Rapid measurement of plasma methadone in a clinical setting using florescence polarization immunoassay.

Currently clinicians using methadone for treatment of opioid dependency or organic pain lack a simple, real-time, analytical method useful for adjusting methadone dose. Therefore, we evaluated the feasibility of measuring plasma methadone concentrations using florescence polarization immunoassay (FPIA) in an outpatient clinical setting. Using this technology, patient plasma methadone concentrations can be determined in approximately 20 minutes. The FPIA calibration curve was found to be linear over the methadone concentration range of 0 to 1600 ng/ml. Day-to-day run coefficient of variation was 5-10%, the within-run coefficient of variation was 1.3-6.3%; the limit of quantification for the assay was 25 ng/ml. Calibration plots of HPLC and GCMS (mass fragment 72) plasma methadone control samples versus FPIA were also linear. A plot of HPLC plasma methadone versus FPIA for patient samples was identical to results for control samples (concentration range 0-1200 ng/ml). No significant amounts of the EDDP methadone metabolite were found in plasma. Based upon these findings, FPIA is clinically useful for monitoring plasma methadone concentration in outpatient settings.

Utilization of plasma and urine methadone concentration measurements to limit narcotics use in methadone maintenance patients: II. Generation of plasma concentration response curves.

Controversy exists still concerning the proper daily dose of methadone required to eliminate illicit narcotics use. To address this, urine methadone and opioid concentrations were measured prospectively (150 maintenance patients, 18 month period, 9250 urine samples) using fluorescence polarization immunoassay. Results demonstrate that current thresholds (EMIT uses 300 micrograms/L) defining opiate positive urines are overly high (FPIA can go as low as 25 micrograms/L), causing severe underestimation of opioid use in the typical clinic. Using this data, plasma methadone concentration and dose response probability curves were generated for illicit opiate use. Results demonstrate a S-shaped, 24 hr trough plasma methadone concentration response curve with effective concentrations, EC90 = 80 micrograms/L, EC98 = 600 micrograms/L. Plotting mean plasma methadone concentration versus dose gives a monotonically increasing function: Conc = 5.367*dose0.858, raw R-squared = 0.967, corrected R-squared = 0.802. Unfortunately, coefficients of variation for plasma concentrations at each prescribed dose are unacceptably large, explaining poor dose response relationships for some patients.

Opiate dependence, comorbidity and seasonality of birth.

OBJECTIVE: In contrast with the non-opiate dependent population, persons biologically-dependent upon opioids display an excess life-time prevalence of affective and anxiety disorders. Many of these addicts state that opiates, particularly methadone, relieve or diminish the severity of their dysphoria. The purpose of this study is to explore this phenomena by analyzing how a specific population of long-term addicts (mean years of addiction 16.9, SD 3.8) differs from a non-opiate dependent population regarding seasonality of birth. METHODS: Birth months were determined for 457 opiate dependent patients, placed onto methadone maintenance for intractable opiate dependence, born between 1930-1970 (sorted by sex, race, year and place of birth), and compared to normal US birth statistics. Affective and anxiety disorders were screened for using psychometric testing, verified by structured clinical intervals. RESULTS: A significant difference was noted when comparing monthly births rates for patients and normals. Grouping the monthly data into birth trimesters (Oct-Jan; Feb-May; Jun-Sep) clearly shows this difference: opioid dependent persons--38.5/29.8/31.8%; normals--33.4/32.0/34.7%. As a group, intractable, opioid dependent patients demonstrate an increased life-time prevalence, relative to normals, of anxiety (27.8 vs. 13.9%), dysthymia (23.4 vs. 6.4%) and combined anxiety + dysthymia (17.9 vs. 1.5%); opioid dependent persons born between Oct-Jan demonstrated more anxiety (32.0 vs. 25.1%), dysthymia (29.3 vs. 19.5) and combined anx + dys (23.3 vs. 14.4) than those born in the other two trimesters. CONCLUSION: Persons entering methadone maintenance for opiate dependence with comorbid anxiety, dysthymia or combined anxiety + dysthymia are more likely to have been born in the period of Oct-Jan. This may be due to a higher risk of developmental aberrations occurring in infants born during the light-limited portion of the year creating a later propensity for intractable, opiate dependence.

Prevalence and improvement in psychopathology in opioid dependent patients participating in methadone maintenance.

