Red-Cell Partitioning And Its Mechanisms

Why Red-Cell Association Matters

A drug measured in whole blood is distributed between plasma and the cellular components of blood, principally red cells. A drug may have a higher concentration per unit volume in red cells than in plasma because it partitions into cell membranes, binds to intracellular constituents, or both. Conversely, a drug can remain mainly in plasma. These patterns affect the relationship between a plasma concentration and a whole-blood concentration, so they matter when interpreting measurements and specifying a blood-to-plasma input for a PBPK model.

The ratio of red-cell concentration to plasma concentration is not the same as the blood-to-plasma ratio. The former describes concentrations in two separate fractions; the latter compares a whole-blood concentration with a plasma concentration. Their relationship depends on the fractions’ volumes. The focus here is on why drugs associate with red cells and what that association implies, rather than on a general analysis of how the ratio varies with hematocrit.

Definition: Let $C_{\mathrm{RBC}}$ be the total drug concentration per unit volume of packed red cells, and let $C_p$ be the total concentration per unit volume of plasma. The red-cell-to-plasma concentration ratio is $R_{\mathrm{RBC:p}}=C_{\mathrm{RBC}}/C_p$. The blood-to-plasma ratio, $R_{\mathrm{b:p}}=C_b/C_p$, instead uses the total concentration $C_b$ per unit volume of whole blood. These ratios are dimensionless when both concentrations use the same mass-per-volume units.

For a sample containing only plasma and red cells, let $H$ denote the red-cell volume fraction. A mass balance gives $C_b=(1-H)C_p+HC_{\mathrm{RBC}}$, and therefore $R_{\mathrm{b:p}}=(1-H)+H R_{\mathrm{RBC:p}}$. This identity clarifies why red-cell association contributes to the blood-to-plasma ratio. The cellular ratio alone does not specify the whole-blood ratio without the blood fractions.

Two Mechanisms of Red-Cell Association

Membrane partitioning is the distribution of drug between the surrounding aqueous phase and the red-cell membrane. Drugs with suitable physicochemical properties may associate with membrane lipids. Greater lipophilicity can favor such association, but lipophilicity alone does not determine the red-cell concentration: ionization, membrane composition, accessibility, and the drug’s interactions with other cell components also matter. A measured red-cell concentration includes drug associated with the membrane if the assay measures the entire cell fraction.

Intracellular binding is association with constituents inside the cell, such as hemoglobin or other proteins. Binding can raise the total amount of drug present in the red-cell fraction relative to the freely dissolved amount. Binding capacity and affinity both matter. When binding sites are finite, their occupancy can change with concentration, so the total red-cell concentration may not increase in direct proportion to the unbound concentration.

These mechanisms can occur together. Membrane association may facilitate accumulation without a specific intracellular binding site; intracellular binding may retain drug after it enters the cell. The measured total red-cell concentration generally does not reveal how much of the drug is free, membrane-associated, or bound to intracellular molecules. Separating these contributions requires additional measurements or a mechanistic model.

Key relationship: Define $f_{u,p}=C_{u,p}/C_p$ and $f_{u,\mathrm{RBC}}=C_{u,\mathrm{RBC}}/C_{\mathrm{RBC}}$, where $C_{u,p}$ and $C_{u,\mathrm{RBC}}$ are the unbound concentrations in plasma and red cells. If $K_u=C_{u,\mathrm{RBC}}/C_{u,p}$, then $$ R_{\mathrm{RBC:p}}=\frac{C_{\mathrm{RBC}}}{C_p} =K_u\frac{f_{u,p}}{f_{u,\mathrm{RBC}}}. $$ This identity follows by writing each total concentration as its unbound concentration divided by its unbound fraction. It does not assume that $K_u=1$.

The relationship helps distinguish two contributors to the observed ratio. A low $f_{u,\mathrm{RBC}}$, consistent with substantial cell association, can increase the total red-cell concentration relative to the unbound concentration. Meanwhile, $K_u$ describes the relationship between unbound concentrations across the plasma and red-cell environments. Equal unbound concentrations are a possible equilibrium condition for a freely permeable drug under appropriate conditions, not a universal rule. Differences in pH, ionization, transport, or other processes can make $K_u$ depart from one.

Worked Example: Separating Unbound Distribution From Cell Binding

Suppose a hypothetical drug has $f_{u,p}=0.10$, $f_{u,\mathrm{RBC}}=0.025$, and $K_u=0.80$ at a specified concentration and sampling time. Calculate its red-cell-to-plasma ratio. If $C_p=8.00\ \mathrm{mg/L}$, also calculate the implied red-cell concentration.

