Method of monitoring markers of bone metabolism

A method of monitoring markers of bone metabolism by continuously collecting a body fluid sample containing bone loss markers therein and analyzing the components of the body fluids for the markers of bone metabolism.

TECHNICAL FIELD 
The present invention relates to a method for detecting markers of bone 
metabolism. More particularly, the present invention relates to method for 
collecting and analyzing sweat for the presence of markers of bone 
metabolism. 
DESCRIPTION OF RELATED ART 
Current methods used to monitor the presence, progress of treatment, or 
disease state for metabolic bone diseases require the measurement of 
markers of bone metabolism found in blood or urine samples. Examples of 
these methods are shown in U.S. Pat. No. 5,283,197 to Robins, U.S. Pat. 
No. 4,973,666 to Eyre, and U.S. Pat. No. 5,140,103 also to Eyre. The most 
commonly measured of these markers include calcium, hydroxyproline, 
alkaline phosphatase, procollagen Type I and its cleavage products, 
osteocalcin, and bone collagen peptides that include crosslinked amino 
acids. The crosslinked amino acids include pyridinoline, hydroxy lysyl 
pyridinoline, lysyl pyridinoline, n-telopeptide, and the peptides that 
contain the former molecules. 
These molecules are specific collagen breakdown products known to be 
produced following bone resorption. Thus, the measurement of these 
crosslinked amino acids (markers of bone metabolism) can provide an 
indication of metabolic bone disease, and can be of use in monitoring the 
progress of medical treatment intended to reduce the loss of bone density 
found in various disease states. 
As with many other molecules of biological interest, the production of 
crosslinked amino acids varies over time in a diurnal cycle.sup.1,2,3 and 
can also vary in concentration from day-to-day.sup.4. Normal biological 
variations in the concentration of these collagen breakdown products in 
healthy individuals can nearly equal the levels of these molecules that 
are obtained in individuals with diagnosed disease states typified by high 
levels of metabolic bone loss over extended periods of time. Such disease 
states include diseases such as osteoporosis.sup.5,6, 
hyperparathyroidism.sup.7,8, Paget's disease.sup.5,8, rheumatoid 
arthritis.sup.9, multiple myeloma.sup.10, tumor-associated 
hypercalcemia.sup.11 and osteoarthritis.sup.10,12. The above-cited U.S. 
patents to Robins and Eyre describe methods for analyzing urine and blood 
samples in order to assay for levels of indicators of bone loss. However, 
the collection of a single blood or urine sample is representative of only 
a single point in time, therefore, any variation in the levels of markers 
of bone metabolism may not be discovered due to the natural diurnal and 
day-to-day variations in concentration inherent with these markers. 
Additionally, these prior art methods typically require the services of a 
technician, such as someone to draw a blood sample or these prior methods 
may require the subject go to a laboratory, a hospital or a doctor's 
office in order to submit a sample for analysis. This can be problematic 
where a subject must submit a series of successive samples for analysis in 
order to obtain enough test results to allow for a meaningful diagnosis. 
That is, since the results derived from the prior art methods are 
representative of only a single point in time; the subject must submit 
multiple blood or urine samples in order reduce the possibility that 
he/she will receive a false positive or false negative test result. This 
problem is not only one of inconvenience, but also represents a 
significant cost to the subject and the potential for misdiagnosis. 
In order to overcome the problems associated with the prior art method of 
analysis, it is necessary that a modality of collection be introduced 
which eliminates the biological variations due to diurnal and day-to-day 
variations by providing a means of collection which is continuous and 
uninterrupted thereby allowing for the collection of a more meaningful 
sample which has the benefit of being integrated over time to reduce the 
effects of biological variations in bone loss marker production. 
Additionally, the method should also increase the diagnostic power of the 
test by reducing the incidence of false positive and false negative test 
results. 
The present invention not only eliminates some of the burden and 
inconvenience associated with the prior art methods, it yields the added 
benefits of improved accuracy and performance by eliminating certain 
long-standing problems such as biological variation, thereby making the 
measurement of the bone loss marker more meaningful and greatly increases 
its significance in diagnosis and treatment of bone resorption disease 
states. The Applicant has found that markers of bone metabolism (collagen 
breakdown products) are present in sweat in detectable levels which are 
indicative of physiological bone loss. Additionally, applicants have found 
that by collecting a sample of sweat over a period of time, quantifiable 
results can be obtained which provide for a more accurate and 
representative assessment of bone resorption. 
