandoned" (12).
In a recent study, it was shown that in 300 subjects tested using both the hydrogen
breath test and the lactose tolerance test, in 40% of the cases, the two tests
did not agree (13). The study suggests, however, that the hydrogen breath test
is better able to identify individuals with lactose malabsorption and those most
likely to have symptoms.
Due to the required lactose loads in these two diagnostic tests and the associated
danger from dehydration resulting from lactose-induced diarrhea, they are generally
not used in infants and very young children. Infants and young children may instead
be given the stool acidity test. Undigested lactose, fermented by bacteria in the
colon, creates lactic acid and other short-chain fatty acids that can be detected
in a stool sample. This test is only effective in completely lactose-dominated
diets (such as infant formula or breast milk) and since the incidence of lactose
maldigestion in infants is very low, it is not often utilized.
Jejunal biopsy is an effective method for establishing a level of a patient's
lactase activity. However, it is highly invasive and used only on rare occasion.
Because lactose maldigestion is not generally considered a dangerous health condition,
such an expensive, invasive and uncomfortable procedure is not a useful alternative.
Thus, what is needed in the art is a reliable, sensitive lactose intolerance
test that is non-invasive, cost effective and accurate. The reverse isotope dilution
test, exemplified herein with respect to lactose intolerance, meets these needs.
SUMMARY OF THE INVENTION
Abbreviations and Definitions
Reverse Tracer—a labeled substrate for a second, constitutive pathway;
exemplified herein as 13C-glucose when used in a lactose intolerance RID
Reverse Tracer metabolite—the labeled metabolite produced by the metabolism
of the reverse tracer in the second, constitutive pathway.
RID—Reverse Isotope Dilution, the assay described herein
wherein a first enzyme is assayed by the dilution of a labeled metabolite produced
by both the first enzyme and a second constitutive enzyme
Substrate—an unlabelled substrate molecule that is metabolized
by the enzyme of interest to produce the same metabolite that is produced by the
metabolism of the reverse tracer; exemplified herein by lactose in the lactose
intolerance RID.
The invention is a reverse isotope dilution assay that can be generally described
as follows: A first enzyme to be assayed is quantified by the diluting effects
of a measurable metabolite produced by the first enzyme (or downstream of the first
enzyme). The metabolite is the same metabolite produced by the action of a second
enzyme (or enzyme pathway). A reverse tracer is co-administered with a substrate
specific to the first enzyme. The reverse tracer molecule is a labeled substrate
molecule that is specific to the second pathway and is quickly and constituitively
metabolized by the second enzyme and/or pathway to produce the labeled metabolite.
Thus, if the labeled metabolite is diluted, it means the first pathway is active.
If it is not diluted, it means that the first enzyme is not active. A typical dilution
curve for a labeled metabolite is illustrated in FIG. 1.
The present invention is exemplified with respect to a lactose intolerance assay,
but can be generally applied to any disease whose course can be traced by tracing
the exhalation of labeled bicarbonate. Such tests include the
Helicobacter pylori
breath test (based on labeled urea), the human liver glycogen metabolism breath
test (using naturally
13C-enriched carbohydrate); the gastric emptying
test (based on labeled octanoate or acetate); the chemotherapy intolerance breath
test (based on labeled erythromycin); the bacterial overgrowth test (based on labeled
xylose or sorbitol); the hepatic function breath test (based on labeled aminopyrine,
methionine, or phenylalanine, for example); and the pancreatic sufficiency breath
test (based on labeled mixed triglycerides or corn starch).
We have shown the combination of the
13C-glucose reverse tracer with
lactose for use in a lactose intolerance assay RID which is based on measuring
CO
2 in the breath. However, other combinations are possible. For example,
13C-acetate reverse tracer and fructose substrate may be combined in
a fructose malabsorption RID CO
2 breath test. An erythromycin breath
test may be converted to RID with the use of
13C-acetate and unlabeled
erythromycin. The
Helicobacter pylori CO
2 breath test could also
be adapted to RID with the use of labeled
13C-glucose and unlabelled
urea. The following table provides additional examples of RID tests.
