WO1999022639A1 - Glucose detector and method - Google Patents

Glucose detector and method Download PDF

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Publication number
WO1999022639A1
WO1999022639A1 PCT/US1998/009345 US9809345W WO9922639A1 WO 1999022639 A1 WO1999022639 A1 WO 1999022639A1 US 9809345 W US9809345 W US 9809345W WO 9922639 A1 WO9922639 A1 WO 9922639A1
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WO
WIPO (PCT)
Prior art keywords
glucose
oral fluid
sample
saliva
collection
Prior art date
Application number
PCT/US1998/009345
Other languages
French (fr)
Inventor
Byron A. Doneen
G. Russel Warnick
Holden H. Harris
Original Assignee
Pacific Biometrics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pacific Biometrics, Inc. filed Critical Pacific Biometrics, Inc.
Priority to AU72932/98A priority Critical patent/AU7293298A/en
Priority to US09/298,398 priority patent/US20010023324A1/en
Publication of WO1999022639A1 publication Critical patent/WO1999022639A1/en

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Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61BDIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00Measuring for diagnostic purposes; Identification of persons
    • A61B5/145Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14532Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring glucose, e.g. by tissue impedance measurement

Definitions

  • the present invention is directed to an apparatus and method for
  • the present invention non-invasively collects oral fluid, oral
  • fluid glucose content is determined, and blood glucose levels are derived based on
  • the pathogenesis of diabetes originates in sustained or periodic
  • Glucose is linked non-enzymatically to accessible
  • the grade of gly cation depends upon glucose
  • Type I persons would be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be able to be .
  • Blood glucose monitoring Blood glucose is monitored non-invasively by
  • An interstitial transudate is
  • the system requires that the sample be taken from the specific
  • non-invasive glucose monitoring device including stimulation means for
  • Detection means operatively
  • the present invention also provides a method of monitoring blood
  • Figure 1 is a perspective view of an oral fluid collection device made
  • Figure 2 is a cross-sectional view based substantially along lines 2-2 of
  • Figure 3 is a perspective view of a second embodiment of the
  • Figure 4 is a perspective view of a third embodiment of the invention.
  • Figure 5 is a schematic plan view of a fourth embodiment of the
  • Figures 6A-B are graphs showing glucose standard curves in buffer or
  • Figure 7A-B are graphs illustrating a glucose standard curve wherein
  • Figure 7A is a standard curve for in phosphate buffer and Figure 7B is a
  • Figure 8 is a graph showing the time to saliva glucose equilibrium in
  • Figure 9 is a graph showing the effect of pH on the glucose assay
  • Figure 10A-B are graphs showing oral glucose contamination of saliva
  • Figure 11 A-C are graphs showing glucose collected by the present
  • Figure 1 IB showing a collection of data from subjects from
  • Figure 12A-B are graphs showing a correspondence between saliva
  • the present invention provides a non-invasive glucose monitoring
  • the device including a mechanism for stimulating salivary
  • a collection apparatus for collecting a
  • a detection mechanism operatively connected to the
  • the elements of the present invention most generally are (1) simulation
  • a detection mechanism such as a qualitative test strip as discussed below in
  • Such a system can be a integrated device wherein
  • stimulation, collection, and quantitation are accomplished on a single strip or
  • the device is non-invasive, so it removes
  • oral fluid is not simply saliva, but rather
  • Oral fluid has a glucose concentration that has approximately 1/200 to
  • measurement of oral glucose can be used to estimate blood glucose.
  • invention is at least useful as a diagnostic for elevated blood glucose and can
  • the collection device generally shown at 10 in Figure 1 and 2 is
  • the collection device is generally an ovoid small disc
  • the chamber can include an osmotic substance 16 which is totally
  • the semi-permeable membrane 12 is made of a substance which has a
  • Such a membrane is Cuprophan® manufactured by Enka AG, a division of
  • the membrane is composed of
  • the molecular weight cut-off also termed the exclusion limit, is
  • membrane is such that molecules larger than 12,000 daltons (such as proteins,
  • filtered saliva more specifically, ultrafiltered saliva
  • a uniform non-semiconductor saliva more specifically, ultrafiltered saliva
  • viscous, particulate or cellular material of oral fluid could be used in the
  • exclusion limits could also be used, provided such membranes are permeable to glucose and allow its transport from whole saliva to the central
  • the osmotic substance 16 is soluble in oral fluid thereby providing an
  • the osmotic substance can be a crystalline or an amorphous
  • the osmotic substance can comprise a high
  • the osmotic substance must be
  • non-toxic in nature and is preferably palatable.
  • the osmotic substance can also take the form of a stimulant of
  • the osmotic substance can be selected from the group
  • the preferable osmotic substance is one which dissolves readily when
  • salts or other substances can also be used.
  • An example is sodium citrate
  • osmotic material is used to collect filtered saliva.
  • adsorbents can be used to collect saliva if they provide a method for the
  • a vacuum could be
  • One example would be a conventional filtration tube in which whole saliva is
  • the-mouth or external device capable of producing a filtered sample of oral
  • citric acid stimulation was made in accordance with the present invention. It
  • blood glucose enters the primary
  • a minor pathway is transcellular mediated by the apocerine secretion of
  • whole saliva was determined after: (1) sonication of sample at 1600 Hz (hertz); (2) freezing and thawing the sample to precipitate large molecular
  • a small device will reduce diffusion distance and destination volume and will also increase surface
  • the preferred stimulant is citric acid.
  • the semi-permeable membrane 12 may be any material. Referring again to figure 2, the semi-permeable membrane 12 may be any material.
  • an outer protective membrane 20 which includes macroscopic
  • outer protective membrane 20 can be made of any material which would be generally pliable, tasteless, and non-toxic. Preferably, silicon materials or
  • the outer membrane can be made from many materials
  • the present invention can include a container 10 as
  • membrane 12 is made of material of sufficient mechanical strength to survive
  • FIG. 3 A preferred embodiment of the subject invention is shown in figure 3
  • a device 24 is in the form of a test strip including a support 26.
  • membrane sac 10' having a structure as described above, is mounted over one
  • the stimulator of salivary gland secretion can include the stimulator of salivary gland secretion, such as sodium citrate.
  • the absorbent matrix 28 is in fluid communication by abutment with a
  • the film contains the enzymes glucose oxidase and horseradish
  • peroxidase (or some other peroxidase) and a combination of dyes and
  • accessory reagents such as buffers and stabilizers, which are capable of
  • Glucose oxidase applied as a dry reagent to the strip hydrolyzes sample glucose to gluconic acid with production of
  • the color intensity is scaled to the amount of glucose
  • the present invention includes any type of solid-phase strip chemistry
  • enzyme-based system e.g., using hexokinase or glucose dehydrogenase
  • chemistry-based systems e.g., a
  • sensors e.g., glucose-specific electrochemistry
  • oral fluid is collected
  • time may be two or fewer minutes.
  • filtered liquid can be a simple pressure-sensitive opening (port), or the rate-of-
  • flow of sample along the test strip can be made sufficiently slow to ensure that
  • a support strip 26' which can be similar to that shown in
  • Figure 3 supports a collection container 10" as described below.
  • thermometer-type indicator film 36 A thermometer
  • type film is one in which the enzymes and dyes required to produce the
  • colorimetric signal are arrayed from proximal to distal on the test section of
  • glucose in the sample is thus proportional to the linear distance of color
  • thermometer-type strip requires that an accurately measured
  • the strip may contain accessory elements, such as sample
  • the indicator film can be graded to provide an indication of blood
  • the film 36 provides a detection mechanism, as well as a quantitation
  • a glucose level can be detected using any detection device known in the art for glucose analysis.
  • a glucose level can be any one of the detection device known in the art for glucose analysis.
