1. The Field of the Invention
The present invention relates generally to techniques for measuring levels of chemical compounds found in biological tissue. More specifically, the invention relates to a method and apparatus for the noninvasive detection and measurement of levels of carotenoids and related chemical substances in biological tissue, which can be used as a diagnostic aid in assessing antioxidant status and detecting malignancy diseases or risk thereof.
2. The Relevant Technology
Carotenoids are plant pigments available from the diet which have important functions in the human body. The role of carotenoids in human health is a rapidly expanding area of research. Much carotenoid research has focused on their role as precursors to retinoids or vitamin A, but current research is also being conducted on other functions of carotenoids. These include antioxidant activities, modulation of the immune response, cell-to-cell communication and gap junction modulation.
It has been demonstrated that carotenoids offer some degree of biologic protection against the formation of malignancies in various tissues. For example, carotenoids have been shown in animal models to prevent carcinoma formation in tissues such as skin, salivary gland, mammary gland, liver, and colon. In addition, low levels of carotenoids and related substances such as retinoids have been assessed as high risk factors for malignant lesions. For example, having low levels of the carotenoid lycopene has been associated with prostate and cervical cancer; the carotenoids lutein, zeaxanthin, alpha-carotene, and beta-carotene with lung cancer; and beta-carotene with oral cancer. Therefore, quantitatively measuring the chemical concentrations of these carotenoids, retinoids and other related substances provides an indicator of the risk or presence of cancer.
The most common cancer in the United States is skin cancer. Despite attempts at patient education, skin cancer rates continue to rise. Methods to provide detection of the levels of chemicals which are associated with skin related malignancies are of great assistance to physicians and medical personnel in the early diagnosis and treatment of skin cancer.
It has been theorized that carotenoids in the skin provide biologic protection from cutaneous malignancy. Most findings, however, have been somewhat compromised by the fact that concentrations of carotenoids in skin and skin malignancies were never measured directly, with data on levels of carotenoids in patients being derived only indirectly from blood plasma.
Prior methods used to detect the presence of chemicals associated with skin cancer have mainly been through the analysis of tissues obtained by biopsies or other invasive procedures. The standard method presently used for measuring carotenoids is through high-performance liquid chromatography (HPLC) techniques. Such techniques require that large amounts of tissue sample be removed from the patient for subsequent analysis and processing, which typically takes at least twenty four hours to complete. In the course of these types of analyses, the tissue is damaged, if not completely destroyed. Therefore, a noninvasive and more rapid technique for measurement is preferred.
A noninvasive method for the measurement of carotenoid levels in the macular tissue of the eye is described in U.S. Pat. No. 5,873,831, the disclosure of which is herein incorporated by reference, in which levels of carotenoids and related substances are measured by a technique known as Raman spectroscopy. This is a technique which can identify the presence and concentration (provided proper calibration is performed) of certain chemical compounds. In this technique, nearly monochromatic light is incident upon the sample to be measured, and the inelastically scattered light which is of a different frequency than the incident light is detected and measured. The frequency shift between the incident and scattered light is known as the Raman shift, and the shift corresponds to an energy which is the "fingerprint" of the vibrational or rotational energy state of certain molecules. Typically, a molecule exhibits several characteristic Raman active vibrational or rotational energy states, and the measurement of the molecule's Raman spectrum thus provides a fingerprint of the molecule, i.e., it provides a molecule-specific series of spectrally sharp vibration or rotation peaks. The intensity of the Raman scattered light corresponds directly to the concentration of the molecule(s) of interest.
One difficulty associated with Raman spectroscopy is the very low signal intensity which is inherent to Raman scattered light. It is well known that the scattered light intensity scales with the frequency raised to the fourth power. The weak Raman signal must be distinguished from Rayleigh scattered light, which is elastically scattered light of the same frequency as the incident light and which constitutes a much greater fraction of the total scattered light. The Raman signal can be separated from Rayleigh scattered light through the use of filters, gratings, or other wavelength separation devices; however, this can have the effect of further weakening the measured Raman signal through the additional attenuation which can occur when the light passes through a wavelength separation device. In practice, the Raman scattered light is extremely difficult to detect. One might attempt to increase the Raman signal by increasing the incident laser power on the tissue sample, but this can cause burning or degradation of the sample.
In order to overcome some of these difficulties, a technique known as resonance Raman spectroscopy has been used, as described in U.S. Pat. No. 5,873,831, referenced hereinabove. Such a technique is also described in U.S. Pat. No. 4,832,483, the disclosure of which is herein incorporated by reference. In resonance Raman spectroscopy, the incident illumination utilized has a frequency which corresponds to the resonance frequency corresponding to electronic energy transitions of the molecules of interest. This has the effect of strongly enhancing the Raman output signal without using a higher intensity input signal, thereby avoiding damage to the sample which can be caused by laser burning. Also, these resonance Raman signals have much higher intensity than off-resonance Raman signals which are virtually invisible. Therefore, in resonance Raman spectroscopy only those Raman signals which belong to the species of interest are obtained.
In the above referenced U.S. Pat. No. 5,873,831, the resonance Raman technique is used to measure the levels of the carotenoids lutein and zeaxanthin, two chemicals which are associated with healthy macular tissue of the human eye. The above referenced U.S. Pat. No. 4,832,483 uses resonance Raman spectroscopy to measure certain carotenoids in blood plasma, and suggests the use of the ratios of the intensities of the Raman spectral peaks as a method of indicating the presence of various malignancy diseases.
Yet another difficulty associated with Raman measurements is that the substances of interest in the skin not only scatter incident light, but can absorb and subsequently fluoresce with substantial intensity. This fluorescence often comprises a very strong, broad signal which tends to "drown out" or overwhelm the Raman spectral peaks, thereby making identification and quantification of the substances of interest practically impossible.
Fluorescence spectroscopy is itself another technique which can be used to measure amounts of chemical compounds in biological tissue. For example, U.S. Pat. No. 5,697,373 discloses use of fluorescence and/or Raman spectroscopy to detect tissue abnormality in the cervix. The disadvantage of fluorescence measurements is that since many different molecules fluoresce in broad bands of frequencies, such measurements cannot be used to conclusively identify the presence or concentration of a particular substance.
It would therefore be a significant advance to provide a method and apparatus for the safe, noninvasive, rapid, accurate, and specific measurement of the levels of carotenoids and other similar chemical compounds which are present in varying degrees in biological tissues, and to use this information to aid in the assessment of cancer risk or disease risk in all types of biological tissue.