The subject invention describes the use of a skin patch for the collection 
of perspiration or sweat which collects a sample for analysis in which the 
diurnal and day-to-day variations of crosslinked amino acids is minimized. 
Perspiration or sweat collected over a period of days accumulates in a 
sample that minimizes the sources of biological variation through 
integration over time, making the measurement of the bone loss marker more 
meaningful value and thereby increasing its significance in the diagnosis 
and treatment of disease states. The use of the skin patch also provides a 
means for specimen collection that can be performed at clinics, in a 
physician's office, or at home, thus, it is suitable for monitoring the 
efficacy of therapeutic regimens for metabolic bone disease and can be 
used in screening for the onset or the progression of bone diseases. 
SUMMARY OF THE INVENTION 
In accordance with the present invention, there is provided a method of 
monitoring markers of bone metabolism by continuously collecting a body 
fluid sample containing markers of bone metabolism therein and analyzing 
the components of the body fluids for the markers of bone metabolism.

DETAILED DESCRIPTION OF THE INVENTION AND ADVANTAGES 
The present invention provides a method for monitoring markers of bone 
metabolism in a sample of bodily fluid continuously collected over a 
period of time. In other words, the subject invention discloses a method 
of continuously collecting a bodily fluid, such as sweat, over a period of 
time in order to collect a sweat sample containing markers indicative of 
diseases involving bone loss or resorption or which indicate normal levels 
of bone metabolism (i.e., normal growth and death of bone). The collected 
sweat sample, then, can be analyzed to determine the presence of these 
markers of bone metabolism and thereby ascertain possible disease states 
or conditions. 
The continuous collection of a bodily fluid is defined to mean the 
uninterrupted collection of a bodily fluid over a given period of time. In 
other words, a sample of a bodily fluid is collected over a period of time 
without any gaps or breaks in the collection of the sample. This is unlike 
a typical blood or urine sample which is only representative of the point 
in time in which the sample was taken. Continuous and uninterrupted 
collection of a bodily fluid allows for the analysis of a sample which is 
representative of a period of time and, therefore, diurnal and other 
variations over time can be reduced, eliminated, or controlled. 
The continuous and uninterrupted collection of a one week long sweat sample 
for analysis poses a novel and, somewhat less cumbersome method than 
collecting a one week long total urine sample. The continuous and 
uninterrupted collection of sweat can be done without any subject 
participation and, therefore, subject compliance can be greatly enhanced. 
In other words, all the subject must do is either apply a collection 
device or have a collection device applied at the beginning of the 
sampling period and wear the collection device for the prescribed 
collection period. After application of the device, the subject is 
required to do nothing else except function normally. Unlike collecting 
total urine samples or successive blood sampling, the subject need only 
visit his or her clinician at the beginning of the collection period for 
application of the device and at the end of the collection period for 
removal and analysis of the sample collected in the device. Alternatively, 
the subject can apply and/or remove the device themselves. Therefore, this 
minimal amount of subject participation greatly increases the subject 
compliance with the sample collection and analysis thereof. 
Perspiration is defined as the functional secretion of sweat due to the 
secretory activity of sweat glands. Sweat is the liquid secreted by the 
sweat glands and primarily contains water and sodium chloride. However, 
sweat can also contain other compounds or analytes present in trace, but 
detectable, amounts such as urea, albumin and markers of bone metabolism. 
Generally, in order to collect a sweat sample, a sweat collection device is 
applied to a subject to be tested. The sweat collection device can be any 
device suitable for the collection of sweat or perspiration which is also 
designed such that the aqueous component (i.e. water) is allowed to 
evaporate from the collection device leaving behind only non-volatile 
matter attached to or trapped within an absorbent pad disposed within the 
device. That is, in order to more precisely maintain normal sweat or 
perspiration rates, the collection device must allow the aqueous component 
of the sweat to evaporate normally. Any collection device which alters the 
normal evaporation of sweat or gas exchange of the skin with its 
surroundings may induce or lead to the production of inaccurate data due 
to abnormal or induced physiological response of the skin to the 
artificial environment created by the application of the collection 
device. 
Typical sweat collection devices can include an adsorbent pad such as a 
cotton gauze, synthetic pad, other permeable materials, a capillary tube, 
or skin patch devices, such as those described in U.S. Pat. Nos. 