  • the strip could be moved to the monitor after the sample is introduced onto
  • the strip, or a small integrated monitor could be created to present a combined
  • the correlation with blood is obtained by solving an equation which
  • equation can be developed for the subject populations; (2) or if individuals
  • blood glucose concentration can be achieved by insertion of a dedicated
  • the container 10'" is mounted at the end of a wicking material
  • the needle can be
  • the device contains a membrane-osmotic driver collection
  • cross-strip and a mechanism in the form of a pin or, alternatively, a pressure-
  • sample of oral fluid to be transferred to the test strip.
  • wicking materials can be used as a means
  • tissue becomes a dominant
  • salivary glucose source or sink of salivary glucose. It can take two hours for saliva glucose to
  • This method uses the enzyme hexokinase to phosphorylate (using
  • the amount of NADPH produced is proportional to the amount of
  • the data reveal a correspondence between finger prick blood glucose
  • Coupled Reagents Sensitivity Response Time to Completion mg/dL OD/mg/dL minutes at 37°
  • Figure 6A summarizes a subset of the visible chromogens used with glucose oxidase-peroxidase in development of a more sensitive glucose assay.
  • Glucose was spiked into two different matrices: 20 mM phosphate buffer (pH 7.0), and whole saliva processed as described above but without heating to 100 c x 10 min (saliva pH, 6.9).
  • the whole saliva used was donated by a single fasting individual and did not have detectable glucose before spiking in any of the assays.
  • the MBTH system compared to other chromogens, showed the greatest sensitivity and steepness of response with acceptable linearity in the target dynamic range.
  • Figure 6B emphasizes the performance of various systems in saliva and shows that the MBTH (in this case, with CTA) system is superior to others (and also that it behaves in saliva as in buffer, with the exception that the limit of detection is slightly higher).
  • Figure 7 shows the results in the final modification made to the MBTH assay; this was in linking color generation to reduction of MBTH and DMAB. This assay could detect 0.04 mg/dL glucose at the two standard deviations criterion (0.06 mg/dL in saliva). The percent coefficient of variation was less than 2% below 1 mg/dL and less than 0.6% when glucose exceeded 1 mg/dL (Figure 7B). Table 2 summarizes composition and methods used for the
  • each step requires its own subset of manipulations, such as centriguration or readjustment of pH to assay optimum.
  • Table 3 shows one experiment in which one sample of whole (unstimulated) saliva was processed according to the sequence outlined. Separate aliquots were spiked with glucose at 1.5 mg/dL or 0.1 mg/dL before sample treatment, and processed in parallel. After each processing step, the product was assayed using the MBTH/DMAB glucose assay. The % CV for
  • each assay (4 replicatesA) is shown in parentheses to indicate variability.
  • Table 4 illustrates an experiment of the type described above in which
  • Stimulation of salivation promotes collection of a filtered sample, collected in accordance with the present invention, which reflects whole saliva glucose in less time than in unstimulated saliva. This advantage apparently originates from reduced viscosity which will increase diffusability of glucose.
  • the deficiency in the large molecular weights mucopolysaccharides and mucoid proteins in stimulated saliva may also prevent "coating" of the sac membrane which could also interfere with flux of analyte.
  • Soluble protein was measured using the Pyrogallol assay; the insoluble material was measured as dry weight of the freeze-thaw pellet.
  • Na-K-ATPase from reduced time of exposure to ducted Na+ pump (Na-K-ATPase; 9).
  • Soluble protein (to the extent it can be discussed as single class) is not lowered
  • pH in the device varied between 6.9 and 8.2. This is an important
  • Figure 9 shows the effect of pH on the Vmax of the assay when it is
  • glucose following oral ingestion of glucose can be explained as an artifact of
  • Figure 10B shows a similar
  • glucose collected in accordance with the present invention referred to as glucose collected in accordance with the present invention
  • the threshold for saliva glucose is a blood glucose of approximately 100 mg/dL or less.
  • the entrance criteria for this study was a blood glucose of greater than or equal to 250 mg/dL. Subjects were not required to fast overnight, but were asked to
  • FIG 11 A shows glucose collected by the present invention plotted
  • SalivaSac and SalSac in the figures indicates use of
  • glucose collected by the present invention values in hyperglycemic subjects
  • Figure 11 combines the data obtained in the study of diabetics with
  • the present invention obtains a saliva sample that corresponds with the blood

Abstract

A non-invasive glucose monitoring device (10') includes a mechanism for stimulating salivary glands secretion of saliva into oral fluid, preferably using citric acid contained in an absorptive matrix (28), prior to collecting a sample of the oral fluid in membrane sac (12'). A test strip mechanism (26) is provided for detecting the amount of glucose in the sample, and for quantitating blood glucose level based on the amount of glucose detected. A method of non-invasive monitoring glucose levels includes the steps of stimulating salivary glands secretion of saliva into oral fluid, collecting a sample of the oral fluid, detecting an amount of glucose in the sample, and then quantitating the blood glucose level based on the amount of glucose detected.

Description

GLUCOSE DETECTOR AND METHOD
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to an apparatus and method for
determining blood glucose content by the collection and analysis of oral fluid.
More particularly, the present invention non-invasively collects oral fluid, oral
fluid glucose content is determined, and blood glucose levels are derived based
upon the amount of glucose detected.
2. Description of the Related Art
The pathogenesis of diabetes originates in sustained or periodic
elevations of blood glucose and glucose in tissues secondary to a deficiency in,
or insensitivity to, insulin. Glucose is linked non-enzymatically to accessible
reactive sites of proteins causing altered structure and function which leads in
time to diseased organs. The grade of gly cation depends upon glucose
concentration and the amount of derivitized protein accumulated depends upon
the lifetime of the individual proteins effected. Accordingly, the significance
of maintaining reduced glucose concentrations is widely accepted. Although early studies focused on Type I patients (Cohen, 1988), it is
generally believed that Type II individuals and others not taking insulin would
benefit from better diabetic control. Although many patients tolerate the pin
prick necessary for the taking of an actual blood sample, followed by blood
analysis, a bloodless, quick and convenient test using saliva can enlist Type II
individuals into an effective, better diabetic control. Type I persons would
also benefit to the extent that a bloodless test would reduce the number of
finger sticks required. The existence of a convenient, non-invasive test can
also permit prescreening of a large number of individuals using the newly
promulgated 126 mg/dL criteria.
Many prior art patents discuss the analysis of glucose in various fluids,
including saliva, but do not discuss the relationship of determining blood
glucose from saliva levels nor do they discuss any specific devices for
obtaining the same. For example, U.S. Patent 3,947,328 to Friedenberg et al.,
issued March 30, 1976, discloses a method, apparatus and test compositions
for a rapid, accurate test of concentration levels of various components of body
fluids, including glucose levels in saliva. An oxidizing test is utilized to
determine the levels, but no relationship is disclosed relating glucose analysis
in saliva to blood levels of glucose. U.S. Patent 5,139,023 to Stanley et al.,
issued August 18, 1992, discloses a method and apparatus for non-invasive
blood glucose monitoring. Blood glucose is monitored non-invasively by
correlation with the amount of glucose which permeates an epithelial membrane, such as skin or a mucosa membrane within the mouth. However,
the Stanley patent specifically states that it is undesirable for such a sample to
be contaminated by oral fluid, specifically saliva. Although the Stanley et al.
patent discloses the step of taking a sample from inside the mouth, the sample
taken is not a sample of oral fluid or saliva.
