Noninvasive in vivo monitoring of methemoglobin formation and reduction with broadband diffuse optical spectroscopy.

We present noninvasive, quantitative in vivo measurements of methemoglobin formation and reduction in a rabbit model using broadband diffuse optical spectroscopy (DOS). Broadband DOS combines multifrequency frequency-domain photon migration (FDPM) with time-independent near infrared (NIR) spectroscopy to quantitatively measure bulk tissue absorption and scattering spectra between 600 nm and 1,000 nm. Tissue concentrations (denoted by brackets) of methemoglobin (MetHb]), deoxyhemoglobin (Hb-R]), and oxyhemoglobin (HbO2]) were determined from absorption spectra acquired in "real time" during nitrite infusions in nine pathogen-free New Zealand White rabbits. As little as 30 nM MetHb] changes were detected for levels of MetHb] that ranged from 0.80 to 5.72 microM, representing 2.2 to 14.9% of the total hemoglobin content (%MetHb). These values agreed well with on-site ex vivo cooximetry data (r2= 0.902, P < 0.0001, n = 4). The reduction of MetHb to functional hemoglobins was also carried out with intravenous injections of methylene blue (MB). As little as 10 nM changes in MB] were detectable at levels of up to 150 nM in tissue. Our results demonstrate, for the first time, the ability of broadband DOS to noninvasively quantify real-time changes in MetHb] and four additional chromophore concentrations (Hb-R], HbO2], H2O], and MB]) despite significant overlapping spectral features. These techniques are expected to be useful in evaluating dynamics of drug delivery and therapeutic efficacy in blood chemistry, human, and preclinical animal models.