| TABLE 1 |
| RID Substrate and Reverse Tracer Combinations |
| Reverse Tracer |
Substrate |
Function/Disease |
| 13C-Acetate/13C-glucose/ |
Amino acids |
Liver Function |
| 13C-bicarbonate |
(methionine, |
| |
phenylalanine, |
| |
lysine) |
| 13C-Acetate/13C-glucose/ |
Carbohydrates |
Small Intestine |
| 13C-bicarbonate |
(xylose, sorbitol) |
Bacterial Overgrowth |
| 13C-Acetate/13C-glucose/ |
Triglycerides |
Pancreatic Function |
| 13C-bicarbonate |
(triolein, |
| |
tripalmitate) |
| 13C-Acetate/13C-glucose/ |
Starch |
Pancreatic Function |
| 13C-bicarbonate |
| 13C-Acetate/13C-glucose/ |
Galactose |
Liver Function |
| 13C-bicarbonate |
| 13C-Acetate/13C-glucose/ |
Urea |
Helicobacter pylori |
| 13C-bicarbonate |
|
Infection |
| 13C-Acetate/13C-glucose/ |
Leucine |
Crohn's Disease |
| 13C-bicarbonate |
The above table shows that either
13C-acetate,
13C-glucose,
or
13C-bicarbonate would work as the reverse tracers. For some tests,
one of these substrate might be preferred for cost or biochemical reasons. Other
13C reverse tracers would function in the invention, such as
13C-glycine,
13C-octanoate,
13C-palmitate,
13C-formate,
13C-propionate,
and
13C-urea; however, their costs are much higher.
The reverse tracer is generally labeled with non-radioactive, stable isotopes
in order to minimize radioactive waste hazards and patient exposure, but other
isotopes may be employed. Generally,
13C isotopes are preferred, but
2D,
15N,
34S, and the like may also be used as
appropriate for the metabolite to be measured. Oxygen-labeled substrates are another
possibility, but the expense of
18O substrates may be so high as to
be unfeasible.
The invention can also be broadened to include reverse isotopic detection of
metabolites other than bicarbonate. For example, it can be employed for
15N-labeled
substrates coupled with the detection of N
2 gas in breath, or NH
3
or urea in blood or urine. For example, the
Helicobacter pylori breath
test could also be adapted to RID with the use of labeled
15N-ammonia
or
15N-ammonium salt and unlabelled urea coupled with the detection
of labeled NH
3 in the urine. Similarly, the method could be employed
with deuterated substrates coupled with the detection of
2H in the breath.
Lactose intolerance, bacterial overgrowth and rapid transit of food through the
small bowel can all be assayed by a hydrogen breath test.
Measurement of labeled metabolites, such as CO
2, in breath
or blood may be made by instruments capable of detecting isotopes such as mass
spectrometry, laser assisted spectrometry, infrared spectrometry or other spectrometry
instruments. Further, the method includes isotopic measure of CO
2 by
continuous monitoring (Katzman et al., U.S. Pat. No. 6,186,958).
The assay herein is exemplified as a breath test, but a blood, fecal, saliva,
urine, or other body fluid specimen test could also be performed, provided the
appropriate reverse tracer and substrate combinations are chosen.
The present invention also provides a method and kit for the assessment of lactose
maldigestion or lactose intolerance in humans. The method is as described above,
and the kit contains at least a labeled tracer and an unlabeled substrate. The
kit may also contain a sample collection device, including a breath collection
device, and instructions for use. Collection devices, such as breath, blood, and
urine collection devices are well known in the art and are not described herein.
One of the major benefits of the RID technology is the reduced cost of the medical
diagnostic test. For example, in the diagnosis of lactose maldigestion, one could
administer
13C-lactose to directly measure lactase enzyme activity.
However,
13C-lactose is a very expensive tracer to synthesize because
it is a disaccharide. Using
13C-glucose and unlabeled lactose in a RID-based
test, the per test cost is only a few dollars compared to greater than $100 for
the usual
13C-lactose-based test.
One embodiment of the invention is a method of assaying enzyme activity in a
subject. The method comprises administering to a subject an effective amount of
a reverse tracer, wherein said reverse tracer is a labeled molecule that is constitutively
metabolized by the subject to produce a labeled metabolite. The subject is co-administering
an effective amount of an unlabeled substrate, wherein said substrate is specifically
metabolized by an enzyme to be assayed and wherein said substrate is metabolized
by said enzyme to produce an unlabeled metabolite that is the same as the metabolite
from the prior step. A specimen is collected from the subject and the amount of
labeled metabolite in the specimen measured to determine the activity of said enzyme
in said subject. The method may also comprise comparing the amount of labeled metabolite
with a standard, whereby said comparing yields a measure of enzyme activity, and
whereby said standard is the mean amount of labelled metabolite produced by a control
population of healthy subjects.
