Gingival crevicular fluid is composed of serum and locally generated materials, including tissue breakdown products, bacterial components and products, and inflammatory and immune mediators. Thus, gingival crevicular fluid contains a wealth of information on periodontal disease processes. A major attraction of gingival crevicular fluid as a diagnostic marker is the site-specific nature of the sample. This allows sophisticated analyses of gingival crevicular fluid components to be linked to clinical assessment at the site of sample collection. Owing to the complexity of the pathogenic tissue alterations associated with periodontitis, which are reflected in gingival crevicular fluid composition, we had hypothesized that IR spectroscopy could reliably differentiate diseased and healthy periodontal sites. We have been able to show that even in unprocressed spectral data (), subtle differences in spectral band intensity and positions arising from the three major components, i.e. lipid, protein and DNA, can be seen in gingival crevicular fluid from healthy, gingivitis and periodontitis groups.
A more sophisticated means with which to extract the relevant biological information hidden in raw infrared spectra is to assess the relative contributions of key functional groups by integrating the band areas, which usually provide more information about concentrations than band intensities. Indeed, as is clearly demonstrated in and , we are able to reliably analyze and compare specific gingival crevicular fluid components among disease groups. For example, the protein concentration in both disease groups is higher than in control samples, in agreement with prior reports of increased total protein levels in periodontitis gingival crevicular fluid (32
), and a significant correlation between total gingival crevicular fluid protein concentration and disease severity (33
). Many gingival crevicular fluid proteins have been extensively explored as potential diagnostic markers that define periodontal inflammation. They include inflammatory mediators, particularly cytokines and matrix metalloproteinases, and tissue breakdown products, such as fibronectin, collagen fragments and hydroxyproline, which should reflect the extent of underlying tissue destruction. Our results suggest that the total protein signal is elevated in periodontal disease, which is consistent with our spectral observations.
Infrared spectroscopy is increasingly recognized as an alternative modality for accurate determination of lipid peroxidation in various biological samples (29
). Increased oxidative stress is the consequence of enhanced production of reactive oxygen species and/or attenuated reactive oxygen species scavenging capacity, resulting in tissue damage reflected in increased lipid peroxidation. Loss of unsaturation during lipid peroxidation reactions was compensated by the presence of double bonds in the lipid peroxidation products such as malondialdehyde, lipid aldehydes and alkyl radicals. It is well recognized that lipid peroxidation markedly increases during periodontal inflammation (36
). For instance, the malondialdehyde contents of chronic apical periodontitis tissues are higher than in healthy tissue of the same individuals (39
). Tsai et al.
) recently reported that the total amount of gingival crevicular fluid lipid peroxidation correlated positively with several clinical disease parameters, indicating that the more severe the inflammation of the periodontal tissue the higher the level of lipid peroxidation. Furthermore, periodontal oxidative stress may even have systemic consequences, with higher blood lipid peroxidation concentrations noted in a rat model of periodontitis compared with periodontally healthy control animals (40
). Several studies have also demonstrated that IR spectroscopic analysis of the integrated area in the 3030–2990 cm−1
region, representing the olefinic band arising from the unsaturated lipids, can be employed to assess lipid peroxidation in various biological samples (29
). In keeping with such prior reports, our spectral data show that significant disease-related changes occur in the lipid content of gingival crevicular fluid, indicative of increased oxidative stress (see ).
We have previously shown that by using the DNA-specific signals embedded in the IR spectra of the leukocytes, one can precisely diagnose leukemia and predict leukocytic apoptosis (20
). We can now apply this newly developed technology to the study of periodontal diseases. Gingival crevicular fluid contains a diverse population of cells, which include bacteria, desquamated epithelia and transmigrating leukocytes (42
). The increased DNA component in gingival crevicular fluid from gingivitis and periodontitis sites, relative to healthy control sites, is likely to be due to a combination of an inflammation-driven increase in leukocyte migration into the gingival crevicular fluid, particularly neutrophils, an increase in epithelial turnover, reflecting ongoing tissue remodelling, and the inflammatory stimulus itself, i.e. plaque bacteria.
Periodontal disease is clearly multi-factorial, yet previous attempts to use gingival crevicular fluid analysis as a diagnostic tool have generally relied on the measurement of only one or two specific gingival crevicular fluid components. Since IR spectroscopy measures the composite molecular content of gingival crevicular fluid, then assuming that molecular alterations do occur during the disease process, the chance for IR spectroscopy to distinguish various stages of periodontitis should be promising, in contrast to previous one-dimensional biochemical approaches. This hypothesis is supported by our LDA studies, which consider multiple components in the gingival crevicular fluid as the basis to designate individual spectra as healthy or diseased. The accuracy of this LDA approach in diagnosing periodontitis was 98.4% for the training set and 93.1% for the validation set. However, the overall accuracy for the gingival crevicular fluid spectral classification between control sampless and gingivitis was 91.4%, and for the training set 72.4%. This discrepancy might reflect fundamental pathological differences between periodontitis and gingivitis, with irreversible tissue damage the hallmark of periodontitis. Also, the changes in three major components (protein, lipid and DNA; and ) in the gingival crevicular fluid from the periodontitis group were much higher than in the gingivitis group. Such molecular data were readily picked up by LDA, leading to optimal diagnostic accuracy for the periodontitis group (93.1%) but only an acceptable level for the gingivitis group (72.4%).
In addition to high accuracy, there are several other advantages to IR spectroscopic screening and diagnosis of periodontitis. Infrared spectroscopy is reagent free and requires only small sample volumes. The gingival crevicular fluid samples are essentially unprocessed and the process is readily automated. Infrared spectroscopy is straightforward, and minimal training is required of operators following automation. Gingival crevicular fluid samples are easily collected by clinicians, with sample collection targeted to specific sites or to a representative set of teeth.
In summary, these studies show that we have established the appropriate IRS technology and the proof of principle that IRS can be used to identify disease-specific molecular signatures in gingival crevicular fluid cross-sectionally. Longitudinal studies are required to ascertain whether IRS is a suitable prognostic tool, long-sought by the periodontal community (4