Most clinical laboratory reports of drug concentrations in plasma (C) represent drug that is bound to plasma protein plus drug that is unbound of free. It is the free or unbound drug that is in equilibrium with the receptor site and is, therefore, the pharmacologically active moiety. Thus, in the case of a drug with significant plasma binding, the reported plasma drug concentration indirectly reflects the concentration of free or active drug. Some disease state are associated with decreased plasma proteins or with decreased binding of drugs to plasma proteins. In these situations, drugs that are usually highly protein bound have a larger percentage of free or unbound drug present in plasma. Therefore, a greater pharmacologic effect can be expected for any given drug concentration in plasma (C). Clinicians must always consider altered protein binding and whether the fraction of free drug concentration or fraction unbound (fu) is altered when interpreting or establishing desired plasma concentrations.

Luckily, the fraction of drug that is unbound (fu) does not vary with the drug concentration for most drugs that are bound primarily to albumin. This is because the number of protein binding sites far exceeds the number of drug molecules available for binding. When the plasma concentrations for drugs bound to albumin exceed 25 to 50 mg/L, however, albumin binding sites can start to become saturated. As a result, fu, or the fraction of drug that is free, will change with the plasma drug concentration.

For those drugs that do not reach serum concentrations capable of saturating protein binding sites, the plasma protein concentration (in many cases, this is albumin) and the binding affinity of the drug for the plasma protein are the two major factors that control the fraction unbound (fu).


Plasma Protein Concentration

Low plasma protein concentrations decrease the plasma concentration of bound drug (C bound); however, the concentration of free drug (C free) generally is unaffected. Therefore, the fraction of drug that is free (fu) increases as plasma protein concentrations decrease. Free or unbound drug concentrations are not significantly increased because the free drug that is released into plasma secondary to low plasma protein concentrations equilibrates with the tissue compartment (see the figures below). Therefore, if the volume of distribution (V) is relatively large, only a minor increase in C free will result.Screen Shot 2014-12-03 at 12.17.45 AM

Screen Shot 2014-12-03 at 12.48.31 AMThe relationship between the new plasma drug concentration (C’), the normal plasma drug concentration (C Normal Binding), the new plasma protein concentration (P’), and the normal plasma protein concentration (P NL) can be expressed as follows:

[Equation 1] C’/C Normal Binding = (1 – fu)[P’/P NL] + fu, whereas fu is the free fraction of the drug associated with “normal plasma protein binding”, or more precisely, associated with the situation when the plasma protein concentration is normal. So when the clinician receives the report of the plasma concentration for a drug, he or she should consider the plasma protein concentration as well. For example, if a drug’s normal free fraction (fu) is 0.1, and a patient with a low serum albumin of 2.2 g/dL (normal albumin, 4.4 g/dL) and an apparently low plasma drug concentration of 5.5 mg/L still has a therapeutically acceptable plasma drug concentration when it is adjusted for the low serum albumin. That is the reported value of actual plasma concentration of phenytoin should be adjusted by [Equation 1] under the condition of low plasma protein concentration. So the adjusted plasma concentration of phenytoin (the normal plasma drug concentration) is,

[Equation 2] C Normal Binding = C’/{ (1 – fu)[P’/P NL] + fu} = 5.5 / [(1 – 0.1 )*(2.2/4.4) + 0.1] = 10 mg/L

While [Equation 2] could be used to adjust for any drug significantly bound to albumin, the degree to which the drug concentration will be adjusted or “normalized” for the alteration in serum albumin between 3.5 and 5.5/dL will be minimal and is generally unwarranted. For example, in the same patient above is given another drug with normal free fraction (fu) of 0.95, and the lab report value of the actual plasma drug concentration is also 5.5 mg/L, then the adjusted plasma drug concentration should be 5.5 / [(1 – 0.95 )*(2.2/4.4) + 0.95] = 5.64 mg/L.

Many other drugs are bound primarily to globulin rather than albumin. Adjustments of plasma drug concentrations for these drugs based on serum albumin concentrations would, therefore, be inappropriate. Unfortunately, adjustments for changes in globulin binding are difficult because drug usually bind to a specific globulin that is only a small fraction of total globulin concentration.

Elevated plasma albumin concentrations are uncommon in the clinical setting, thus the use of [Equation 2] for high serum albumin would be rare. Many basic drugs, however, are bound to the acute phase reactive protein, α1-acid glycoprotein (AAG). This plasma protein has been known to be significantly decreased and increased under certain clinical conditions. For example, increases in plasma quinidine concentrations have been observed following surgery or trauma. The change in the quinidine concentration is the result of increased concentrations of the plasma binding proteins (AAGs) and increased bound concentrations of quinidine. There appears to be little or no change in the free quinidine level because re-equilibration with the larger tissue stores occurs. In this situation, there would be a decrease in unbound free fraction (fu), and the therapeutic levels of free or unbound drug should correlate with higher-than-usual drug concentration (bound plus free).