In another embodiment, the method is a method of assessing metabolic dysfunction
in a subject using steps similar to those above. The reverse tracer, substrate
and metabolic dysfunction can be as described throughout or as listed in Table
1. In one particular embodiment, the reverse tracer is labeled glucose, the substrate
is lactose and the RID is for lactose intolerance. In another embodiment, the reverse
tracer is labeled glucose, the substrate is fructose and the RID is for fructose malabsorbtion.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. is a CO
2 Dilution Curve (Lactose Intolerance)
FIG. 2. is a Schematic Diagram of RID Concept
FIG. 3. is a CO
2 Dilution Curve (Fructose Malabsorption)
DETAILED DESCRIPTION OF THE INVENTION
Provided herein is a method of assessing lactose maldigestion using reverse
isotope dilution (RID) to measure enzyme rates directly by combining a labeled
tracer and an unlabeled probe. The method uses a co-administration of 1-
13C-glucose
(25 to 1000 milligrams) as a reverse tracer and unlabeled lactose (500 milligrams
to 100 grams) as the test substrate. Unlabeled lactose is metabolized to glucose
and galactose, which are subsequently converted rapidly to carbon dioxide. The
administered 1-
13C-glucose is also rapidly metabolized to
13CO
2.
The amount of dilution of
13CO
2 in the breath or blood
is indicative of the lactase enzyme activity. For the lactose maldigester, the
1-
13C-glucose tracer will appear undiluted in the breath as
13CO
2.
That is, the results of the breath test, in the case the maldigester, will be the
same whether lactose is administered or not. This is due to the fact that in the
maldigester, lactose is minimally, if at all, converted to glucose and galactose.
The normal digester on the other hand will generate unlabeled CO
2 from
the lactose load (after processing through glucose and galactose) given with the
test. This test demonstrates the degree of lactose maldigestion by measuring the
amount of lactose digested via the amplitude of
13CO
2 arising
from the 1-
13C-glucose reverse tracer in the breath.
The method further comprises comparing said amount of labeled carbon dioxide
with a standard, whereby said comparing yields a measure of lactose maldigestion.
The standard comprises the mean isotopic value of CO
2 in a control population
without lactose maldigestion or lactose intolerance.
The following examples serve to illustrate specific embodiments of the invention,
but should not be considered as a limitation on the scope of the invention.
EXAMPLE 1
Administration of the Test
All breath tests are performed after a minimum of 8 hours of fasting. Prior to
the detection substrate administration, a baseline breath sample is collected using
an alveolar gas collection system (QUINTRON GASAMPLER COLLECTION BAG™, QUINTRON
INSTRUMENT COMPANY™, Milwaukee, Wis.). Subjects are administered a 10% aqueous
lactose solution containing 25 grams of orange-flavored lactose (QUINTRON INSTRUMENT
COMPANY™) in 250 ml tap water. In addition to the lactose, subjects consume
100 milligrams of 1-
13C-glucose (CAMBRIDGE ISOTOPE LABORATORIES™,
Andover, Mass.) which is added to the aqueous lactose solution. End-alveolar breath
samples are at evaluated for
13C enrichment in carbon dioxide at 0,
60 and 90 minutes.
EXAMPLE 2
Bicarbonate Measurement
The amount of
13CO
2 in breath storage tubes are measured
with a EUROPA SCIENTIFIC™ 20/20 gas isotope ratio mass spectrometer (EUROPA
SCIENTIFIC™, Cincinnati, Ohio). The ratio of
13CO
2 to
12CO
2 (mass 45 to 44) is measured in the sample and compared
to a reference gas (5% CO
2, balance 75% N
2, 20% O
2).
The reference gas is calibrated with international standards. The units of measurement
are atom %
13C and defined by:
Standards of carbon dioxide gas at 3 different levels of atom %
13C
are run before and after each daily run to check instrument performance. The analytical
precision of the instrument is 0.0001 atom %
13C.
The atom %
13C value of each breath sample is used to calculate the
percent of the dose recovered in the breath during each time period. The area under
the curve (AUC) for each time period, is calculated by the linear trapezoid method,
using the atom %
13C for the two points during time period. The percent
of the dose metabolized within each time period is calculated as:
EXAMPLE 3
Test Validity
Initial investigations established the validity of the test. One hundred
twenty (120) subjects (51 males and 69 females) of ages greater than 18 years were
evaluated for lactose maldigestion. Each subject was tested on two occasions following
an overnight fast. The subject underwent a physical exam and was interviewed concerning
their experience with dairy consumption. On Day 1, a 100 mg dose of D-glucose (1-
13C,
99%), (CAMBRIDGE ISOTOPE LABORATORIES™, Andover, Mass.) was diluted to with
25 ml with tap water. A 50 g dose of Lactose (QUINTRON, INC.™, Milwaukee
Wis.) was simultaneously administered. Breath samples were collected for
13CO
2/
12CO
2
ratio measurement were collected at 5, 15, 30, 45, 60, 75, 90, 105 and 120 minutes
from dosing. The samples were analyzed on A FINNIGAN BREATHMAT PLUS™ gas
isotope ratio analyzer for the
13C/
12C ratio of the exhaled
CO
2. All of the breath test results were then converted to % dose metabolized
per unit time.