4,957,108, 5,076,273, and 5,203,327 all to Schoendorfer et al. and all 
assigned Sudor Partners, Inc., all of which are incorporated herein by 
reference. The sweat collection devices disclosed in these patents are all 
trans-dermal sweat collection devices which allow for the collection of 
one or more analytes in a bodily fluid expressed through the skin to be 
collected in a patch and concentrated by active and passive driving off a 
substantial portion of the water fraction under the influence of body heat 
(active) and air evaporation (passive). Said another way, these sweat 
collection devices allow for the recovery of the analytes present in the 
sweat while allowing for the normal evaporation of fluids and gas exchange 
of the skin with the surrounding atmosphere. 
Each of the devices described in the Schoendorfer et al. references refers 
to a transdermal sweat collection patch which contains an absorbent pad 
for collection of analytes present in the collected sweat samples which is 
affixed to the subject by means of an adhesive such as adhesive tape or 
other means known to those skilled in the art. Analytes present in the 
sweat samples are deposited on the absorbent pad and the aqueous and 
volatile contents of the sweat are eliminated by evaporation and normal 
gaseous exchange with the environment, respectively. 
The sweat collection patches are applied over the prepared area of skin and 
are worn by the subject for a prescribed collection time period. 
After the patch has been worn for a sufficient period of time, the patch is 
removed from the subject and is prepared for analysis. The absorbent pad 
is separated from an adhesive backing and is prepared for analysis by 
placing the absorbent pad into a vacuum desiccator to ensure uniform 
dehydration. The analytes, i.e. markers of bone metabolism, are then 
extracted from the absorbent pad in a suitable extraction buffer in order 
to remove the analytes containing the markers of bone metabolism from the 
pad area by methods described below. 
Markers of bone metabolism are understood to include any products produced 
or given off during normal or abnormal growth and/or death of bone. These 
markers of bone metabolism can be used to determine normal, healthy 
conditions as well disease states. The markers of bone metabolism may be 
any of a group including crosslinked amino acids, such as pyridinoline, 
hydroxy lysyl pyridinoline, lysyl pyridinoline, substituted pyridinolines, 
n-telopeptide, procollagen Type I and its cleavage products, and 
osteocalcin. The preferred markers for use in the method of the present 
invention are pyridinoline and n-telopeptide since they are compounds 
known to be indicative of bone resorption and are present in the sweat in 
amounts which are detectable and representative of resorptive bone 
diseases. 
After the markers of bone metabolism, i.e., pyridinoline, have been 
extracted from the absorbent pad, the extract containing the markers of 
bone metabolism is analyzed by enzyme immunoassay (EIA), or by any other 
method well known in the art which is capable of both quantitatively and 
qualitatively detecting the presence of desired markers, in order to 
quantitatively determine the concentration of the bone loss marker and 
qualitatively determine the presence of markers of bone metabolism. 
In order to more accurately determine the concentration of the bone loss 
marker present in a given sweat sample, the volume of sweat is normalized 
by quantitatively analyzing the concentration of a reference marker in the 
sweat collected concurrently with the sweat sample from the subject. The 
reference marker may be any analyte secreted transdermally at a relatively 
constant and known rate. Such a reference analyte can typically include 
creatinine, urea, potassium or any other analyte secreted at a constant 
rate. By quantitatively measuring the concentration of a specific 
reference marker, it is possible to determine the volume of sweat which 
has been transdermally secreted over the given collection period. Knowing 
the approximate volume of sweat secreted during this time period provides 
an accurate estimate of the volume of sweat collected thereby allowing an 
accurate calculation of the concentration of the bone loss marker present 
in the sample. Therefore, a quantitative value for the concentration of a 
bone loss marker, integrated over a given time period, is obtained which 
is free of diurnal and day-to-day variations. This method provides for the 
continuous and uninterrupted collection of a sample over a period of time 
which eliminates the uncertainty and variability associated with a "point 
in time" sampling method such as a blood or urine analysis. 
EXAMPLES 
Materials and Methods.sup.1 
FNT .sup.1 Abbreviations: EB, extraction buffer (10 mM NaPO.sub.4 pH 7.4, 0.02% 
thimerosal, 0.1% Triton X-100); PYD, pyridinoline. 
