The analysis of spiked mice blood confirmed the hypothesis that TCC concentrates in the cellular fraction of blood. After 10 min of incubation, the TCC plasma concentration was only 57.6 ± 5.8 nM of the 100 nM TCC spiked to the blood (). Accordingly the cellular fraction contained a higher TCC concentration of 141.8 nM ± 3.9 nM. Taking a hematocrit of murine blood of 0.4– 0.6 into account (Hedrich, 2004
), TCC mass balance was very good with a calculated whole blood concentration of 99 ± 8 nM. Comparing the measured whole blood concentrations, a factor of 1.9 ± 0.2 between whole blood concentration and plasma resulted, which remained stable over the incubation time (). The same distribution was found in vivo
in mice after oral gavage of TCC, with a blood/plasma concentration ratio of 1.9 ± 0.2, though the observed TCC whole blood levels between mice varied 202 ± 7 nM and 792 ± 34 nM (). By contrast, the investigation of rat blood led to a varying blood/plasma ratio between 2 and 15, trending toward a higher factor with lower TCC concentrations. This indicated that binding of TCC to the cellular fraction in blood is concentration and species dependent, which warrants further investigation. This preliminary study clearly demonstrated that the whole blood concentration of TCC better reflect the total amount of TCC present in blood than the plasma concentration, which is at least 2-fold lower.
Fig. 1 Distribution of TCC in mice blood. In vitro A: Fresh blood from Swiss Webster mice was spiked with 100 nM TCC and incubated at 37 °C. In vivo B: The TCC blood and plasma levels 4 h after oral gavage. Concentrations are presented as mean and SD (more ...)
Whole blood analysis was therefore utilized for the bio-monitoring of TCC after exposing human subjects by a single shower with antibacterial soap. As shown in , this analysis demonstrated the detection of TCC in whole blood after human exposure with TCC-containing personal care products. In accordance to the previously reported urine concentration of TCC-N
-glucuronides(Schebb et al., 2011c
), Volunteer A and B showed the highest TCC concentration with a Cmax
of 530 nM and 240 nM TCC, respectively detected 2–3 h after exposure (). A surprisingly high level 285 ± 5 nM TCC was observed in the blood of volunteer A at baseline. This volunteer regularly used TCC-containing soap. This indicates that frequent users of these personal care products may have a significant body burden of TCC.
TCC whole blood concentrations of human subjects after showering with TCC containing soap: (A) TCC levels in the blood of volunteer A (a regular TCC-soap user) and volunteer B. (B) The TCC blood concentration of volunteers C–F.
Similarly as described for the urinary excretion (Schebb et al., 2011c
), strong inter-individual differences were observed in blood levels of the subjects (). Despite a standardized exposure protocol, the blood levels of volunteer C–F were about 10-fold lower than those of volunteer A–B. Moreover, the observed pharmacokinetics varied widely among the six volunteers. Because of these inter-individual variations, human exposure to TCC by the use of antibacterial soaps cannot be broadly extrapolated to the general population from such a small group of volunteers. Nevertheless, a single shower with an antibacterial soap led to a detectable TCC whole blood concentration in all exposed volunteers (Cmax
between 23 and 530 nM, ). This clearly demonstrates that measuring the systemic TCC levels is feasible by analyzing whole blood as sample matrix. Several convenient techniques are available for sample preparation of whole blood, such as dried-blood-spot analysis (Li and Tse, 2010
), or the mixing with an excess of water followed by liquid/liquid or solid phase extraction (Schebb et al., 2010
). These techniques shall be used in combination with sensitive analysis by LC–MS (Sapkota et al., 2007
; Liu et al., 2011
; Schebb et al., 2011c
) for further studies of human TCC exposure. The possible off-target effects of TCC on mammals merit a comprehensive bio-monitoring of TCC in the population. With preliminary results about human TCC exposure by soaps, we demonstrate that whole blood is the sample matrix of choice for these investigations.