At the same time, the hydrogen breath test (QUINTRON, INC.™, Milwaukee
Wis.) and the Lactose Tolerance Test (blood glucose levels) were administered according
to standard protocols. Further, urine was collected for the determination of galactose
levels in the urine as another measure of lactose digestion. The next day (Day
2), the experiment was repeated but with load of lactose changing from 50 g to
25 g.
A major limitation to the analysis of the subsequent data was the absence of a
"gold standard" for the diagnosis of lactose malabsorption. Even the most reliable
test, the hydrogen breath test, reports accuracy at no better than 85%. Therefore,
a new test, even if perfectly accurate, can not have an accuracy score above that
of the gold standard (85%). For our study, in an attempt to address this limitation,
a diagnosis of lactase status was determined by combining all of the reference
methods and drawing a unifying diagnosis from the collection of results based on
majority diagnostic opinion (2 of 3 tests). The Lactose Maldigestion Breath Test
(LMBT) and each reference method (hydrogen breath test, blood glucose test and
urinary galactose) were individually evaluated versus the unifying diagnosis. The
following performance characteristics are shown in table 2. Note, the 25 gram hydrogen
breath test was done on only 59 subjects while the other tests were performed on
120 subjects.
| TABLE 2 |
| Lactose Intolerance Validation |
| Test |
Sensitivity |
Specificity |
PPV |
NPV |
Accuracy |
| LMBT |
87% |
79% |
82% |
85% |
83% |
| Hydrogen Breath Test |
87% |
80% |
83% |
85% |
84% |
| 50 grams |
| Hydrogen Breath Test |
75% |
100% |
100% |
82% |
88% |
| 25 grams |
| Blood Glucose Test |
78% |
84% |
84% |
77% |
81% |
| Urinary Galactose Test |
55% |
82% |
77% |
62% |
66% |
| PPV = Positive Predictive Value |
| NPV = Negative Predictive Value |
Although samples were collected every 15 minutes, we were able to differentiate
lactose digesters from maldigesters using only the baseline, 60 and 90-minute samples
without any loss of accuracy. If the test was positive at 60 minutes, in theory,
the test could be stopped. The fact that the test can be completed within 90 minutes
prevents potential problems associated with glucose being metabolized by the colonic
flora. Generally intestinal transit or oro-cecal time is normally at least 75 minutes
or more.
Based on these studies with adults, the following cutoff values can be used
to define lactose malabsorption:
At 60 minutes, greater than 1.50% 13C glucose metabolized
At 90 minutes, greater than 4.50% 13C glucose metabolized
At 120 minutes, greater than 7.50% 13C glucose metabolized
EXAMPLE 4
Fructose Malabsorbance RID
Although not yet fully validated, a RID has been exemplified for a fructose
malabsorbance breath test. In the test, the patient is co-administered labeled
acetate and unlabelled fructose. If the patient is unable to absorb and utilize
the fructose, the
13CO
2 levels remain high whereas in the
normal patient the
13CO
2 levels are diluted by concomitant
absorption and metabolism of the unlabelled fructose.
FIG. 3 shows the results of one subject administered 100 mg sodium 1-
13C-acetate
with and without 25 grams fructose. The graph shows the plot of the percent acetate
metabolized to carbon dioxide per unit time. In this subject, since fructose is
absorbed and itself converted to carbon dioxide, it dilutes the amount of
13C
appearing in breath carbon dioxide. For a subject who does not absorb fructose,
the two breath
13C excretion curves would be identical.
Each reference is listed herein for convenience, and is incorporated by reference
in its entirety.
1. Suarez, F. L., et al., (1995) N. Engl. J. Med. 333:1-4.
2. Saltzman, J. R., et al., (1999) Am. J. Clin. Nutr. 69:140-6.
3. Peuhkuri, K., et al., (2000) Am. J. Clin. Nutr. 71:600-1.
4. Stallings, V. A., (1997) Am J. Ther. 4: 259-273.
5. Montes, R. G. and Perman, J. A., (1991) Postgrad. Med. 89: 175-184.
6. Srinivasan, R. and Minocha, A., (1998) Postgrad. Med. 104: 109-123.
7. Carrocio, A., et al., (1998) J. Am. Coll. Nutr. 17:631-36.
8. Davidson, G. P., et al., (1984) J. Pediatr. 105:587-90.
9. Douwes, A. C., et al., (1985) Arch. Dis. Child. 60: 333-7.
10. Arola, H. (1994) Scand. J. Gastroenterol. 29 (Suppl 202):26-35.
11. Douwes, A. C., et al., (1978) Arch. Dis. Child. 53: 939-942.
12. Harrison, M. and Walker-Smith, J. A., (1977) Gut 18: 48-52.
13. Hermans, M. M., et al., (1997) Am. J. Gastroenterol. 92:981-4.
14. U.S. Pat. No. 6,186,958
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