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Description  |
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BACKGROUND OF THE INVENTION
This invention relates to a method and apparatus to monitor, detect and
determine cholesterol levels and more particularly, it relates to a method
to monitor high cholesterol levels in a non-invasive manner with photon
correlation spectroscopy.
Cholesterol is a steroid alcohol and is present in animal cells and body
fluids. It is known that high levels of cholesterol of the blood lead to a
build up of plaque on the wall of arteries and veins thereby restricting
blood flow and causing a dangerous health situation. The condition of an
excessive amount of plaque build up in the arteries and veins is known as
atherosclerosis. Build up of excessive amount of plaque in the arteries is
called artereosclerosis.
There is a school of thought which indicates that high cholesterol levels
in the blood are caused by a person's diet. Those foods which have a
significant cholesterol content are meat, poultry, fish, eggs, dairy
products (including cheese, cream, milk and yogurt), fats and oils
(including butter, lard, shortening, margarine, mayonnaise, peanut butter,
palm oil, coconut oil and soybean oil). See Winston, M., A. Owen,
Measurement and patterns of food consumption in the U.S., Cholesterol and
Coronary Disease . . . Reducing the Risk, 1:5 (1987). Diet is also
responsible for raising low-density lipoprotein (LDL) levels and can
contribute to hypercholesterolemia. See Grundy, S.M., The effects of diet
on plasma cholesterol, Cholesterol and Coronary Disease . . . Reducing the
Risk, 1:1 (1987). Most LDL is derived from the catabolism of very
low-density lipoproteins (VLDL) which are secreted by the liver. Saturated
fatty acids, which are found in animal and plant fats, generally raise the
plasma LDL level. The most potent cholesterol raising fats are coconut
oil, butter fat and palm oil. Meat fats, such as beef, pork and chicken,
and cocoa butter, the fat in chocolate, raise the plasma cholesterol level
approximately half as much as palm oil.
Early detection of an abnormally high cholesterol level is important in
order to prevent the more serious problems associated with
artereosclerosis. Early detection is important because if detected early,
proper diet can be prescribed by a physician which will prevent the build
up of the condition which leads to atherosclerosis.
A significant drawback with monitoring or measuring cholesterol levels from
blood samples is that this technique obviously requires the withdrawal of
blood from a patient. It also requires a significant amount of time for
the blood to be analyzed and the results returned to the physician for
diagnosis and prescription of a proper diet.
SUMMARY OF THE INVENTION
In accordance with the present invention, it has been discovered that light
scattering characteristics of both the lens and the aqueous of a human eye
change in relation to the cholesterol level in a patient. Furthermore,
equipment presently exists which with modification is capable of measuring
these light scattering characteristics and correlating those measurements
into a cholesterol level reading.
Accordingly, the present invention is a non-invasive technique to monitor
the level of blood cholesterol in a patient by measuring light scattering
characteristics of the anterior chamber of the eye. A reading is taken
from the aqueous using the technique of quasi-elastic light spectroscopy.
Any increase in the level of cholesterol is observed by a marked increase
in scattering of the aqueous as analyzed from these readings. In this
manner, early detection of an elevated blood cholesterol level can be
made, and appropriate steps can be observed to decrease that level.
It is therefore an object of the present invention to provide a method to
monitor the levels of cholesterol in a patient.
It is another object of the present invention to provide a method to detect
and/or determine and/or monitor the levels of cholesterol in a patient in
a non-invasive manner.
It is a further object of the present invention to provide a method to
detect and monitor the presence and level of cholesterol in a patient in a
non-invasive manner by examination of the aqueous or anterior chamber of
the eye to detect any subtle changes in light scattering characteristics
of the aqueous.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a diagram of an eye showing the aqueous (labeled as aqueous
humor) and the lens;
FIG. 2 is a schematic diagram of the quasi-elastic light spectroscopy
instrumentation used in the method of the present invention;
FIG. 3 illustrates the average decay constant obtained using the method of
the present invention from normal rabbits over a period of 380 days at the
5 microsecond (.mu.s) sample time;
FIG. 4 illustrates the average decay constant obtained using the method of
the present invention from normal rabbits over a period of 380 days at the
100 .mu.s sample time;
FIG. 5 is a standard curve showing the dependence of the aqueous scattering
factor on the total blood cholesterol level in the method of the present
invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
At the outset, the invention is described in its broadest overall aspects
with a more detailed description following. The broadest aspects of the
invention involve passing a beam of light through the anterior chamber of
the patient's eye and measuring the light scattering characteristics from
the lens and correlating those light scattering characteristics to
determine a patient's cholesterol level. The technique is non-invasive and
safe as light levels are low and will not be directly incident on the
retina. Furthermore, the results are immediately obtainable by a
physician.
