Millipore Technical Publications |
 | Application Note AN1002EN00 | |
Assay of Free Anticonvulsant Drugs With separation by Centrifree® Micropartition Device | ||
| Lit No: | AN1002EN00 | |
| Year: | 2000 | |
The binding of anticonvulsant drugs (phenytoin, valproic acid and carbamazepine) to a patient's blood proteins can have a significant effect on the pharmacokinetic and pharmacodynamic characteristics of the drug. This, of course, can considerably affect how the patient responds to the drug as well as the physician's approach and adjustment of drug therapy. For drugs largely bound to proteins in the blood plasma (mostly albumin), it is the unbound (free) concentration of the drugs which is biologically active and is, therefore, significant for the patient's pharmacologic response and for the physician's determination of drug administration.
Normal changes in physiology and diseases as well as the drug therapy itself can alter the degree of protein binding, thereby changing the important concentration of free drug in the blood stream. When patient response to anticonvulsant drug therapy does not correspond to the measured total drug concentration, knowledge of the unbound drug concentration enables the clinician to make appropriate adjustments of the patient's therapeutic regimen.
Indications For Free Drug Measurement
A good rule of thumb: Whenever the patient's clinical status does not correlate with what is indicated by given total drug levels, altered protein binding and altered free drug concentration should be suspected.
The following patient conditions suggest the need for determination of free (as well as total) anticonvulsant drug levels:
- Hypoalbuminemia: Low albumin concentrations lead to increased free drug levels which may produce undesirable effects.
- Hyperalbuminemia: High albumin concentrations lead to decreased free drug levels which may cause therapeutic failure.
- Renal failure: Albumin/protein binding sites are altered. Increased free drug levels can produce toxicity even though total concentrations are within the therapeutic range.
- Drug/drug interactions: Drugs with greater affinity for protein binding sites displace an anticonvulsant drug from its protein binding sites and increase free drug concentrations. For example, valproate displaces other anticonvulsant drugs from their protein binding sites.
- Saturation kinetics: Valproic acid and disopyramide exhibit nonlinear (zero order) binding kinetics. The free level can vary remarkably between patients, at the same total drug concentration.
- Change in normal physiological status: Any age-related change in physiologic factors (pregnancy, disease, aging) can alter free drugs. These changes can also alter how a receptor responds to a given drug concentration
Centrifree Disposable Micropartion Device |
Glossary
The terminology of drug proteinbinding can be confusing. Note the distinction between free drug concentration and fraction:
- Free drug concentration is the actual concentration of drug not bound to protein, in plasma.
- Free drug fraction is the portion of the drug concentration in plasma which is not bound to protein. It is determined by dividing free drug concentration by total drug concentration. Multiplying the free drug fraction by 100 gives the % of free drug present in the specimen. Percent of free drug (percent free) is often used inter-changeably with "free drug fraction" in the older scientific literature.
Free/Bound Separation Now Easy
In the past, free drug levels in serum or plasma samples were not widely measured, partly for want of a convenient means of separating free from protein-bound drugs. The technique of choice was generally equilibrium dialysis, a time-consuming procedure, subject to effects of dilution and buffers. It does not directly indicate the free drug concentration in the sample. Such other techniques as ultracentrifugation and gel filtration are no less time-consuming and there is inadequate standardization of results.
Today's better alternative for free/bound separation is ultrafiltra-ion (UF) with Millipore's Centrifree micropartition device. Centrifree employs a membrane filter with controlled porosity which retains more that 99.9% of serum protein and lets free drugs readily pass the membrane for collection and analysis. This is done rapidly in a standard laboratory centrifuge (preferably angle-head), with samples in the range of 0.15 to 1 mL. The sample is not diluted in the process. Multiple samples are conveniently handled, typically in 10 minute runs per set. With the availability of Centrifree micropartition, combined with assay calibration and control kits, there is no longer any practical impediment to the ready availability of unbound as well as total anticonvulsant drug concentration measurement. As stated by Dr. C. E. Pippenger, "Any clinical
laboratory can perform free level analyses for antiepileptic drugs, using a simple, one-step filtration process: Centrifree micropartition."
Free Drug Assay Kits Readily Available
Determinations of free phenytoin, valproic acid and carbamazepine levels are just as easy to run via TDx as total level assays. The reagent system is actually identical. Only calibrator and control kits are different. TDx assays for the quantitative measurement of the unbound fractions of anticonvulsant drugs in serum or plasma samples are available from Abbott Laboratories. They utilize fluorescence polarization immunoassay technology, as discussed in the TDx operation manual. Other standard analytical methods may also be used, with low-level standards.