Using the relationship between total and unbound concentrations, $$ R_{\mathrm{RBC:p}}=K_u\frac{f_{u,p}}{f_{u,\mathrm{RBC}}} =0.80\frac{0.10}{0.025}=3.20. $$ Thus, under the stated conditions, the total concentration per unit volume of red cells is 3.20 times the total concentration per unit volume of plasma.

The implied red-cell concentration is $$ C_{\mathrm{RBC}}=R_{\mathrm{RBC:p}}C_p =3.20(8.00\ \mathrm{mg/L})=25.6\ \mathrm{mg/L}. $$ This result is consistent with the specified unbound concentrations: $C_{u,p}=0.10(8.00)=0.800\ \mathrm{mg/L}$, while $C_{u,\mathrm{RBC}}=0.80(0.800)=0.640\ \mathrm{mg/L}$. Dividing $0.640\ \mathrm{mg/L}$ by $f_{u,\mathrm{RBC}}=0.025$ gives $25.6\ \mathrm{mg/L}$. The example shows that a red-cell total concentration can exceed the plasma total concentration even when the unbound red-cell concentration is lower.

Concentration Dependence From Intracellular Binding

A simple binding model illustrates why a single red-cell-to-plasma ratio may not describe all concentrations. Suppose one class of intracellular binding sites is at equilibrium, with total site capacity $B_{\max}$, free intracellular concentration $C_{u,\mathrm{RBC}}$, and dissociation constant $K_d$. The bound concentration is $$ C_{\mathrm{bound}}=\frac{B_{\max}C_{u,\mathrm{RBC}}}{K_d+C_{u,\mathrm{RBC}}}. $$ If the free concentration is measured in the same cell-volume units as the bound concentration, total red-cell concentration in this simplified model is $C_{\mathrm{RBC}}=C_{u,\mathrm{RBC}}+C_{\mathrm{bound}}$. This model assumes a single reversible binding class, equilibrium, and a fixed site capacity. It omits membrane association and other cell processes.

Worked Example: Saturable Binding Changes the Ratio

Consider a hypothetical drug with $K_u=0.50$, $f_{u,p}=1$, $B_{\max}=6.00\ \mathrm{mg/L}$ of red-cell volume, and $K_d=1.00\ \mathrm{mg/L}$. Assume the plasma drug is entirely unbound, so $C_p=C_{u,p}$, and use the binding model above. Compare $R_{\mathrm{RBC:p}}$ at plasma concentrations of $2.00$ and $18.0\ \mathrm{mg/L}$.

At $C_p=2.00\ \mathrm{mg/L}$, the unbound red-cell concentration is $C_{u,\mathrm{RBC}}=K_u C_{u,p}=0.50(2.00)=1.00\ \mathrm{mg/L}$. The bound concentration is $$ C_{\mathrm{bound}}=\frac{6.00(1.00)}{1.00+1.00}=3.00\ \mathrm{mg/L}. $$ Thus $C_{\mathrm{RBC}}=1.00+3.00=4.00\ \mathrm{mg/L}$, and $R_{\mathrm{RBC:p}}=4.00/2.00=2.00$.

At $C_p=18.0\ \mathrm{mg/L}$, $C_{u,\mathrm{RBC}}=0.50(18.0)=9.00\ \mathrm{mg/L}$. Then $$ C_{\mathrm{bound}}=\frac{6.00(9.00)}{1.00+9.00}=5.40\ \mathrm{mg/L}, $$ so $C_{\mathrm{RBC}}=9.00+5.40=14.4\ \mathrm{mg/L}$, and $R_{\mathrm{RBC:p}}=14.4/18.0=0.800$. The ratio falls from 2.00 to 0.800 because bound concentration approaches its finite capacity while plasma and free intracellular concentrations continue to rise. The example is a model-based illustration, not evidence that any particular drug follows this binding model.

Deriving the Whole-Blood Contribution

The conversion from cell and plasma concentrations to a whole-blood concentration follows directly from the definition of concentration. For a unit volume of whole blood, plasma occupies volume $1-H$ and red cells occupy volume $H$. The amount of drug in that unit is the plasma concentration multiplied by plasma volume, plus the red-cell concentration multiplied by red-cell volume: $$ \text{amount per unit blood volume}=(1-H)C_p+H C_{\mathrm{RBC}}. $$ Since the left side is $C_b$, division by $C_p$ yields the blood-to-plasma ratio. This derivation assumes that the sample is represented by the plasma and red-cell fractions and that concentrations are expressed per volume of the corresponding fraction.

Worked Example: Translating Red-Cell Partitioning to Blood-to-Plasma Ratio

In a hypothetical sample, let $H=0.45$, $C_p=8.00\ \mathrm{mg/L}$, and $C_{\mathrm{RBC}}=25.6\ \mathrm{mg/L}$, as in the earlier calculation. Find the blood-to-plasma ratio and whole-blood concentration.