The subject's eye will be at no risk during this procedure as the light
level from the laser is low (1.5 mW). a short working distance lens causes
the beam of light to rapidly diverge beyond the focal point so that
overall illuminance posterior to the focal point is low. In addition, even
if the beam from the laser was directly incident on the retina, this beam
would have to be stationalry on the retina for over one (1) hour before
there was any risk of harm to the retina. The measurement itself is
performed in 5 seconds. It is highly unlikely that this beam will be
incident on the retina as the pupil will not be dilated and this light
beam will travel across the aqueous from the temporal to the nasal side.
Thus the light beam will travel across the pupil of the eye rather than
through it.
The present invention builds on a prior discovery that diabetes can be
diagnosed by measuring the light scattering characteristics of the lens. A
brief discussion of this procedure follows.
In simplest terms it is known that the disease diabetes causes a condition
in which the lens becomes less transparent; thus, a measurement of the
opacification of the lens can be correlated to determine the diabetic
condition.
Recently, researchers have used a method of photon correlation spectroscopy
or quasi-elastic light scattering spectroscopy to provide a non-invasive
probe of the development of lens opacities associated with diabetes.
The lens of the eye is an encapsulated, avascular, continually-growing
organ in the anterior of the eye. For purposes of this specification and
claims, the term aqueous is intended to describe the aqueous humor of the
eye. As shown in FIG. 1, the aqueous humor is anterior to the lens of the
eye. It is a transparent, watery fluid that is continually being replaced.
The vitreous humor is a similar fluid which is posterior to the lens and
gives the eye its shape.
The maintenance of lens transparency is essential for normal vision.
Traditional techniques for assessing lens transparency rely on slit lamp
observation. This is not an acceptable index as a lens region can appear
perfectly clear on slit lamp examination, yet may exhibit significant
cellular abnormalities.
Lens transparency depends on the spatial ordering of lens proteins in the
lens fiber cells. Thus, any localized alteration in the density and/or
structural integrity of this ordering due to aggregation, conformational
changes, or changes in lens hydration can result in regional changes in
refractive index and lens transparency leading to the development of
opacities. The prevention or reversal of the development of opacities or
cataracts therefore requires a sensitive method of in vivo detection, at
the molecular level, of the subtle lens changes that initiate the
opacification process.
Conventional techniques for examination of the eye do not provide such a
method. Scheimpflug photography, a non-invasive technique for the
detection of lens changes, provides quantitation of the optical density
changes arising from opacification in the lens. Cataractic changes
measured by this technique do not, however, explain the primary causes of
opacification such as changes in lens protein conformation. Laser Raman
spectroscopy offers the potential of quantitating some of these early lens
changes such as the quantitation of sulfhydryl and disulfide bond
formation implicated in the lens protein aggregation process. This method
is currently restricted to in vitro measurement.
With modifications in accordance with the present invention, equipment and
techniques used in the past to measure the opacity of a lens can be used
to measure the scattering of light through the aqueous. The changes in the
levels of cholesterol in the blood affect the scattering of light as it
passes through the aqueous. These changes can be accurately quantitated.
At the outset, it should be noted, that it is not readily clear whether
this light scattering is due to the presence of the ingested cholesterol
molecule itself or whether it is the result of some other metabolic
activity taking place in the body which manifests itself by increased
light scattering in the aqueous and in the lens of the eye. At this point,
it should be noted that no correlation has ever been made between high
cholesterol and vision, therefore it was an unexpected discovery that high
cholesterol would manifest itself as changes in the scattering properties
of the aqueous.