U.S. Patent No. 5,056,521 to Parsons et al, issued October 15, 1991,
discloses an absorbent non-reactive collecting swab which is brought into
contact with a favorable surface of the oral cavity. An interstitial transudate is
selectively collected from the vestibule region of the oral cavity at the
conjunction of the superior labal mucous membrane and the superior gingivae
between the upper canine teeth. The fluid collected is then squeezed out from
the swab into a monitoring instrument located off site. The patent goes into
great detail to note that, although general statements are made with regard to
oral fluid, the system requires that the sample be taken from the specific
mucous membrane described above so that the sample is devoid of
uncontrolled oral fluid that might distort the glucose level in the sample by the
diluting the desired fluid (namely, interstitial transudate, column 3, lines 35-
40, of the Parsons et al. patent). From this sample, glucose levels of the
sample itself are determined, the specification being devoid of any teaching of
how blood levels of glucose can then be obtained. Hence, the Parsons et al.
patent does not disclose any method or apparatus for utilizing whole oral fluid to determine blood glucose levels and, in fact, teaches away from using the
same or from diluting a sample with such oral fluid.
In view of the above, it would be desirable to develop a non-invasive
means for determining blood glucose levels. It is also desirable to provide a
simple means for doing so which does not require exclusion of oral fluid from
a bucual cavity device.
SUMMARY OF THE INVENTION AND ADVANTAGES
In accordance with the present invention, there is provided a
non-invasive glucose monitoring device including stimulation means for
stimulating salivary gland secretion of saliva into oral fluid and collection
means for collecting a sample of the oral fluid. Detection means, operatively
connected to the collection means, detects an amount of glucose in the sample
and quantitation means operatively connected to the detection means
quantitates blood glucose levels based on the amount of the glucose detected.
The present invention also provides a method of monitoring blood
glucose by stimulating salivary gland secretions of saliva into oral fluid,
collecting a sample of the oral fluid, detecting an amount of glucose in the
sample, and finally quantitating blood glucose level based on the amount of
glucose detected. DESCRIPTION OF THE DRAWINGS
Other advantages of the present invention will be readily appreciated as
the same becomes better understood by reference to the following detailed
description when considered in connection with the accompanying drawings
wherein:
Figure 1 is a perspective view of an oral fluid collection device made
in accordance with the invention;
Figure 2 is a cross-sectional view based substantially along lines 2-2 of
figure 1;
Figure 3 is a perspective view of a second embodiment of the
invention;
Figure 4 is a perspective view of a third embodiment of the invention;
Figure 5 is a schematic plan view of a fourth embodiment of the
present invention;
Figures 6A-B are graphs showing glucose standard curves in buffer or
saliva indicating a comparison of selected chromogens wherein Figure 6A
shows spiked buffers and saliva and Figure 6B shows only spiked saliva;
Figure 7A-B are graphs illustrating a glucose standard curve wherein
Figure 7A is a standard curve for in phosphate buffer and Figure 7B is a
standard curve and assay variation;
Figure 8 is a graph showing the time to saliva glucose equilibrium in
the subject invention; Figure 9 is a graph showing the effect of pH on the glucose assay;
Figure 10A-B are graphs showing oral glucose contamination of saliva
following ingestion, wherein Figure 10A shows oral glucose ingestion being
present and Figure 10B are results where there was no ingestion of glucose;
Figure 11 A-C are graphs showing glucose collected by the present
invention compared to finger stick glucose (A and C) and venipuncture (C) in
hyperglycemic and normal subjects, Figure 11A showing the results of 13
diabetic subjects, Figure 1 IB showing a collection of data from subjects from
the present study and an earlier study as described in the specification; Figure
11C showing glucose collected by venipuncture vs. SalivaSac® glucose, and
Figure 12A-B are graphs showing a correspondence between saliva
glucose and venipuncture blood, Figures 12A showing venipuncture vs.
stimulated subject using the SalivaSac® (present invention) for collection of
saliva, and Figure 12B shows the same comparison but using all subjects, not
only stimulated subjects using the SalivaSac®.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a non-invasive glucose monitoring
device and method, the device including a mechanism for stimulating salivary
gland secretion of saliva into oral fluid, a collection apparatus for collecting a
sample of the oral fluid, a detection mechanism operatively connected to the
collection device for detecting an amount of glucose in the sample, and a quantitation mechanism operatively connected to the detection mechanism for
quantitating blood glucose level based on the amount of glucose detected.
Thus, the elements of the present invention most generally are (1) simulation
of salivation; (2) insertion of a collection device into the mouth for the period
of time required for the contents to reach equilibrium with whole saliva; (3)
withdrawal from the mouth of the collection device and transfer of the sample
to a detection mechanism, such as a qualitative test strip as discussed below in
which glucose concentration is estimated; and (4) means for calculation of
estimated blood glucose. Such a system can be a integrated device wherein
stimulation, collection, and quantitation are accomplished on a single strip or
can be a non-integrated device, for disposal, in or out of the mouth, as
discussed in greater detail below. The device is non-invasive, so it removes
resistance to testing and can be used in public. It can be made inexpensively,
thereby lowering economic barriers to benefits of the device. It can be a single
use device and thereby avoid the spread of an infection and is also easily
transportable. It is also a simple device thereby requiring little to no training
for its use. Hence, the present invention, as most broadly defined, provides
significant improvements over the prior art.
More specifically, the term "oral fluid" is not simply saliva, but rather
the liquid contents of the mouth which include cellular secretions, components
from food, saliva, as well as other components which may be secreted into the mouth, regurgitated into the mouth, or brought into the mouth by airborne
means.
Oral fluid has a glucose concentration that has approximately 1/200 to
1/100 of the contemporaneous blood concentration. Accordingly,
measurement of oral glucose can be used to estimate blood glucose.
Prior to the development of the present invention, there were few
reports in the literature concluding that a general correspondence between
concentration of blood and saliva glucose or whole oral fluid glucose exists.
As stated above, many prior art devices excluded saliva and other oral fluid,
maintaining that the inclusion of such would cause inaccuracies in glucose
measurements. Borg and Berkhed (1988) demonstrated the correlation
following oral loading with 75 grams of glucose. In accordance with the
present invention, it is proved that Borg and Berkhed were measuring an
artifact in which contamination of oral mucosa in the interval following
ingestion of glucose falsely mirrored the rise in blood. Reuterving et al.
(1987) measured glucose secretions of three individual salivary glands and
showed that the closest correspondence with blood is in fluid from the parotid.
These investigators also claimed the existence of a threshold for the spill over
of the plasma glucose into the saliva of 10-15 mmL/L (180-260 mg/dL). A
threshold of this type is analogous to well characterized glucose threshold of
renal tubules. If this threshold identified by Reuterving et al. is accurate, then
saliva cannot be used to detect glucose below about 200 mg/dL. The data disclosed in the example section below shows that if a
threshold exists, it must occur at blood concentrations substantially less than
200 mg/dL. The problem with the published work cited above is that the
investigators used a standard Trinder assay, and the analytical variations seen
in whole saliva, particularly at the lowest concentrations, render conclusions
on detection of "zero" saliva glucose highly suspect. It is concluded based
upon the present work that a new, more sensitive glucose oxidase-peroxidise
chemistry in combination with the present invention makes it possible to
follow saliva glucose concentrations to the lower concentrations secreted as
blood declined to hypoglycemic levels. The results set forth herein show a
threshold for saliva glucose to exist at least as low as 70-100 mg/dL,
depending on the subject, approximately at least one half of the blood
concentration specified by Reuterving et al. Based on the above, the present
invention is at least useful as a diagnostic for elevated blood glucose and can
certainly be predicted to be useful for lower blood glucose as well.
The collection device, generally shown at 10 in Figure 1 and 2 is
preferably an oral fluid collection article disclosed in detail in U.S. Patent No.
4,817,632 to Schramm, issued April 4, 1989, and assigned to the assignee of
the present invention. The collection device is generally an ovoid small disc
or pillow-shaped article adapted to fit in the mouth of a patient. The article
includes a semi-permeable membrane 12 which defines an enclosed chamber 14. The chamber can include an osmotic substance 16 which is totally
enclosed by the semi-permeable membrane 12.