Use of Centrifree Is Simple, Efficient
The Centrifree micropartition device is a disposable unit. It contains Millipore's Amicon YMT hydrophilic ultrafiltration membrane which has virtually no non-specific adsorptivity for anticonvulsant drugs. The pressure generated by centrifugation forces plasma water and free drug molecules (typically < 1,000 MW) convectively through the ultrafilter, then into a filtrate collection cup. The larger protein molecules remain in the sample reservoir above the membrane. Free drug concentration in the collected ultrafiltrate (plasma water) is quantified by routine analytical techniques. Operation at an angle causes the protein layer that normally forms on the membrane to slide radially during centrifugation, maintaining high filtratiion efficiency (see Fig. 3).
Figure 1. Before ultrafiltration, the drug (small dots) is partly free in the serum, partly bound to serum protein (large bodies). | |
Figure 2. During ultrafiltration in Centrifree, free drug permeates the membrane. Its concentration in the filtrate is the same as in the sample above the membrane. The drug/protein molar binding ratio is unchanged. During centrifugation in a fixed-angle rotor, the proteins on the membrane surface are forced to slide laterally, maintaining relatively free access to the membrane. | |
Figure 3. Polarization control, using a 35¡ fixed-angle rotor. |
Figures 1 and 2 illustrate the relationship between free and bound drug before and during UF. During ultrafiltration, convective transport of only free drug assures that equilibrium between free and protein-bound drug is maintained. This equilibrium is largely a function of the free drug concentration which does not change during filtration and to a lesser degree of the bound fraction which will not play a role unless large filtrate volumes are removed. Although total drug concentration in the sample reservoir increases during UF, the drug/protein molar binding ratio remains constant, as does the drug concentration in the filtrate. Therefore, drug concentration in the filtrate is the same as free drug concentration at equilibrium in the plasma sample before UF. Because the drug/protein molar binding ratio remains constant, drug concentrations in the ultrafiltrate are also independent of the volume collected.
Technical Considerations
When determining free drug concentrations by UF, one should remember factors that can alter drug/protein binding in-vitro. They include:
- Temperature: Drug binding decreases with rising temperature. The centrifuge should produce minimal temperature rise in its chamber. Generally, run times are so short that this is not a problem.
pH: Protein binding of some drugs is pH-dependent. Exposure of plasma to air can raise its pH, increasing free drug concentration. pH changes can be prevented by closing the sample reservoir with the provided cap before the run. - Fatty acids: Free fatty acids are released by lipolysis of triglycerides during prolonged plasma storage. They can displace drugs from their binding sites, increasing free drug concentration. Sample UF prevents those effects.
TDx Anticonvulsant Drug Assays
All Abbott calibrations, controls and assay systems are run on the TDx system.
| Item | Quantity | Abbott Catalog No. |
| Free phenytoin | ||
| Free phenytoin calibrators | 50 runs, 6 calibrators per kit | 9530-01 |
| Free phenytoin assay system | 100 test kit | 9507-69 |
| Free phenytoin control | 50 runs, 3 controls per kit | 9530-10 |
| Free valproic acid | ||
| Free valproic acid calibrators | 50 runs, 6 calibrators per kit | 9537-01 |
| Free valproic acid assay system | 100 test kit | 9514-69 |
| Free valproic acid control | 50 runs, 3 controls per kit | 9537-10 |
| Free carbamazepine | ||
| Free carbamazepine calibrators | 50 runs, 6 calibrators per kit | 9538-01 |
| Free carbamazepine assay system | 100 test kit | 9515-69 |
| Free carbamazepine control | 50 runs, 3 controls per kit | 9538-10 |
For more information regarding the above contact Abbott laboratories, North Chicago, IL 60064 (phone: 1-800-527-1869)
Low Cost
The only difference in laboratory cost between performing free and total drug assays is the cost of the disposable Centrifree device and the time required to ultrafilter the specimen by centrifugation, typically 10 minutes. The analytical system for measuring both total and free drug level is the same, except that free level controls are run to verify system performance.
How To Order
For the Physician:
To order tests, please contact your clinical laboratory. If the lab is not familiar with the contents of this publication, please ask them to order it.