Other basic compounds with significant binding to AAGs would be expected to be similarly affected. Unfortunately, AAG concentrations are seldom assayed in the clinical setting, making it difficult to evaluate the relationship between the total drug concentration and the unbound or free fraction. For this reason, evaluation of plasma levels for basic drugs that are significantly protein bound is often difficult. A careful evaluation of the patient’s clinical response to a measured drug level, as well as an evaluation of any concurrent medical problems (such as surgery, trauma, or inflammatory disease) that could influence plasma protein concentrations and drug binding, is required.

Patients with cirrhosis vary considerably in their plasma protein binding characteristics. Some patients have significantly elevated binding capabilities, whereas others have significantly decreased binding capabilities. This variation probably reflects the fact that some cirrhotic patients have a strong stimulus for the production of AAGs, whereas others with more serious hepatic disease are unable to manufacture these binding proteins.

Binding Affinity

The binding affinity of plasma protein for a drug also alter the fraction of drug which is free (fu). For example, the plasma proteins in patients with uraemia (severe end-stage renal failure) have less affinity for phenytoin than do proteins present in nonnumeric individuals (different from general drug-receptor binding, other factors except binding affinity such as efficiency of the occupancy-response, degree of spareness [please refer detail at http://www.tomhsiung.com/wordpress/2014/06/the-properties-of-drugs-receptor-rationale/]). As a result, the fu for phenytoin in ureic patients is estimate to be in the range of 0.2 to 0.3 in contrast to the normal value of 0.1. However, the plasma concentration of free or unbound drug is little changed (due to the same reasons as previously discussed). So the total drug concentrations (free or unbound plus bound drug) are decreased compared to that in nonnumeric patients and the fu is increased.Screen Shot 2014-12-05 at 10.16.26 PM

Let’s see this example. In normal individuals, the fu of phenytoin is 0.1 and when a TDM report of 10 mg/L is achieved, the free or unbound drug should be 10*0.1 = 1 mg/L. So in the same patient if he or she were with low binding affinity (uremic syndrome) and with the same dose which achieves 10 mg/L of phenytoin just as said before, the serum concentration of free or unbound phenytoin is nearly the same – 1 mg/L. Because the low binding affinity and according to the references the fu should be 0.3 here, so the total serum drug concentration or the TDM report should be 1/0.3 = 3.3 mg/L. As a general rule, if fraction unbound is increased in any given situation, the clinician should reduced the desired C by the same proportion.

So to clinicians, a careful interoperation of the lab report for therapeutic drug monitor is very necessary. In patients with low plasma concentration protein, or elevated plasma concentration protein, or in patients with significant change in protein binding affinity, the lab report value for a specific drug may puzzle clinicians’ sight.


Plasma Protein Binding and Clearance

Clearance can be thought of as the intrinsic ability of the body or its organs of elimination to remove drug from the blood or plasma. Clearance is expressed as a volume per unit of time. It is important to emphasise that clearance is not an indicator of how much drug is being removed; it only represents the theoretical volume of blood or plasma which is completely cleared of drug in a given period. The amount of drug removed depends on the plasma concentration of drug and the clearance.

For highly protein-bound drugs, diminished plasma protein binding is associated with a decrease in reported steady-state plasma drug concentration (total of unbound plus free drug) for any given dose that is administered. According to this equation of Cl=(S)(F)(Dose/τ)/(Css ave) [Equation 3], a decrease in the denominator, Css ave, increases the calculated clearance. It would be misleading, however, to assume that because the calculated clearance is increased, the amount eliminated per unit of time has increased. Equation 3 assumes that when Css ave (total of bound plus free drug) changes, the free drug concentration, which is available for metabolism and renal elimination, changes proportionately. In actuality, the free or unbound fraction of drug in the plasma generally increases with diminished plasma protein binding. As a result, the amount of drug administered per unit of time remains unchanged.

This (low plasma protein binding) lower plasma concentration (C bound plus C free) is associated with a decreased C bound, no change in C free, and as a result there is an increase in the fraction of unbound drug (fu). Therefore, the pharmacologic effect achieved will be similar to that produced by the higher serum concentration observed under normal protein binding conditions. This example re-emphasizes the principle that clearance alone is not a good indicator of the amount of drug eliminated per unit of time (RE).

The direct proportionality between calculated clearance and fraction unbound (fu) does not apply to drugs that are so efficiently metabolised or excreted that some (perhaps all) of the drug bound to plasma protein is removed as it passes through the elimination organ. In this situation the plasma protein acts as a “transport system” for the drug, carrying it to the eliminating organs, and clearance becomes dependent on the blood or plasma flow to the eliminating organ. To determine whether the clearance for a drug with significant plasma binding will be influenced primarily by blood flow or plasma protein binding, its extraction ratio is estimated and compared to its (fu) value.

The extraction ratio is the fraction of the drug presented to the eliminating organ that is cleared after a single pass through that organ. It can be estimated by dividing the blood or plasma clearance of a drug by the blood or plasma flow to the eliminating organ. If the extraction ratio exceeds the (fu), then the plasma proteins are acting as a transport system and clearance will not change in proportion to (fu). If, however, the extraction ratio is less than (fu), clearance is likely to increase by the same proportion that (fu) changes. This approach does not take into account other factors that may affect clearance such as red blood cell binding, elimination from red blood cells, or changes in metabolic function.