REAGENTS 
From Sigma Chemical Co. (St. Louis, Mo.) sodium phosphate, thimerosal, 
Triton X-100, Tween 20 and assay kits for the determination of creatinine 
and urea. The osmolarity of samples was measured by use of a vapor 
pressure osmometer (Wescor model 5500, Logan Utah.). 
SWEAT SAMPLE COLLECTION 
The area of the skin to which skin patches were applied was wiped for 
.about.30 seconds with a sterile alcohol prep pad (Professional 
Disposables, Inc., Orangeburg, N.Y.), and the skin was allowed to dry 
completely (.about.2 min.) before patch application. Care was taken not to 
touch the absorbent pad of the skin patch at any time. Patches were 
applied by stretching the skin slightly to eliminate wrinkles. Each patch 
was placed over the prepared area and was smoothed from the center toward 
the periphery. The time of skin patch application was recorded, and 
patches typically were removed at 24 hour intervals, .+-.1/2 hour. Pads 
were separated from their adhesive backing and were stored in plastic bags 
at 4.degree. C. 
EXTRACTION 
Skin patches (Sudormed, Inc., Santa Ana, Calif.) were dried in a vacuum 
desiccator overnight to assure uniform hydration. The patches were placed 
in 3 mL syringes (Becton Dickinson Company, Rutherford, N.J.) along with 
1.0 mL (1.0-2.5 ml) of the extraction buffer (EB; 10 mM NaPO.sub.4 pH 7.4, 
0.02% thimerosal, 0.1% Triton X-100). The syringes were agitated on a 
rotary platform shaker at 180 rpm for 3 hours at room temperature in the 
dark, and the extract was expelled for analysis. Patch extracts were 
filtered by centrifugation through 0.1 .mu.m Whatman (Clifton, N.J.) spin 
filters at 2000.times.g for 10 min. Extracts were stored at 4.degree. C. 
in the dark. 
RECOVERY FROM SKIN PATCHES 
100 .mu.L aliquots of solutions containing pyridinoline and creatinine were 
placed onto duplicate skin patches. The patches were dried in a vacuum 
desiccator overnight and then were extracted with 1.0 mL of EB. Samples 
were analyzed for pyridinoline by EIA and for creatinine using alkaline 
picrate. 
ANALYTICAL METHODS 
Pyridinoline. Pyridinoline in sweat was determined by modifying an enzyme 
immunoassay kit designed to measure pyridinoline in serum (Special Edition 
Collagen Crosslinks.TM. Kit; Metra Biosystems, Inc. Mountain View, 
Calif.). Pyridinoline standards and controls from the kit were diluted 
500-fold with extraction buffer. In coated microliter wells, 100 .mu.L 
(50-200 .mu.L) of standard, control or sample was reacted overnight at 
4.degree. C. with 50 .mu.L (25-150 .mu.L) of primary antibody. After 
washing the wells, 150 .mu.L of second antibody-enzyme conjugate was 
added. Following one hour incubation at room temperature, the wells were 
washed and 150 .mu.L of substrate (p-nitrophenol) solution was added. The 
absorbance at 405 nm was measured in a plate reader (Titertek Multiskan 
Plus, ICN Biomedicals, Costa Mesa, Calif.) after a one hour incubation. 
Creatinine. Creatinine was measured using minor variations of a common 
clinical method using the Jaffe reaction (alkaline picrate; Metra 
Biosystems, Mountain View, Calif.). The creatinine standard was diluted 
10, 100 and 1000-fold to include the low level of the analyte found in 
sweat samples. In microliter wells we placed 50 .mu.L of standards or 
sample and 100 .mu.L alkaline picrate, and absorbance at 492 nm was 
measured in the Titertek plate reader. 
Urea. The urea nitrogen assay kit (Sigma) was used to measure both urea and 
ammonia in sweat samples, based on the hydrolysis of urea into ammonia and 
carbon dioxide. The total concentration of urea and ammonia (T) was 
measured by reacting samples with urease, followed by reaction with 
hypochlorite and phenol which, in the presence of sodium nitroprusside, 
forms the blue chromophore, indophenol. Indophenol was quantified by 
absorbance at 600 nm. Ammonia (A) was measured by omitting urease in the 
same reaction scheme. The amount of urea in a sample prior to urea 
hydrolysis (U) was calculated by subtracting the ammonia value from the 
total value found for both ammonia and urea (U=T-A). 
