Measuring zinc in biological nanovesicles by multiple analytical approaches

Exosomes are nanovesicles known to mediate intercellular communication. Although it is established that zinc ions can act as intracellular signaling factors, the measurement of zinc in circulating nanovesicles has not yet been attempted. Providing evidence of the existence of this zinc fraction and methods for its measurement might be important to advance our knowledge of zinc status and its relevance in diseases. Exosomes from 0.5 ml of either fresh or frozen human plasma were isolated by differential centrifugation. A morphological and dimensional evaluation at the nanoscale level was performed by atomic force microscopy (AFM) and Transmission Electron Microscopy (TEM). Energy Dispersive X-Ray Microanalysis (EDX) revealed the elemental composition of exosomes and their respective total Zinc content on a quantitative basis. The zinc mole fraction (in at%) was correlated to the phosphorous mole fraction, which is indicative for exosomal membrane material. Both fresh (Zn/P 0.09 +/- 0.01) and frozen exosomes (Zn/P 0.08 +/- 0.02) had a significant zinc content, which increased up to 1.09 +/- 0.12 for frozen exosomes when treated with increasing amounts of zinc (100-500 mu M; each p < 0.05). Interestingly, after zinc addition, the Calcium mole fractions decreased accordingly suggesting a possible exchange by zinc. In order to estimate the infra-exosomal labile zinc content, an Imaging Flow Cytometry approach was developed by using the specific membrane permeable zinc-probe Fluozin-3AM. A labile zinc content of 0.59 0.27 nM was calculated but it is likely that the measurement may be affected by purification and isolation conditions. This study suggests that circulating nano-vesicular-zinc can represent a newly discovered zinc fraction in the blood plasma whose functional and biological properties will have to be further investigated in future studies.