Total and Unbound Drug Concentrations: What a Fasting Study Could Not Resolve
A laboratory can quantify total drug in plasma while struggling to quantify the much smaller unbound portion. That difference can determine whether a pharmacokinetic study answers its question. A result reported as not statistically significant does not resolve the question when the measurements are too limited to detect a meaningful difference.
Why measure the unbound fraction?
Lammers and colleagues investigated whether changes in plasma protein binding could help explain previously observed changes in drug disposition during short-term fasting. Total plasma concentration includes protein-bound and unbound drug. The unbound fraction is the ratio of unbound concentration to total concentration. A change in that ratio can complicate an attempt to attribute a change in total-drug clearance solely to altered enzyme activity.
The researchers analyzed samples from nine healthy men in a randomized crossover study. Each received an intravenous five-drug probe cocktail after an overnight fast and after 36 hours of fasting. The comparison was between those two conditions, not between fed and fasted oral dosing. Because administration was intravenous, this experiment did not test a meal’s effect on gastrointestinal absorption.
The analytical limit changed the strength of the answer
Free fatty acid concentrations increased during the longer fast. The investigators found no statistically significant change in the unbound fractions of caffeine, metoprolol or omeprazole. They interpreted those findings as evidence against altered binding explaining the earlier disposition findings for those drugs.
Midazolam and S-warfarin required a different conclusion. Most unbound measurements were below the lower limit of quantification. Only samples taken at the observed concentration peak could be used in the free-fraction comparison, and some of those measurements were still below that limit. The authors substituted half the quantification limit for those remaining values. This is an analytical assumption, not a measurement of the missing concentration.
The authors expressly concluded that the midazolam and S-warfarin analyses were underpowered and could not establish how fasting affected their protein binding. A nonsignificant comparison in that setting does not demonstrate unchanged binding.
Below quantification does not mean absent
A concentration below the lower limit of quantification cannot be assigned the same numerical certainty as a validated quantitative result. It does not establish zero drug. When the unbound concentration is already small, differences hidden below the analytical limit can still represent substantial relative differences in the calculated unbound fraction.
The study therefore illustrates two separate requirements: collecting the relevant samples and using an assay sensitive enough for the particular fraction being measured. Reliable total-drug measurements do not automatically validate conclusions about unbound drug.
Keep the conclusion within the experiment
These were low-dose probe-drug observations in nine healthy men. They do not establish the effects of chronic malnutrition, critical illness, every fasting practice, or an individual patient’s treatment. The paper supports a precise pharmacokinetic distinction: a study may answer its binding question for some drugs while leaving it unresolved for others. It provides no basis to infer an individual’s impairment or clinical response from total concentration alone.
Source
Lammers LA, Achterbergh R, Romijn JA, Mathôt RAA. Short-Term Fasting Alters Pharmacokinetics of Cytochrome P450 Probe Drugs: Does Protein Binding Play a Role? European Journal of Drug Metabolism and Pharmacokinetics. 2018;43:251-257. Read the primary study. Original commentary on the supplied paper; no figures or tables reproduced.