The instrumentation used in the method of the present invention is as
follows and is illustrated in FIG. 2. The quasi-elastic light spectroscopy
apparatus 10 is based on a modified Haag-Striet slit lamp biomicroscope
which is used to deliver the incident illumination to a focus in aqueous
of the eye 14 and to collect the resulting scattered intensity. The
incident illumination is provided by a 5 mW Helium/Neon laser (632.8 nm
wavelength light) 20. Beam 18 from laser 20 is passed through an
attenuator to reduce the laser beam power and is focused onto a 100 .mu.m
core optical fiber 24 using a 40 x microscope objective lens 22. The
optical fiber 24 delivers the illumination light to the slit lamp 16. The
output beam from this fiber is brought to a 37 .mu.m diameter focal spot
in the lens of the eye using a short-working distance lens 23. The power
incident on the cornea is 1.5 mW. The illumination optics are mounted on
the slit lamp using a third arm designed to attach to the central rotation
axis of the slit lamp. This arm fixes the scattering angle between
illumination and observation light paths at 130%. Micrometers attached to
the arm allow adjustments in the vertical and axial directions in addition
to rotation about the vertical axis. These adjustments ensure that the
laser light illumination is parfocal with the regular slit lamp
illumination and observation optics. Divergence of the rapid beam from the
focal plane ensures low retinal illuminance, approximately 20 mW/cm.sup.2,
giving a maximum permissable retinal exposure time of 1000 seconds.
The optical section of the laser beam in the aqueous is viewed through the
standard binocular observation eyepiece of the slit lamp. A 150 .mu.m
diameter optical fiber is positioned at the image plane in one of the
observation oculars and centered over the image of the focal spot in the
aqueous. Vertical micrometer adjustments of the incident laser beams
facilitates the centering of the image of the focal spot of the beam in
the aqueous of the eye over the collecting fiber optic in the eyepiece.
The light backscattered from the focal spot in the aqueous is collected
through this fiber optic. This optical geometry restricts the measurements
to one coherence area in the aqueous 12. An aperture positioned in front
of the input to the observation optics limits the collection angle for the
scattered light and discriminates against light scattered from regions
other than the measurement site. The collection fiber optic is coupled to
a fiber optic bundle mounted in the eyepiece which delivers the back
scattered light to a photomultiplier for detection and processing.
A photon counting photomultiplier (PMT) 26 detects the scattered light
signals and the resulting photocurrent pulses are processed through a
pre-amplifier discriminator (PAD) 28 to provide a digital photopulse
output. This digital signal is processed using a 128 channel digital
autocorrelator 30. The resulting intensity autocorrelation function is
stored and analyzed. The IBM PC computer 32 controls the measurement
acquisition process and stores the results. The system was calibrated
routinely using solutions of microspheres of known size.
The light scattered from the aqueous is analyzed in the form of an
intensity autocorrelation function using the methodology of quasi-elastic
light scattering spectroscopy. This technique measures the temporal
fluctuations and scattered light intensity resulting from Brownian motion
of the aqueous protein scattering elements. The temporal fluctuations and
scattered light intensity are proportional to the photocurrent fluctuation
output of the photomultiplier and is analyzed using a 128 channel digital
autocorrelator. The autocorrelator provides an intensity autocorrelation
function of these fluctuations in the form:
g(T)=(i(t)i(t+T))
For T varying from .DELTA.T to 128 x .DELTA.T where .DELTA.T the sample
time can be chosen from 100 ns to 1 s.
In the simplest case of light being scattered from a single scattering
species undergoing translational Brownian motion, the resulting
autocorrelation functions will have the form:
g(T)=i.sup.2 (1+exp(-2T/T.sub.1))
Where T.sub.1 is the characteristic decay time and i is the scattered light
intensity detected by the PMT. The decay constant .GAMMA.=1/T.sub.1 is
related to the diffusion coefficient, D, of the scatterers by
.GAMMA.=Dq.sup.2
where q=(4.pi./.lambda.) sin .theta./2 and .theta. is the scattering angle.
.lambda. is the wavelength light (632.8 nm). The diffusion coefficient of
the scatterers is related to their hydrodynamic radius (r) by the
Stokes-Einstein relation, assuming that the scatterers are
non-interacting, where
D=K.sub.B T.sub.A /6.pi..eta.r
Where K.sub.B is Boltzman's constant, T.sub.A is the absolute temperature
in degrees K, and .eta. is the viscosity of the medium.