Questions continue in the literature concerning potential cause and effect relationships between opiate dependency and several organically-based psychiatric disorders. For example, does opiate dependency produce secondary anxiety and dysthymic syndromes in otherwise healthy persons? or is narcotics misuse by a patient an attempt to self-medicate pre-existing psychopathology? Does the severity of psychopathologic symptoms decrease with time in treatment? To resolve such questions, we routinely conduct psychiatric evaluations on all opioid dependent patients enrolled into methadone maintenance. In this study, we report upon treatment outcomes for a cohort of 71 patients evaluated for psychopathology upon intake and followed up after being in treatment for a mean time of 24.5 (SD 8.0) months. Based upon objective psychometric testing with confirmatory clinical interview, significant, longitudinal improvements were seen in the symptom severity of anxiety and dysthymia present upon intake evaluation. Personality profiles also improved with treatment. In general, patients presenting with more severe psychopathology required more visits with professional staff in order to stabilize their life situations and personal relationships. No correlation was noted between drug use and severity of psychopathology. Data support the thesis that many opioid dependent patients are self-medicating themselves for preexisting organic psychopathology, most commonly, a combined anxiety-dysthymia syndrome. Improvement seems to occur secondary to the mood stabilizing properties of methadone in disorders thought to be mediated or moderated by endogenous endorphins rather than because of psychotherapeutic interventions.

Utilization of plasma and urine methadone concentrations to optimize treatment in maintenance clinics: I. Measurement techniques for a clinical setting.

One difficulty for methadone maintenance treatment programs is the absence of a simple, analytical method useful for guiding adjustment of methadone dose. Currently, dose adjustment decisions are based upon: documentation of continuing opioid use by qualitative urine drug screens, patient complaints of dose not holding or physiological evidence of opioid withdrawal. Although decisions utilizing clinical parameters are helpful in adjustment of dose, a sizeable number of patients continue to receive inadequate doses. Incorrectly, many clinicians assume that low doses prevent toxicity and favor eventual abstinence. One solution to the question of efficacious dose adjustment would be utilization of repetitive blood sampling for monitoring plasma methadone concentrations to insure they remain within established therapeutic windows. Unfortunately, it is impractical to obtain ongoing blood samples from these patients because of poor venous access and the unpleasantness of frequent phlebotomy. To circumvent these problems, we developed analytical methods useful for estimating plasma methadone concentrations from random, spot urine samples. Over the past several years, we have analyzed approximately 16,000 samples (urine plus plasma) drawn from 200 methadone maintenance patients. These data have allowed generation of methadone dose vs. plasma methadone concentration curves, demonstrating why dose adjustment decisions should be buttressed with objective laboratory data. In addition, these methods are useful for uncovering covert methadone supplementation and diversion. These improvements in clinical care have been accomplished using a computerized, pharmacokinetics program which accounts for compounding effects of urine pH, specific gravity, volume of distribution and gender upon renal methadone excretion. The program allows calculation of total plasma methadone concentrations from concurrent urine measurements, which are within 5-10% of actually measured values. This system has eliminated the need for venous blood sampling, resulted in optimization of patient doses and helped uncover supplementing or diverting of methadone.

Surface charge near the cardiac inward-rectifier channel measured from single-channel conductance.

The conductance of a channel to permeable ions depends on the number of ions near the mouth of the pore. Surface charge controls the local concentration, and impermeable cations can modify this charge. Correlating channel conductance with the concentration of impermeable cations therefore determines the local charge near the open pore. This paper presents data from cell-attached patches on embryonic chick ventricle cells, and it uses the conductance of inward-rectifier channels in the patch (in 100 mM K, with various concentrations of Na, Ca, Ba, and Mg) to estimate the local surface potential. The results indicate the presence of ionized residues near the mouth of the channel. Using the Boltzmann equation and the Gouy-Chapman relation, the surface potential due to these residues (in 100K/33Na/0Ca/0Ba/0Mg) is -40 mV, and the charge density is -0.25 e/nm2.

Sperm-activated currents in ascidian oocytes.

Using patch electrodes and the whole-cell recording technique to study fertilization currents in ascidian oocytes under voltage clamp, this paper shows that between -85 and 0 mV the currents are inward with an initial peak ranging from 50 to 600 pA. Voltages more positive than 0 mV inhibit initiation of the fertilization current, but by allowing the oocyte to return briefly to its resting potential fertilization occurs and fertilization currents are outward at positive potentials. By comparison with previous single-channel work, a fertilizing spermatozoon opens about 300 large-conductance channels with zero reversal potential.

Equilibrium energy analysis of freeze-fracture planes in membranes.

We have used equilibrium energy calculations to determine the most probable freeze-fracture planes in a lipid bilayer. Using a pairwise-summation computer method, we have generated numerical values for the Van der Waals potentials (electron shell repulsion, dispersion forces and electrostatic interactions) between molecules. We have compared our theoretical predictions with the experimental conclusion that the fracture planes occur normally between lipid molecules. These calculations also provide information about the composition of intramembranous particles, the potential for local clustering of single lipid types in the fluid membrane, and the importance of lipid molecules to the function of membrane proteins such as voltage-sensitive ion channels.