The semi-permeable membrane 12 is made of a substance which has a
plurality of pores which are of a suitable size to allow for the collection of oral
fluid or which acts as a filter for filtering out unwanted particulate matter or
larger molecules such as binding proteins from the sample. An example of
such a membrane is Cuprophan® manufactured by Enka AG, a division of
Akzo, Inc. This membrane is available as flat sheets or in a tubular form, both
of which can be cut to the appropriate size. The membrane is composed of
regenerated cellulose and has a nominal molecular weight cut-off of 12,000
daltons. The molecular weight cut-off, also termed the exclusion limit, is
central to the function of the semi-permeable membrane. The pore size of the
membrane is such that molecules larger than 12,000 daltons (such as proteins,
polysaccharides and particulate matter) cannot cross the membrane 12 to enter
the central compartment 14. In this way, the fluid obtained by the collection
device is filtered saliva (more specifically, ultrafiltered saliva), a uniform non-
viscous sample required for accurate measurement of glucose (molecular
weight, 180 daltons). Any membrane, filter, fabric, paper, mineral, plastic or
other material capable of allowing the passage of glucose while excluding the
viscous, particulate or cellular material of oral fluid, could be used in the
collection of filtered saliva. Other dialysis membranes having a range or
exclusion limits could also be used, provided such membranes are permeable to glucose and allow its transport from whole saliva to the central
compartment.
The osmotic substance 16 is soluble in oral fluid thereby providing an
osmotic pressure inside the chamber 14 for drawing oral fluid from the
mucosal cavity of the patient into the chamber 14. The membrane 12 retains
at least a portion of the oral fluid in the chamber 14 for later removal, as
discussed below. The osmotic substance can be a crystalline or an amorphous
material which is soluble in saliva and allows interference-free analysis of the
sample for whatever particular analysis is being undertaken to determine the
glucose levels. Alternatively, the osmotic substance can comprise a high
molarity solution of a crystalline or amorphous material which is dissolved in
water or some other non-interfering solute. The osmotic substance must be
non-toxic in nature and is preferably palatable.
The osmotic substance can also take the form of a stimulant of
salivation. For example, the osmotic substance can be selected from the group
including salts, sugars, amino acids, other organic acids and small peptides.
The preferable osmotic substance is one which dissolves readily when
hydrated by the moisture in oral fluid, establishes, when dissolved, an osmotic
pressure capable of drawing additional fluid across the filtering surface, and is
compatible with subsequent measurement of glucose in the sample obtained.
In example, the osmotic substance used in collection of samples forming of the
data presented in Figures 7-12 is sodium citrate. This salt also has the effect of stimulating salivation, the first element of the present invention. A mixture of
salts or other substances can also be used. An example is sodium citrate
mixed with a small amount of citric acid, the latter acting to further stimulate
salivation.
The basic elements of the present invention are retained if a non-
osmotic material is used to collect filtered saliva. For example, absorbents or
adsorbents can be used to collect saliva if they provide a method for the
separation of glucose from the viscous large molecular- weight materials of
whole saliva. Completely different physical forces and methods could also be
used to obtain a filtered sample of oral fluid. For example, a vacuum could be
created to draw oral liquid by aspiration through a filtering surface with
deposition of the glucose-containing fluid in a sink. Or a positive pressure
could be exerted on a saliva sample, forcing liquid through a rigid filtering
surface with elaboration of filtered liquid into a central or lower compartment.
One example would be a conventional filtration tube in which whole saliva is
forced from an upper to a lower camber by positive pressure or by
centrifugation, or by application of a negative pressure or vacuum to the lower
chamber. Though the preferred embodiments illustrated in Figure 1-5 are
based on the patent SalivaSac® with its features which allow direct insertion
into the mouth, the claims of the present invention are also extended to any in-
the-mouth or external device capable of producing a filtered sample of oral
fluid containing a concentration of glucose equivalent to that in whole oral fluid. The expanded claims embody specifically any device or method in
which expectorated saliva or oral fluid is processed further by a device
external to the oral cavity which obtains an accurate measure of glucose.
Stimulation of salivation has been found to be critical. Preliminary
data set forth herein is indicative that much of the controversy surrounding the
correspondence between blood and saliva glucose or full oral fluid glucose can
be traced to analytical imprecision associated with the sticky, viscous, and
generally variable qualities of whole saliva or whole oral fluid. Testing in a
limited number of subjects indicated that the blood-saliva relationship was
improved by the use of the ultrafilfrate obtained by the collection device after
citric acid stimulation was made in accordance with the present invention. It
was felt necessary to show that the contents of the collection device accurately
reflect whole saliva glucose concentration since it is whole saliva that is
derived in the first instance from blood; blood glucose enters the primary
secretion of salivary glands principally by paracellular diffusion through leaky
epithelial cellular junctions. The rate of diffusion (and thus the amount of
glucose transported per unit of time) will be increased as blood glucose rises.
A minor pathway is transcellular mediated by the apocerine secretion of
glandular cells (Baum, 1993). Accurate measurement of glucose in whole
saliva is possible provided numerous processing steps are first employed to
produce the equivalent of a filtered sample. Thus, glucose concentration in
whole saliva was determined after: (1) sonication of sample at 1600 Hz (hertz); (2) freezing and thawing the sample to precipitate large molecular
weight interferences; (3) centrifugation at 3000 x g for 10 minutes; (4) heating
the sample to 100° C x 10 minutes to eliminate glucose- and carbohydrate-
hydrolyzing activities (enzymes); (5) adjusting pH to optimal assay pH (pH
6.5-7.5). This procedure produced accurate measurement of glucose to 0.06
mg/dL, as shown by quantitative recovery of glucose spiked into such
samples. (It can be noted that the filtration properties of the preferred
embodiment of the present invention produce a sample that is equivalent to the
five-step processed whole saliva described immediately above).
The ability to measure glucose in saliva allowed for the reexamination
of the time necessary for the container made in accordance with the present
invention to reach equilibrium with whole saliva glucose. Subjects were
observed and were found to be variable in the time to reach equilibrium, but it
could require as much as 20 minutes. The more viscous and protein that
enriched the saliva, the longer the time needed to reach equilibrium.
Individuals with copious salvia, clear in appearance and relatively
impoverished in protein often reached equilibrium at approximately the time
the last of the crystalline osmotic driver in the container dissolved, six to seven
minutes.
As a consequence of these results, the desirability of a dilute saliva by
stimulation of salivation was recognized. The desirability of scaling down to a
smaller size of the collection device was also recognized. A small device will reduce diffusion distance and destination volume and will also increase surface
to volume ratio. These three factors are the principal determinants of the time
required to reach equilibrium with surrounding whole oral fluid.
It was further found, as is demonstrated in the experimental section
below, that stimulated saliva glucose more closely parallels blood glucose then
did unstimulated saliva. This was a critical discovery. It was also found that
stimulation of saliva secretion also reduced protein content of saliva and
elevated sodium concentration while having a modest effect on potassium.
The conclusion from the physiological finding above is that
stimulation forces saliva quickly through salivary ducts and this minimizes
reabsorption of glucose, water, and sodium ions by salivary gland ductal
transport systems. Therefore, stimulated saliva more nearly reflects the
composition of the primary filtrate-secretion elaborated by the secretory
portion of the salivary glands and it is this fluid that is derived by passive
diffusion from blood. Accordingly, as described above, it is preferred to
provide a stimulatory component. This is preferably accomplished, as stated
above, by stimulatory component being disposed within the container 10 for
release therefrom. As also stated above, the preferred stimulant is citric acid.
Referring again to figure 2, the semi-permeable membrane 12 may be
enclosed by an outer protective membrane 20 which includes macroscopic
pores and is disposed about and completely exposes the membrane 12. The
outer protective membrane 20 can be made of any material which would be generally pliable, tasteless, and non-toxic. Preferably, silicon materials or
other materials are selected which have substantial mechanical strength to
protect the inner membrane from damage due to biting by a patient and similar
hazards which may be associated with the use of the present invention in a
patient's mouth. The outer membrane can be made from many materials
whereby saliva can pass through easily, the material having microscopic pores
22. Alternatively, the present invention can include a container 10 as
described above without the use of the outer membrane 20, wherein the inner
membrane 12 is made of material of sufficient mechanical strength to survive
in the environment of the mouth of a patient.