In the lens of the eye, the autocorrelation function reflects a
polydispersed sized distribution of protein. The decay of the
autocorrelation function can no longer be described by a single
exponential function. The method of cumulant analysis has been used to
analyze these autocorrelation functions. A second order function was fit
to the measured function. Higher order terms provide no useful information
as the errors associated with the fitted parameters were large. The form
of the function fit to the data is given by
a(T)=-2.GAMMA.T+{(.GAMMA.-.GAMMA.).sup.2 }T.sup.2
where a(T) is the natural logarithm of the normalized intensity
autocorrelation function. The coefficient of the second order term denotes
an average over a distribution of decay times. The degree of
polydispersity of "Quality" (Q) is given by
Q={(.GAMMA.- .GAMMA.).sup.2 } .GAMMA..sup.2 ]1/2
which is the ratio of the half width at half height to the average value of
the distribution. For a monodispersed distribution of scatterers, the
value of Q will equal zero.
Animal trials using rabbits illustrate correlations existing over a broad
range of sample times. Measurements were made at different sample times.
The results show that the value of Q exhibited local minima at sample
times of 5.mu.s and 100.mu.s. This indicated that at these sample times,
the scattered signal contributing to the measured autocorrelation function
was more nearly monoexponential in nature suggesting that, at these sample
times, the scattered signal came from a predominantly well-defined size
distribution of lens protein. Thus, the measurements at the 5.mu.s and
100.mu.s sample times allowed the quantification of two distinct sizes of
lens protein scatterers. The measurements at the 5.mu.s sample time were
characteristic of smaller, more mobile lens proteins, while at the
100.mu.s sample time were characteristic of slower moving, larger lens
proteins.
Three sets of age matched albino New Zealand rabbits were used in this
study. A set of three normal rabbits were measured at different times over
the course of a year. A second set of four rabbits were used to
investigate both chronic and acute effects of a high cholesterol diet. To
investigate the acute effects of the diet, measurements were made everyday
for five days after the start of the diet. The third set of four rabbits
were used to provide additional data for the investigation of the chronic
effects of the cholesterol diet on the lens of the eye. Blood samples were
taken when possible, prior to the quasi-elastic light spectroscopy (QLS)
measurement, to determine levels of total cholesterol in the blood.
Prior to the measurements, each rabbit eye was dilated using a drop of
topically administered 1% Mydriacyl. The unanesthetized rabbit was then
comfortably positioned on a specially designed device which left the eyes
accessible for the QLS measurement. During the measurements, a drop of
tear substitute was applied to prevent dehydration of the eye. The animals
were maintained in accordance with the guidelines of the Committee on
Animals of the Harvard Medical School. Measurements were made from the
center of the lens nucleus on the optical axis of the lens. Four
measurements were made at each sample time. The duration of each
measurement was 5 seconds. The corresponding decay constants, determined
using cumulant analysis, were averaged to provide a mean decay constant
for each rabbit lens at each sample time. The effects of eye movements in
the course of a measurement have been shown to have little effect on the
measurements. However, at the longer sample time used here, eye movements
tended to increase the variance in the calculated average decay constant.
Measurements were made from the three normal rabbits to assess the
reproducibility of the measurements from the lens. The measurement
sessions were separated in time by two hours. The results are presented in
Table 1 for the average decay constants obtained at each sample time at
the constants obtained at each sample time at the two different
measurement times. There were no statistically significant differences
between the two reading at each sample time for each rabbit.
TABLE 1
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REPRODUCIBILITY
AVERAGE DECAY AT
AVERAGE DECAY AT
RABBIT 5 .mu.S ST (.+-. SD) (S.sup.-1)
100 .mu.S ST (.+-. SD) (S.sup.-1)
__________________________________________________________________________
J 25 183 .+-. 45 82 .+-. 19
J 25 (2 HOURS LATER)
192 .+-. 39 96 .+-. 13
J 26 160 .+-. 39 90 .+-. 19
J 26 (2 HOURS LATER)
191 .+-. 48 90 .+-. 27
J 27 156 .+-. 46 128 .+-. 18
J 27 (2 HOURS LATER)
164 .+-. 43 101 .+-. 24
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FIGS. 3 and 4 illustrate the average decay constant obtained from the
normal rabbits over a period of 380 days at the 5.mu.s and 100.mu.s sample
times, respectively. The results in the 5.mu.s sample time demonstrates
the significant decrease in the decay constant or diffusion coefficient
with increasing age of the rabbit. There were no marked changes in the
decay constants measured at the 100.mu.s sample time over the same period.
On comparing the two sets of results, it can be noted that the decay
constants at the 5.mu.s sample times became comparable to those determined
at the 100.mu.s sample time in the older rabbits. This would indicate
that, at these later times, most of the population of smaller protein
scatterers had undergone aggregation to form larger protein molecules
characteristic of those measured at the 100.mu.s sample time.
