SummaryObesity is a very common condition; however, the effect of excess body weight on the appropriate dose of immunoglobulin has not been defined empirically. The proposed pharmacokinetic differences between lean and obese patients and the opportunity to reduce costs has led to the proposition that obese patients should receive proportionally lower doses of immunoglobulin once a certain threshold is reached. Here the theoretical factors which could affect dosing in obese patients are considered alongside the available empirical evidence. The available evidence indicates that obesity may affect the pharmacokinetics of immunoglobulin; however, the effect is likely to be too small to have a clinically important effect on dosing. Wide interpatient individuality and highly variable clinical need mean that obesity should not play a major factor in dosing considerations. However, patients who are obese are more likely to have multiple cardiovascular risk factors and their weight indicates a large dose. This puts these patients at a higher risk of adverse reactions, and therefore caution is advised.

The light chain of the major histocompatibility complex class 1 (MHC-1), the protein β2-microglobulin (β2m), has amyloidogenic properties that arise only upon its dissociation from the MHC-1. Here hydrogen/deuterium exchange electrospray ionization mass spectrometry (HDX-ESI-MS) has been used to compare the solution dynamics of β2m in its MHC-1 bound state compared with those of β2m as a free monomer. The capability of tandem mass spectrometry to dissociate the MHC-1 into its individual constituents in the gas phase following deuterium incorporation in solution has permitted the direct observation of the exchange properties of MHC-1 bound β2m for the first time. The HDX-ESI-MS data show clearly that the H→D exchange of MHC-1 bound β2m follows EX2 kinetics and that about 20 protons remain protected from exchange after 17 days. Free from the MHC-1, monomeric β2m exhibits significantly different HDX behavior, which encompasses both EX1 and EX2 kinetics. The EX2 kinetics indicate a tenfold increase in the rate of exchange compared with MHC-1 bound β2m, with just 10 protons remaining protected from EX2 exchange and therefore exchanging only via the EX1 mechanism. The EX1 kinetics observed for unbound β2m are consistent with unfolding of its exchange-protected core with a t1/2 of 68 min (pH 7, 37° C). Thus, upon dissociation from the stabilizing influence of the MHC-1, free β2m becomes highly dynamic and undergoes unfolding transitions that result in an aggregation-competent protein.

RATIONALE Amyloid formation is implicated in a number of human diseases. β2-microglobulin (β2m) is the precursor protein in dialysis-related amyloidosis and it has been shown that partial, or more complete, unfolding is key to amyloid fibril formation in this pathology. Here the relationship between conformational flexibility and β2m amyloid formation at physiological pH has been investigated.METHODS HDX-ESI-MS was used to study the conformational dynamics of β2m. Protein engineering, or the addition of Cu2+ ions, sodium dodecyl sulphate, trifluoroethanol, heparin, or protein stabilisers, was employed to perturb the conformational dynamics of β2m. The fibril-forming propensities of the protein variants and the wild-type protein in the presence of additives, which resulted in >5-fold increase in the EX1 rate of HDX, were investigated further.RESULTS ESI-MS revealed that HDX occurs via a mixed EX1/EX2 mechanism under all conditions. Urea denaturation and tryptophan fluorescence indicated that EX1 exchange occurred from a globally unfolded state in wild-type β2m. Although >30-fold increase in the HDX exchange rate was observed both for the protein variants and for the wild-type protein in the presence of specific additives, large increases in exchange rate did not necessarily result in extensive de novo fibril formation.CONCLUSIONS The conformational dynamics measured by the EX1 rate of HDX do not predict the ability of β2m to form amyloid fibrils de novo at neutral pH. This suggests that the formation of amyloid fibrils from β2m at neutral pH is dependent on the generation of one or more specific aggregation-competent species which facilitate self-assembly. Copyright © 2012 John Wiley & Sons, Ltd.