We developed an optical bound water index (BWI) from quantitative tissue absorption spectra in the NIR. In order to validate the accuracy and sensitivity of BWI for water state measurements, bound water phantoms were fabricated and tested using MRS and a conductivity measurement cell.
demonstrates that BWI increases linearly with phantom gelatin content (R=0.98). Since gelatin fraction was the only variable in all phantoms, we conclude that BWI measures water disposition changes caused by gelatin.
In order to confirm that the phantoms indeed have protein bound water, each was measured using MRS and a conductivity measurement cell. T2 relaxation time is strongly affected by the number of water binding sites in tissue (Fullerton and Dornbluth, 1982
; Moser et al., 1996
). Many studies have shown the ability of T2 for measuring water mobility in biological tissues such as breast, brain and lung (Assaf et al., 1997
; Brauer, 2003
; Shioya et al., 1997
; Tan et al., 2008
). The T2 value increases as freely mobile water increases. In , T2 relaxation time decreased mono-exponentially as protein concentration increased. This result is due to reduced excitation of nuclei in bound states and consequent fast recovery. Also the mono-exponential decrease of T2 values satisfies a two-state model with a rapid exchange between the bound and bulk water (Fukuzaki et al., 1995
; Lambelet et al., 1988
; Moser et al., 1996
; Oakes, 1976a
). T2 values smaller than T1 relaxation times (less than half of T2 values, not shown) signify the prevalence of heterogeneous water-proton environments in our phantoms, which is associated with bound water molecules (Odajima, 1959
; Sasaki et al., 1960
). High correlation between T2 relaxation rates (R2) and protein concentration has been reported in other studies (Lambelet et al., 1988
; Moser et al., 1996
; Oakes, 1976a
) which is also from the two-state model. Thus, the high correlation between R2 and BWI () suggests that BWI measures macromolecular bound water accurately.
Careri et al
. have observed increasing conductivity with increasing bound water fraction as shown in . By analyzing the relationship between dielectric constant and conductivity, they found that the protons involved in the conductivity at 100kHz are not the free hydrogen ions of the solvent, but the ions bound to ionizable groups of proteins (Careri et al., 1985
). The high correlation between BWI and conductivity shown in further demonstrates the reliability of BWI as an index for bound water fraction.
DW-MRI has been used to measure water state in in-vivo
tissues using apparent diffusion coefficients (ADC) of water. In , ADC and BWI from homogeneous phantoms demonstrated an inverse correlation suggesting that as more water is bound to macromolecules the degree of water diffusion decreases. Based on our clinical data where the BWI decreased in tumor tissues ( and ), this would suggest that ADC and BWI are complementary and ADC should increase in tumors. Interestingly, previous MRI studies have demonstrated lower ADC in malignant tumors compared to both normal tissue and benign tumors (Englander et al., 1997
; Guo et al., 2002
; Partridge et al., 2001
). However, in these studies the ADC is typically measured on regions of interest (ROI) carefully drawn to exclude necrotic and cystic areas. If any of the necrotic, cystic or fat regions are included, ADC values change significantly. Paran et al.(Paran et al., 2004
) acquired heterogeneous diffusion parameters on large, progressing tumors due to partial and full necrotic loci spread throughout the tumor tissues. It is challenging to select an ROI that only represents the cellular part of a tumor due to the low resolution of DW-MRI images. In addition, the diffusion of free water can be obstructed by the presence of macromolecules and fibrous structures unbound to water (Callaghan et al., 1993
; Cho et al., 1996
; Nusbaum et al., 2000
; Putz et al., 1992
). Thus, the interpretation of bound water state in tissues using ADC will vary substantially depending on tissue micro-structure and the precise ROI selected. In contrast, BWI represents an average bulk property of the tissue and is a relatively simple measurement to acquire and interpret.
In normal well-differentiated tissues, we expect a relatively narrow range of BWI and water content values due to the absence of significant disorder and the expectation that normal tissues have an intrinsic maximum water content and binding capacity (). With the appearance of cancer and loss of differentiation, water content increases dramatically and the tissue structural diversity increases. This phenomenon is communicated by the wide range of water content values and reductions in BWI seen in .
Overall, significantly lower BWI was observed in tumor vs. normal tissues (–). This indicates that the water associated with tumor tissues is more like free water. This could be from necrotic regions in the tumors where free water can fill. The measured free water increase might also be related to alterations in extracellular matrix (ECM). Hyaluronan (or, hyaluronic acid, HA), one of the components of the ECM, is a large, negatively charged polysaccharide that participates in transducing signals in proliferating and migrating cells, and it is closely related to tumor growth (Vignal et al., 2002
), metastasis (Hayen et al., 1999
), increased drug resistance (Baumgartner et al., 1998
), cellular invasiveness (Toole, 2001
) and angiogenesis in malignant tumors.(West et al., 1985
) In breast cancer patients, high levels of HA in the stroma are associated with low survival rate (Auvinen et al., 2000
; Vignal et al., 2002
). HA has the ability to retain water and the meshwork structure exerts swelling pressure because of increased mutual repulsion between and within HA molecules (Toole, 2004
). Therefore, the finding that water binding state correlates inversely with tumor histopathological grade maybe related to the increased presence of HA and necrotic regions in malignant tumors. This inverse correlation between BWI and tumor grade complements our previous observation that overall water content positively correlates with histopathological scores (Cerussi et al., 2006
). BWI appears to convey additional information about tissue pathological changes specifically related to the molecular disposition of water, possibly due to alterations in cellularity and extracellular matrix. Although preliminary, these results suggest that both water content and disposition as measured by DOS may provide clinical prognostic information related to metastatic potential and therapeutic outcome
In conclusion, we provide evidence that water state is communicated effectively by the optical measurement of BWI. The accuracy of BWI as a water state index has been validated in bound water phantoms by comparing broadband DOS to MRS and a conductivity measurement cell. Breast cancers were found to possess significantly more free than bound water and BWI correlated inversely with tumor histopathological grade. These results highlight broadband DOS sensitivity to tissue water content and state, and underscore the potential for BWI to be used as a complementary index to MRI relaxation time and ADC measurements.