The chronic effects of a high cholesterol diet on the lens of the eye were
investigated by comparing the group of normal rabbits with an age-matched
group of rabbits at a high cholesterol diet after 100 days. In this case,
the rabbits on the high cholesterol diet had been on the diet for 94 days.
The results at the 5.mu.s sample time are presented in Table 2.
TABLE 2
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COMPARISON BETWEEN NORMAL AND AGE MATCHED CHOLESTEROLEMIC
RABBITS AT 100 DAYS
AVERAGE DECAYS (S.sup.-1) AT
5 .mu.S SAMPLE TIME
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NORMAL GROUP
J25 624 .+-. 59
J26 790 .+-. 48
J27 810 .+-. 63
AVERAGE DECAY = 741.3 .+-. 102.1
s.sup.-1
CHOLESTEROLEMIC GROUP
K1 268 .+-. 55
L6 202 .+-. 58
L7 149 .+-. 56
L8 249 .+-. 52
L10 299 .+-. 63
AVERAGE DECAY = 233.4 .+-. 58.4
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s.sup.-1
There was a statistically significant difference in lens decay constants
between the normal and cholesterol-fed groups. The decay constants of the
cholesterol-fed group were approximately 3 times lower than those from the
normal group. There was no statistically significant difference between
the two groups at the 100.mu.s sample time.
It was noted, while performing measurements on the lenses of the
cholesterol fed rabbits, that the aqueous became quite highly scattering.
It was decided to perform QLS measurements from the aqueous of the normal
and cholesterolemic rabbits. The scattered intensity from the aqueous was
measured from 13 rabbits on cholesterol diets. Blood cholesterol levels
were determined from blood levels drawn before the measurements. The
scattered light intensity from the aqueous was normalized with respect to
the measured scattered intensity from the lens to provide an "aqueous
scattering factor". FIG. 5 demonstrates the dependence of this factor on
the total blood cholesterol level. As the cholesterol levels increase, we
note a slow increase in the aqueous scattering factor. At the higher
cholesterol level, the scattered intensity from the aqueous increases very
rapdily and becomes greater than that from the lens. In addition,
autocorrelation functions were measured from the aqueous once the aqueous
scattering became more prominant. Autocorrelation functions measured at
different sample times, show that the scattered signal originated from a
fairly monodispersed distribution of scatterers. Based on cumulant
analysis, a sample time of 5.mu.s was chosen as optimum for characterizing
the decay constant of these scatterers. Two different decay constants were
measured depending on the relative magnitude of the scattered intensity
from the aqueous. For the more intensely scattering aqueous the average
decay constant determined from 7 rabbits measured at 18 different times
was 7390.+-.462 s.sup.-1. The corresponding diffusion coefficient was
1.14+0.07.times.10.sup.-7 cm.sup.2 /s and the resulting hydrodynamic
radius was calculated to be 190.+-.12 .ANG. assuming that the aqueous had
a viscosity equal to that of water. For the less intensely scattering
aqueous the average decay constant obtained from 7 rabbits measured at 12
different times was 4268.+-.882 s.sup.-1. The diffusion coefficient and
hydrodynamic radius of these scatterers was calculated to be
6.59.+-.1.36.times.10.sup.-8 cm.sup.2 /s and 330 +68 A respectively.
The measurements made from the aqueous show that scattering contributions
arise from two different species. The average hydrodynamic radii of 190
.ANG. and 330 .ANG. suggest that the scattering results from different
types of lipoproteins which are known to have sizes in the range between
200 and 400 .ANG.. The reduction in backscattered intensity from the
larger particles is possibly a consequence of a more efficient forward
scattering in the case of the larger particles.
The results on the effects of high cholesterol on the rabbit eye indicate
that QLS measurements can provide a useful, non-invasive method for
quantitating systemic cholesterol effects. The measurements from the
aqueous may provide a useful, clinical method for the non-invasive
monitoring of blood cholesterol levels.
The invention may be embodied in other specific forms without departing
from the spirit or essential characteristics thereof. The present
embodiments are therefore to be considered in all respects as illustrative
and not restrictive, the scope of the invention being indicated by the
appended claims rather than by the foregoing description, and there is no
intention to exclude any equivalence thereof. Hence, it is recognized that
various modifications are possible and within the scope of the present
invention as claimed.
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