A preferred embodiment of the subject invention is shown in figure 3
and 4. A device 24 is in the form of a test strip including a support 26. A
membrane sac 10', having a structure as described above, is mounted over one
end of the strip 26 and contains an absorptive matrix 28. Absoφtive matrix 28
can include the stimulator of salivary gland secretion, such as sodium citrate.
The absorbent matrix 28 is in fluid communication by abutment with a
threshold-type indicator film 30.
The film contains the enzymes glucose oxidase and horseradish
peroxidase (or some other peroxidase) and a combination of dyes and
accessory reagents, such as buffers and stabilizers, which are capable of
producing a colored spot or line in which color intensity is proportional to the
amount of glucose in the sample. Glucose oxidase applied as a dry reagent to the strip hydrolyzes sample glucose to gluconic acid with production of
hydrogen peroxide The peroxidase converts the peroxide product to water and
uses the electrons produced to react with the dyes to form a colored
compound. The color intensity, as noted, is scaled to the amount of glucose
initially present in the sample. Numerous enzyme-based glucose-sensitive
strips of the general type described exist. Various dyes have been used to
generate the final color product. Some of these are described in the
experimental section herein.
The present invention includes any type of solid-phase strip chemistry
capable of determining glucose at the concentrations existing in filtered saliva
or oral fluid. Moreover, as the essential elements of this invention are the use
of a filtered and stimulated saliva, any method of glucose measurement could
be associated with the processed sample. These include, but are not limited to,
other enzyme-based system (e.g., using hexokinase or glucose dehydrogenase
or any glucose metabolizing enzyme), chemistry-based systems (e.g., a
specific glucose reagent producing some quantifiable signal), and glucose
sensors (e.g., glucose-specific electrochemistry).
Utilizing this embodiment of the invention, oral fluid is collected
within the container 10' by the absorbent matrix 28. Upon contact with the
oral fluid, sodium citrate is dissolved and released through the container 10'
thereby stimulating saliva secretion. The collected oral fluid is retained in the
central compartment 28 for the period required for contents to reach equilibrium with whole oral fluid glucose. In a small collection device, this
time may be two or fewer minutes. The contents of the sac are then exposed
to one end of the colorimetric glucose strip. The mechanism retaining of the
filtered liquid can be a simple pressure-sensitive opening (port), or the rate-of-
flow of sample along the test strip can be made sufficiently slow to ensure that
sample has reached glucose equilibrium.
An alternative embodiment of the present invention is generally shown
at 32 in figure 4. A support strip 26', which can be similar to that shown in
Figure 3 supports a collection container 10" as described below. The
collection container, containing the absorbent matrix 28' which can also
contain the sodium citrate, is mounted adjacent a wicking material 34 in
communication with a thermometer-type indicator film 36. A thermometer
type film is one in which the enzymes and dyes required to produce the
colorimetric signal are arrayed from proximal to distal on the test section of
the strip. As sample moves through the test zone, glucose is depleted and
colored products are formed. When glucose is exhausted from the sample, no
further color development can occur in the distal enzyme field. The amount of
glucose in the sample is thus proportional to the linear distance of color
development. A thermometer-type strip requires that an accurately measured
fixed volume of sample be applied to the strip. This can be achieved in this
embodiment by creation of a saturable strip having a limited (and fixed)
capacity for liquid absoφtion, by timing the reaction to allow a known volume of sample to enter the test zone, or by application of a known sample volume
obtained by a chamber of defined volume between sample and strip. The flow
of liquid and its glucose up the strip proceeds by capillarity according to well
known principles. The strip may contain accessory elements, such as sample
volume adequacy indicators, as shown in Figure 5, additional filtration
materials, and test sections to check quality of reagents.
The indicator film can be graded to provide an indication of blood
glucose level correlated from the glucose content of the collected oral fluid.
Thus, the film 36 provides a detection mechanism, as well as a quantitation
mechanism. Alternatively, a container 10, 10' or 10", mounted on a strip
26, 26' or independent thereof shown in figure 1 and 2, can be transferred to a
detection device known in the art for glucose analysis. A glucose level can
then be correlated to blood glucose levels.
One such embodiment would require placing the strip into a reflectance
spectrophotometer similar to those currently used in monitoring blood glucose.
The strip could be moved to the monitor after the sample is introduced onto
the strip, or a small integrated monitor could be created to present a combined
replaceable strip-plus-collection device into the mouth or sample receptacle
(for the embodiment using a device external to the mouth to process the saliva
sample).
The correlation with blood is obtained by solving an equation which
relates blood glucose to oral fluid glucose concentration. For example, solving the linear equations shown in figures 11 and 12 for "x", will produce the blood
glucose concentration when the oral fluid glucose (y) is known. The exact
quantitative values of the constants in this equation have not yet been
determined. The nature of these constants could take one of two forms: (1) if
most individuals show the same saliva to blood glucose ratios, a single
equation can be developed for the subject populations; (2) or if individuals
show different ratios, then each individual will be required to calibrate the
saliva test against periodic measurements of their own blood glucose. In each
situation, a simple equation is produced. It is understood that in actual use, the
solution to the equation may be translated into an easily readable table or color
chart. In the embodiment in which the collection device and strip test are
incoφorated into a reflectance spectrophotometer, the computation of the
blood glucose concentration can be achieved by insertion of a dedicated
computational chip into the monitor. These electronics thus convert a
spectrophotometric signal into an estimated blood glucose value.
The preferred embodiment of the present invention is shown
schematically in figure 5. Again, the container 10'" includes an osmotic
component 40 contained within an inner membrane 12' and a citric acid
component 42. The container 10'" is mounted at the end of a wicking material
44 supporting a plunger 46 containing a needle 48 therein. The needle can be
used to puncture the outer and inner membranes 12', 42 to release the collected
oral fluid therefrom onto the wicking material 44. The fluid wicks across the material 44 to the indicator portion 46 . This embodiment allows for a
retention of the sample in the central compartment until the user elects to
admit the sample to the strip. Thus, the voluntary act of breaking a seal or
barrier is required. This embodiment would be used if it takes an unusually
long time for the collection device to reach glucose equilibrium in some
subjects, or if the subject prefers to analyze the sample at a later time.
Therefore, the device contains a membrane-osmotic driver collection
component, a dispenser of citric acid, a mount for attachment of the disposable
cross-strip, and a mechanism in the form of a pin or, alternatively, a pressure-
sensitive valve, to penetrate or open the container to allow a measured volume
of sample of oral fluid to be transferred to the test strip. Alternatively, as
shown in the various embodiments, wicking materials can be used as a means
for transferring an adequate sample as indicated by an adjacent indicator on
the strip.
The following experimentation demonstrates the usefulness of the
present invention.
Experiment I
As observed by Borg and Birkhed (1988), saliva glucose in whole oral
fluid rose and fell in concert with blood glucose from a finger stick following
an ingestion of bollus glucose (25, 50, and 75g) in non-diabetic volunteers.
However, utilizing the present invention, duplication was achieved of the oral elevation by having subjects dissolve glucose tablets in the mouth, followed
by expectoration without swallowing. In this experiment, there was no, or at
least only minor, elevations in blood glucose. It can be concluded that
following absoφtion of glucose by oral mucosea, tissue becomes a dominant
source or sink of salivary glucose. It can take two hours for saliva glucose to
reach precontamination baseline values. Various rinsing protocols using
water, concentrated sodium chloride, and glucose-free astringent mouthwashes
only modestly reduced the time to baseline. It was also determined that
routine meals not regulated for content have the same effect as glucose tablets,
though the degree of oral contamination was reduced compared with tablets or
concentrated liquid glucose.