First, $R_{\mathrm{RBC:p}}=25.6/8.00=3.20$. The whole-blood concentration is $$ C_b=(1-0.45)(8.00\ \mathrm{mg/L})+(0.45)(25.6\ \mathrm{mg/L}) =4.40+11.52=15.92\ \mathrm{mg/L}. $$ Therefore, $$ R_{\mathrm{b:p}}=\frac{15.92}{8.00}=1.99, $$ which also follows from $R_{\mathrm{b:p}}=0.55+0.45(3.20)=1.99$. The whole-blood ratio is below the red-cell-to-plasma ratio because whole blood contains both plasma and red cells, not red cells alone.

Assumptions, Diagnostics, and Alternatives

Mechanistic interpretation requires more than a ratio. It is useful to ask whether the measurements describe comparable conditions: the same drug concentration range, sampling time, species, temperature, and relevant physiological state. A claim of equilibrium needs support that distribution has had time to stabilize. The simple free-concentration relationship also assumes that the unbound concentrations and fractions are defined consistently and that the sampled fractions represent the system being interpreted.

Several observations can signal that a simple, constant partition ratio is inadequate. If $R_{\mathrm{RBC:p}}$ changes with concentration, investigate saturable binding, concentration-dependent transport, or assay effects. If it changes with time, distribution may not have equilibrated, or processes such as uptake, efflux, or metabolism may be relevant. Differences across conditions may also reflect changes in pH or cell properties. These patterns are diagnostic prompts, not proof of a particular mechanism. For example, saturable binding is a plausible explanation for a falling ratio, but the concentration trend alone cannot distinguish it from other concentration-dependent processes.

A measured ratio and a mechanistic model answer different questions. A measured $R_{\mathrm{RBC:p}}$ summarizes the observed distribution under specified experimental conditions; it does not identify the mechanism. A mechanistic representation can help explain or predict changes across conditions, but it relies on assumptions and additional parameters, such as binding capacity or unbound fractions. Direct experimental measurement is often preferable when the relevant conditions can be reproduced. A model is useful when extrapolation is needed, provided its assumptions and uncertainty are made explicit.

A major limitation is that total concentrations alone cannot separate membrane partitioning, intracellular binding, and freely dissolved drug. Treating $R_{\mathrm{RBC:p}}$ as a universal constant can therefore fail when concentration or biological conditions change. Nor does a large ratio by itself prove strong binding to a particular cellular constituent: membrane association, ionization effects, or other processes can also contribute. Interpretation should remain tied to what was measured and under which conditions.

Common pitfall: “The red-cell-to-plasma ratio is 3, so the blood-to-plasma ratio is 3.” This is incorrect because the two ratios use different concentration denominators. The red-cell ratio compares red cells with plasma; the blood ratio reflects the mixture of red cells and plasma. Under the two-fraction mass balance, $R_{\mathrm{b:p}}=(1-H)+H R_{\mathrm{RBC:p}}$, not simply $R_{\mathrm{RBC:p}}$.

Check Your Understanding

Answer each question, then compare your reasoning with the brief solutions.

If $C_{\mathrm{RBC}}=12\ \mathrm{mg/L}$ and $C_p=4\ \mathrm{mg/L}$, what is $R_{\mathrm{RBC:p}}$? Does that value alone give $R_{\mathrm{b:p}}$?
In the relationship $R_{\mathrm{RBC:p}}=K_u f_{u,p}/f_{u,\mathrm{RBC}}$, what does a low $f_{u,\mathrm{RBC}}$ imply, and what does it not establish about mechanism?
For $B_{\max}=4\ \mathrm{mg/L}$, $K_d=2\ \mathrm{mg/L}$, and $C_{u,\mathrm{RBC}}=2\ \mathrm{mg/L}$, calculate $C_{\mathrm{bound}}$.
Name two possible explanations for a red-cell-to-plasma ratio that changes with concentration.

Solutions. (1) $R_{\mathrm{RBC:p}}=12/4=3.00$. It does not alone give $R_{\mathrm{b:p}}$; the red-cell volume fraction is also needed. (2) A low $f_{u,\mathrm{RBC}}$ means a small fraction of total red-cell drug is unbound, and can contribute to a larger total red-cell concentration. It does not identify whether membrane partitioning, intracellular binding, or another process caused the association. (3) $C_{\mathrm{bound}}=4(2)/(2+2)=2.00\ \mathrm{mg/L}$. (4) Possible explanations include saturable binding and concentration-dependent transport; changes in assay behavior should also be considered.