Here is an excerpt from Kollmann et al. (2020, J. Physiol.):

This ring was placed in a recording chamber continuously perfused with 37°C aerated Hepes solution containing (in mM) 136 NaCl, 10 glucose, 5 KCl, 10 Hepes, 1.2 MgCl2, 2.5 CaCl2 (pH 7.40) at a rate of 11 ml min-1. Hypoosmolality of the Hepes solution was established by reduction of the NaCl content to 33 mM (94 mOsm kg-1 H2O), 58 mM (144 mOsm kg-1 H2O) or 83 mM (194 mOsm kg-1 H2O).

For validation, I tried to calculate the osmolality by myself. For example, when the NaCl content is changed to 33 mM, the osmolality should be $$ 33\times2~(\mathrm{NaCl})+10\times1~(\mathrm{Glucose})+5\times2~(\mathrm{KCl})+10\times1~(\mathrm{Hepes})+1.2\times3~(\mathrm{MgCl_2})+2.5\times3(\mathrm{CaCl_2}) $$ which gives 107.1 mOsm/L, i.e., 107.1 mOsm/kg. But 107.1 is largely different from what the author stated, namely 94 mOsm/kg.

When the NaCl content is changed to 58 mM or 83 mM, the osmolality should increase by (58-33)*2=50 mM, or (83-33)*2=100 mM. This is consistent with the author's calculation, i.e., 144-94=50 mOsm/kg, or 194-94=100 mOsm/kg. So, my calculation is correct at least in terms of NaCl. But how can I calculate the contributions of the remaining solutes correctly?

Reference: Kollmann, P. et al. Submucosal enteric neurons of the cavine distal colon are sensitive to hypoosmolar stimuli. J. Physiol. 598, 5317–5332 (2020).


1 Answer 1


I assume this is a measured osmolality rather than a calculated one. It's challenging to calculate actual osmolality because of incomplete dissociation of the solutes, see for example https://en.wikipedia.org/wiki/Osmotic_coefficient. There's also a subtle difference between osmolality (particles per mass) and osmolarity (particles per volume).

I believe HEPES can contribute to this effect because it will bond with free ions. Sugars do, too. The gap still seems a bit larger than I'd expect, some of that might involve the temperature of water when originally mixed versus when measured. If you're trying to replicate their methods, I'd get yourself an osmometer.


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