Experiment II
It was previously hypothesized that saliva glucose could be detected
even in periods of hypoglycemia given the development of a highly sensitive
glucose assay. When such an assay was perfected, it was used to confirm the
existence saliva glucose threshold, though at about one-half the blood glucose
concentration claimed by Reuterving et al. (1987). The confirmation of the
threshold of 70 to 100 mg/dL in eight non-diabetic subjects led to the
investigation of saliva glucose levels in normal to hyperglycemic persons. A study in 18 diabetic subjects was initiated, the subjects being screened by
finger stick to ensure the existence of the study criteria of greater than 250
mg/dL. Subjects contributed whole saliva samples and samples collected
by a device made in accordance with the present invention. Subjects also
provided venipuncture blood for measurement of glucose by the reference
method. This method uses the enzyme hexokinase to phosphorylate (using
ATP) glucose to glucose-6-phosphate. Glucose-6-phosphate is next
converted to 6-phosphogluconate with reduction of NADP+ to NADPH, the
latter reaction read with a spectrophotometer (340 run) after a specified period
of time; the amount of NADPH produced is proportional to the amount of
glucose in the deproteinized sample.
The data reveal a correspondence between finger prick blood glucose
and glucose derived by the device made in accordance with the present
invention when blood and saliva samples are taken at the same time. The
correspondence with venipuncture glucose is also high, shown in Figure 12.
Experiment HI
A highly sensitive assay for saliva glucose was derived. Table 1 lists
most visible wavelength chromogens investigated, identifies the limits of
glucose detection (+2 standard deviations of blank in phosphate buffer), and
tabulates time to complete assay (high standard OD 1.2-1.8). These assays
were done in solution (96 well plate, sample volume 100 μL) at 37°C with
samples added last. Table 1. Visible Wavelength Chromogens in GO/HRP Glucose Assays
Investigated
* Coupled Reagents: Sensitivity Response Time to Completion mg/dL OD/mg/dL minutes at 37°
MBTH--
DMAB 0.04 0.18 15
CTA 0.06 0.13 20
3,6 CTA 0.12 0.08 >30
5,7 CTA 0.17 0.12 25
4-AA--
4-HBS 0.16 0.14 15
* Single Reagents:
O-Di 0.29 0.04 >30
OPD 0.16 0.08 not done
5-AS 0.25 0.02 >30
ABTS 0.15 0.08 15
TMB 0.22 0.11 20
*Names of compounds used in Appendix I. Assays done in phosphate or Tris buffers.
Figure 6A summarizes a subset of the visible chromogens used with glucose oxidase-peroxidase in development of a more sensitive glucose assay. Glucose was spiked into two different matrices: 20 mM phosphate buffer (pH 7.0), and whole saliva processed as described above but without heating to 100c x 10 min (saliva pH, 6.9). The whole saliva used was donated by a single fasting individual and did not have detectable glucose before spiking in any of the assays. The MBTH system, compared to other chromogens, showed the greatest sensitivity and steepness of response with acceptable linearity in the target dynamic range. Figure 6B emphasizes the performance of various systems in saliva and shows that the MBTH (in this case, with CTA) system is superior to others (and also that it behaves in saliva as in buffer, with the exception that the limit of detection is slightly higher). Figure 7 shows the results in the final modification made to the MBTH assay; this was in linking color generation to reduction of MBTH and DMAB. This assay could detect 0.04 mg/dL glucose at the two standard deviations criterion (0.06 mg/dL in saliva). The percent coefficient of variation was less than 2% below 1 mg/dL and less than 0.6% when glucose exceeded 1 mg/dL (Figure 7B). Table 2 summarizes composition and methods used for the
GO/HRP-MBTH/DMAB glucose assay in the remaining studies shown.
Table 2. Composition of GO/MBTH Glucose Assay
Solution Enzyme Chromogen Buffer
Horseradish DMAB* 100 mM PO4
Peroxidase 30 mM pH 7.5
12.5 U/ml
Glucose MBTH** 100 mM PO4
Oxidase 1.5 mM
37.5 U/mL
100 μL sample; 20 μL Solution 1; 20 μL Solution 2; Incubate 15 min at 37° or
25 min at room temperature. Read OD @ 600 nm.
* 3-dimethylaminobenzoic acid
** 3-methyl-2-benzo-thiazolinone hydrazone (dissolved in methanol at 15 mM) Experiment IV A series of experiments was performed to learn if whole saliva could be processed in a manner that would reduce variability and improve accuracy in assay of glucose. As summarized above, it was determined that both goals could be achieved only after treatment of saliva using four separate procedures:
sonication, mucoprotein precipitation using freeze-thawing, precipitation f soluble proteins using 10% TCA (trichloracetic acid), and heating processed saliva to 100°C for 10 minutes. In most cases, each step requires its own subset of manipulations, such as centriguration or readjustment of pH to assay optimum.
Table 3 shows one experiment in which one sample of whole (unstimulated) saliva was processed according to the sequence outlined. Separate aliquots were spiked with glucose at 1.5 mg/dL or 0.1 mg/dL before sample treatment, and processed in parallel. After each processing step, the product was assayed using the MBTH/DMAB glucose assay. The % CV for
each assay (4 replicatesA) is shown in parentheses to indicate variability.
Table 3. Effect of Processing Whole Saliva on Accuracy of Glucose Assay
Sample 0 mg/dL Spike(% CV) 1.5 mg/dL( % CV) 0.1 mg/dL(% CV)
Whole 1.71 (18.3) 3.63 (28.1) 1.92 (25.0)
Sonicated 1.94 (20.4) 2.79 (16.3) 1.37 (14.6)
Freeze-Thaw 0.75 (10.6) 1.48 (12.9) 0.86 (13.5)
TCA Ppt 0.64 (5.6) 1.63 (8.2) 0.68 (11.5)
Heat 100° 0.62 (5.8) 1.77 (7.3) 0.70 (9.9)
% Expected n Final Step: 83.5% 97.2% Spiked glucose was measured in saliva with approximately 80-110% recovery.
However, the saliva of individuals is quite different with less viscous samples
being less variable and requiring less processing. Capacity of the present
invention to obtain a sample which accurately reflects, at equilibrium, whole
saliva glucose was examined. One such experiment is shown in Figure 8. In
these four nondiabetic subjects, glucose collected and measured in accordance
with the present invention reached approximately ( ± 12%) the whole saliva
concentration in 26 minutes (each subject placed two devices in the mouth and
these were removed at 12 minutes and 26 minutes: The arrowheads on the
right indicate the glucose concentration measured in whole saliva collected
between minutes 26-29). These results showed that the contents collected did
equal concentration in whole saliva, though the time required was somewhat
longer than the earlier estimate. The longest times required seemed to be in
those individuals with the thickest whole saliva.
Subsequently, it became possible to obtain a less viscous saliva in all
subjects by stimulation with citric acid. With citric acid, time to reach
equilibrium with whole saliva glucose appeared to be reduced to 12 minutes.
Table 4 illustrates an experiment of the type described above in which
three nondiabetic subjects and one diabetic subjects had two devices, made in accordance with the present invention placed in the mouth, but on this occasion, following citric acid. When compared to whole saliva (collected after removal of the last device and reapplication of citric acid), most subjects showed glucose values from fluid collected by the subject device approximately equal to whole saliva by 12 minutes, but at least one required longer.
Table 4. Time to Reach the Equilibrium Glucose Concentration in a Device Made in Accordance with the Present Invention Following Citric Acid Stimulation of Salivation
Subject Time SalivaSac End Whole Saliva (minutes) Glucose Glucose (mg/dL) (mg/dL)
1 12 0.85 0.94 20 0.88
2 12 1.14 1.43 20 1.56
3 12 0.43 0.52 20 0.57
4* 12 3.27 3.67 20 3.35
Values are means of replicate determinations with Standard Errors less than 7.3% (SalivaSac) or 11.8% (whole saliva) of the mean. * Diabetic subject.