Potassium. Potassium in sweat was determined by means of an ion specific 
electrode (Cole Parmer Instrument Company, Niles, Ill.). The electrode was 
calibrated against dilutions of a 1.0 g/L potassium standard (Cole 
Parmer). 
Results 
Recovery of pyridinoline spiked onto unworn skin patches. Pyridinoline 
recovery in the patch extracts was nearly quantitative when patches were 
extracted with EB (FIG. 1). Similar results were obtained using 0.1% Tween 
20 instead of Triton X-100 and also when the buffer was omitted. We 
selected EB as the standard method due to the stabilizing effect of the 
buffer and the lack of interference of Triton X-100 on the immunoassay 
(data not shown). 
SOURCES OF ASSAY INTERFERENCE 
It is possible that artificially low or high levels of pyridinoline or of 
reference markers was measured due to sample instability, contamination or 
other assay interference. Significant potential sources of error include: 
a) photolysis of pyridinoline by ultraviolet light; b) the effect of skin 
enzymes or bacterial action on analyte levels; and c) the effect of shed 
epidermis on measurements. 
Elimination of photolysis. Pyridinoline was spiked onto patches that were 
then exposed to direct sunlight for 21 hours (FIG. 2). Patches that were 
shaded by aluminum foil gave no significant loss of analyte. Most 
pyridinoline in uncovered patches was lost, and covering patches with thin 
cloth did not completely protect the pyridinoline. Thus, for the purpose 
of measuring pyridinoline in sweat, the skin patch should include a layer 
of material that is opaque to ultraviolet light to prevent photolysis of 
pyridinoline. 
Effect of bacterial action. Compounds that accumulate in the skin patch are 
exposed to conditions that might favor growth of microorganisms. 
Effect of cellular debris. Pyridinoline is found in collagen from a variety 
of tissues, but it has not been found in skin. While there may be no 
pyridinoline in skin, it is possible that cellular debris collected in the 
patch will interfere non-specifically in the pyridinoline assay. To test 
for the presence of such assay interference, skin patches were worn by 
volunteers, the patches were extracted and aliquots of the extracts were 
filtered through Whatman nitrocellulose spin filters (0.1 .mu.m) and 
assayed for pyridinoline. Results of the comparison of filtered and 
nonfiltered samples (FIG. 3) suggest that there may be no significant 
assay interference due to protein and cellular debris in the extract; the 
calculated values average 103% of the measured values for nonfiltered 
samples. 
MEASUREMENT OF PYRIDINOLINE IN SWEAT SAMPLES. 
Pyridinoline levels based on location on the body. Levels of pyridinoline 
in sweat collected in skin patches worn on the trunk (abdomen and lower 
back) and on the extremities (upper arms and legs). In all individuals 
tested, the mass of pyridinoline was greater in patches worn on the trunk 
than those on the extremities (FIG. 4). 
Pyridinoline levels based on age. Levels of pyridinoline measured in sweat 
collected in from a variety of individuals, ranging in age from 2 to 50 
years. In all cases, skin patches were worn on the lower back. The mass of 
pyridinoline was greater in very young children, and, among adults, 
highest among females (FIG. 5). 
Pyridinoline levels based patch contact time. Skin patches worn for longer 
time accumulate a greater mass of pyridinoline (FIG. 6). In all cases, 
skin patches were worn on the lower back. The rate of accumulation of 
pyridinoline was similar in many cases. 
MEASUREMENT OF REFERENCE MARKERS. 
Three candidate compounds were selected creatinine, urea and potassium, 
based on their high concentrations in sweat and relatively constant 
concentrations at varying sweat rates. It is possible that none of these 
analytes will suffice as a sweat volume marker, and there are others we 
might need to test, but this will be known only after sufficient data have 
been collected. In addition to these analytes, we will test the utility of 
specific gravity of patch extracts as an index of sweat volume. 
Creatinine in sweat. Levels of creatinine in sweat did not increase rapidly 
with an increase in the length of time a patch was worn (FIG. 7). Urea in 
sweat. The mass of urea increased at about the same rate for all 
individuals tested, leveling off when patches were applied for greater 
than five days (FIG. 8). 
Potassium in sweat. As with urea, the levels of potassium in sweat from 
several individuals increased with the length a patch was worn, and the 
accumulation of potassium dropped off when patches were worn for greater 
than five days (FIG. 9). 
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 
description rather than of limitation. 
Obviously, many modifications and variations of the present invention are 
possible in light of the above teachings. 
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