Stimulation of salivation promotes collection of a filtered sample, collected in accordance with the present invention, which reflects whole saliva glucose in less time than in unstimulated saliva. This advantage apparently originates from reduced viscosity which will increase diffusability of glucose.
The deficiency in the large molecular weights mucopolysaccharides and mucoid proteins in stimulated saliva may also prevent "coating" of the sac membrane which could also interfere with flux of analyte.
Subsequent investigation unexpectedly showed that glucose in stimulated saliva (whole processed saliva or when collected by the present invention) also showed closer parallelism with blood glucose than did unstimulated saliva.
Some explanation for this improved correspondence was gained by examination of certain biochemical properties of saliva which relate to mechanisms of secretion. In particular, it was investigated as to whether glucose absoφtion from the primary filtrate by salivary ducts might be minimized when flow through the ducts was maximized by stimulation. It was inferred that this is the case from the data presented in Table 5. It compares mean content of glucose, Na+, K+, soluble protein and total protein (and polysaccharides) in five individuals who contributed whole unstimulated and stimulated saliva within a
20 minute period. Soluble protein was measured using the Pyrogallol assay; the insoluble material was measured as dry weight of the freeze-thaw pellet.
Table 5. Concentrations of Protein, Sodium, Potassium and Glucose in Citric Acid Stimulated and Unstimulated Whole Saliva
Insoluble Soluble Na+ K+ (mM) Glucose Protein Protein (mM) (mg/dL)
(mg/mL) (mg/mL)
Unstimulated 7.5±1.2 0.4±0.2 10.4+2.9 5.7±1.9 0.6+0.4
Stimulated 3.3±0.9* 0.5±0.1 37.2±5.9* 8.8±2.5 1.3±0.5*
Values are means ± SEM; n=5. *p< 0.05, t-test. Increased glucose concentration in stimulated saliva is consistent with
reduced net reabsoφtion by the ducts. Likewise, the elevation in Na+ results
from reduced time of exposure to ducted Na+ pump (Na-K-ATPase; 9).
Stimulation of flow rate through the ducts would reduce net effect of any
reabsoφtive systems. The reality of a glucose reabsoφtive system is also
supported by existence of the saliva glucose threshold; the reduced amount of
glucose diffusing from plasma when its concentration is low can apparently be
completely cleared by the duct, provided flow rate is sufficiently slow.
Interestingly, the concentration of soluble protein is not significantly
effected by stimulation, whereas insoluble materials are reduced. The reduced
components are in the viscous, sticky material normally precipitated (in our
method) by freeze-thawing and centrifugation. Its lower content can be
observed in the "watery" saliva elaborated immediately upon stimulation.
Soluble protein (to the extent it can be discussed as single class) is not lowered
by stimulation; apparently secretion of some macromolecules is matched to
the volume discharged, and others (especially the larger moieties) are not.
There is no ready explanation for the elevation in K+ upon stimulation.
(3). It seems reasonable that with a reduction in reabsoφtion, saliva glucose
will more precisely reflect the concentration of glucose deposited in the primary filtrate of salivary secretions. And this concentration will, in turn, be
set by the free glucose concentration in plasma from which saliva glucose is
ultimately derived.
The performance of the GO-HRO-MBTH/DMAB assay in the device
of the present invention matrix following the dissolution of the Na3Citrate
osmotic drives was next examined. The focus was in the pH of this medium
and the possible consequences of the elevated sodium ion and citrate
concentrations. As sodium citrate is a weak base, it was found that in most
subjects, pH in the device varied between 6.9 and 8.2. This is an important
finding because the pH of the stimulated whole saliva is typically between 2
and 4, an effect of the acidic stimulant. Thus, Na3Citrate in the sac and citric
acid in the stimulant are acting as the conjugate pairs of a buffer, the effect in
the sac producing a pH in the optimal range of the enzyme assay.
Figure 9 shows the effect of pH on the Vmax of the assay when it is
performed in 500 mM Na3Citrate. The concentration of osmotic driver was
arrived at by measuring Na+ concentration (flame photometry) in several
samples after the equilibration period of 20 minutes in the mouth. Na+
concentration was approximately 1.5 M(range, 1.25-1.8 mM) and the citrate
concentrations was computed assuming that the ratio of Na/Citrate was
maintained at 3. Conditions prevailing in the sample collected by the present
invention are compatible with sensitive and accurate performance of the
solution version of the strip assay. Earlier research centered on development of a new sensitive glucose
assay and in defining conditions in whole saliva and in samples obtained by
the present invention that permitted accurate quantitation of saliva glucose. In
the remaining results shown, the assay was used in human subjects to establish
the basic feasibility of a saliva test as a potential substitute for blood tests.
Experiment V
Factors Influencing Diagnostic Specificity of Saliva:
Two requirements of this noninvasive approach are that saliva glucose
reflect blood glucose, and that the time lag in saliva be limited to minutes.
The latter point is proven in the literature [Reuterving et al., 1987). The
findings of Borg and Birkhed (1988) in which they showed a rise in saliva
glucose following oral ingestion of glucose can be explained as an artifact of
mucosal contamination which ostensibly duplicates the elevation in blood.
The basis of the skepticism was that previous papers reported levels of saliva
glucose that exceeded values observed and reported herein in subjects, even
when blood glucose was relatively high. These are shown in Figure 10 A
which illustrates the rise in saliva and blood glucose in one nondiabetic subject
undergoing a modified Oral Glucose Tolerance Test, in which 50g of glucose
(in 200 mL H2O)) was taken orally and whole saliva and blood (finger-stick)
collected for assay of glucose at 15 minute intervals. This experiment did not
use the present invention as shown in Figures 1 and 2 as it was necessary to sample frequently at intervals less than the device equilibration period. Both
blood and saliva glucose rise in the early period. Figure 10B shows a similar
experiment done in the same individual. In this case, however, the subject
rotated two 5g glucose tablets within his mouth for four minutes, and next
expectorated imdissolved tablets and saliva, and rinsed mouth once with water
prior to contributing whole saliva and blood samples. In this case, there was a
transient elevation in saliva glucose as in the earlier experiment, but this one
was not paralleled by blood glucose.
It is evident that glucose contamination of tissues of the mouth,
especially when oral glucose load is high, can be the dominant source of
glucose measured in saliva. The same contamination could apply when
glucose loading is reduced to the content in an average meal. Table 6 shows
that glucose collected in accordance with the present invention (referred to as
"SalivaSac") tends to be higher in some individuals one to two hours after than
immediately before lunch, even when blood concentrations increase only
modestly between sampling periods.
Table 6. SalivaSac Glucose and Blood Glucose Before and After Lunch
Subject Before Saliva Before Blood After Saliva After Blood
1 <0.12 77 1.22 103
2 0.18 89 5.13 98
3 <0.12 102 1.38 107
4 0.43 101 1.49 103
5 0.17 98 0.51 132
6 0.49 96 1.64 142
7 1.32 100 1.26 74
8 <0.12 82 0.46 92
SalivaSac® in mouth for 20 minutes after citric acid stimulation; blood glucose measured using the same finger-stick strips and monitor (One-Touch®). Meals unregulated for glucose content; after lunch samples taken 1-2 hour after meal. 0.12 mg/dL was the LOD (2 standard deviations criterion) in SalSac samples at the time this assay was done.
Experiment VI As discussed above, the threshold for saliva glucose is a blood glucose of approximately 100 mg/dL or less. A study in hyperglycemic individuals in which whole saliva and glucose collected by the present invention were compared with blood glucose in finger-stick and venipuncture samples. These data are presented as evidence in support of the contention that the present invention is feasible when blood concentrations are normal to elevated. The entrance criteria for this study was a blood glucose of greater than or equal to 250 mg/dL. Subjects were not required to fast overnight, but were asked to
refrain from eating for the three house before samples were taken in mid-
morning or mid-afternoon. Adult subjects meeting criteria placed a device
made in accordance with the present invention in the mouth after stimulation
with citric acid. The collection period was 20 minutes, after which subjects
also donated whole saliva and venipuncture blood.
Figure 11 A shows glucose collected by the present invention plotted
against finger stick glucose. Each subject used their own monitor to obtain the
finger stick glucose value. SalivaSac and SalSac in the figures indicates use of
the present invention. Variation in blood measurements by use of several
monitors of unknown precision or calibration might have contributed to scatter
in the correlation (Li et al., 1994). Nonetheless, there is a general
correspondence between glucose and blood glucose. It is also evident that
glucose collected by the present invention values in hyperglycemic subjects
exceeded the typical concentrations observed in normoglycemic persons.
Figure 11 combines the data obtained in the study of diabetics with
data previously obtained using four nondiabetic and one diabetic subject.
Each of the earlier subjects were sampled twice, once before lunch and once
after , and both measurements are included in the figure.
Figure 12 presents data from the same experiment with diabetic and
non-diabetic subjects. In this figure, the correlation between saliva glucose
collected by the present invention and glucose in venipuncture blood measured by the reference method (Hexokinase; see above) is shown. When blood
glucose exceeds approximately 70 mg/dL (in the normal range), there is a
close parallel between blood and filtered saliva. Data on Figures 11 and 12
show that whether blood glucose is measured by the finger stick method (as is
typical among diabetics) or by venipuncture (as occurs in medical practice),
the present invention obtains a saliva sample that corresponds with the blood
values. As noted above, the precise nature of the computation to estimate
blood glucose has not yet been determined, though its general form is shown
by the equations in Figures 11 and 12.
In view of the above, it can be concluded that glucose in saliva is
quantitatively related to glucose concentration in plasma from which it is
derived. The relationship is only effective to individuals and situations in
which blood glucose is greater than 70-100 mg/dL.
Further, the above data demonstrate the feasibility and utility of the
subject method wherein, generally, blood glucose is monitored by most
generally, stimulating salivary glands secretion of saliva into oral fluid,
collecting a sample of the oral fluid, detecting an amount of glucose in the
sample and then quantitating blood glucose level based on the amount of
glucose detected.
Throughout this application, various publication are referenced by
authors and years. Full citations for the publication are listed below. The
disclosure of these publications in their entireties are hereby incoφorated by reference into this application in order to more fully describe the state of the
art to which this invention pertains.
The invention has been described in an illustrative manner, and it is to
be understood the terminology used is intended to be in the nature of the
description rather than of limitation.
Obviously, many modifications and variations of the present invention
are possible in light of the above teachings. Therefore, it is to be understood
that within the scope of the appended claims, reference numerals are numerals
are merely for convenience and are not to be in any way limiting, the invention
may be practiced otherwise than as specifically described.
REFERENCES CITED
BAUM, 1993, "Principles of saliva secretion", Acad. Sci., 694:17-23
BORG and BIRKHED, 1988, "Secretion of glucose in human paroitid saliva after carbohydrate intake", Scand. J. Dent. Res., 96:551-556.
COHEN, 1988, "Non-enzymatic glycosylation proteins", Diabet. Ann., 4:469-484
LI, 1994, "Comparing self-monitoring blood glucose devices", Lab. Med., 25:585-590
REUTERVING et al., 1987, "Salivary flow rate and salivary glucose concentration in patients with diabetes mellitus", Diabet. Metab., 13:457-462.

Claims

CLAIMSWhat is claimed is:
1. A noninvasive glucose monitoring device comprising:
stimulation means for stimulating salivary gland secretion of saliva
into oral fluid;
collection means for collecting a sample of the oral fluid;
detection means operatively connected to said collection means
for detecting an amount of glucose in the sample; and
quantitation means operatively connected to said detection
means for quantitating blood glucose level based on the amount of glucose
detected.
2. A noninvasive glucose monitoring device of claim 1 including
a housing defining said collection means, said housing containing said
stimulation means for release into a buccal cavity.
3. A noninvasive glucose monitoring device of claim 2 wherein at
least a portion of said housing includes a porous material or means of injection
for dispensing said stimulation means therefrom and collection of oral fluid
therein.
4. A noninvasive glucose monitoring device of claim 3 wherein
said porous material is a dialysis membrane.
5. A noninvasive glucose monitoring device of claim 2 wherein
said housing consists of a semi-permeable membrane or some filtering surface
container defining an enclosed chamber.
6. A noninvasive glucose monitoring device of claim 5 further
including outer protective means disposed over said membrane container for
containing and protecting said membrane container.
7. A noninvasive glucose monitoring device of claim 3 wherein
said housing contains osmotic means for drawing the oral fluid into said
housing.
8. A noninvasive glucose monitoring device of claim 7 wherein
said stimulating means stimulates salivary gland secretion and draws the oral
fluid into said housing.
9. A noninvasive glucose monitoring device of claim23
8 wherein said stimulating means is citric acid.
10. A noninvasive glucose monitoring device of claim 3 including
a support strip including said housing mounted at a first end thereof, said strip
supporting said detection means and quantitation means therein and fluid
transfer means for transferring oral fluid from said housing to said detection
means and quantitation means.
11. A noninvasive glucose monitoring device of claim 10 wherein
said housing includes a sealed membrane container, said device including
piercing means for piercing an opening in said container to release the sample
into said transfer means.
12. A noninvasive monitoring device comprising:
oral fluid collection means for collecting a sample of oral fluid
and
blood glucose determining means for determining blood
glucose level from the sample of oral fluid.
13. A noninvasive fluid collection device comprising:
oral fluid collection means for collecting a sample of oral fluid
and
saliva stimulating means for stimulating salivary gland
secretion of glucose.
14. A noninvasive collection device comprising oral fluid
collection means for collecting a sample of oral fluid and oral fluid dilution
means for diluting the oral fluid to increase the rate of uptake of the oral fluid
into said collection means.
15. A method of monitoring blood glucose by :
stimulating salivary gland secretion of saliva into oral fluid;
collecting a sample of the oral fluid;
detecting an amount of glucose in the sample; and
quantitating a blood glucose level based on the amount of
glucose detected.
16. A method of monitoring blood glucose of claim 15 wherein
said collecting step is further defined as osmotically driving the oral fluid into
a collection container; or
used some other physical force to drive oral fluid through a
filtering surface into the sample receptacle.
17. A method of monitoring blood glucose of claim 16 wherein the
container is a sealed, puncturable container, said method including the further
step of puncturing an opening in the container to release the oral fluid
collected therein onto a detection step, said detecting step being further
defined as producing a readable signal on the strip indicative of the blood
glucose level.
18. A method of noninvasively monitoring blood glucose levels by
collecting a sample of oral fluid and determining a blood glucose levels
therefrom.
19. A method of noninvasively collecting oral fluid by collecting a
sample of oral fluid while stimulating saliva secretion.
20. A method of noninvasively collecting oral fluid by first diluting
oral fluid to increase oral fluid uptake in a collection device and then
collecting a sample of the oral fluid.
21. A noninvasive glucose monitoring device comprising:
collection means for collecting a sample of the oral fluid;
detection means operatively connected to said collection means
for detecting an amount of glucose in the sample; and
quantitation means operatively connected to said detection
means for quantitating blood glucose level based on the amount of glucose
detected.
PCT/US1998/009345 1997-11-03 1998-05-07 Glucose detector and method WO1999022639A1 (en)

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US09/298,398 US20010023324A1 (en) 1997-11-03 1999-04-23 Glucose detector and method for diagnosing diabetes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US6406797P 1997-11-03 1997-11-03
US60/064,067 1997-11-03
US09/072,115 US6102872A (en) 1997-11-03 1998-05-04 Glucose detector and method
US09/072,115 1998-05-04

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