Association of busulfan area under the curve with veno-occlusive disease following BMT.

Busulfan pharmacokinetics, specifically area under the concentration curve (AUC), have been correlated with the occurrence of veno-occlusive disease (VOD) following BMT. To evaluate the risk of VOD, we studied 66 patients who received pharmacotherapeutically monitored busulfan regimens in combination with CY, etoposide (VP16) and/or Ara-C in preparation for BMT. These patients received a total of 16 doses of busulfan dosed as 1 mg/kg/dose q 6 h beginning at 09.00 (n = 39), 18.00 (n = 2), 21.00 (n = 1) or 24.00 (n = 24) h. With the first dose, blood samples were obtained at baseline, every 15-30 min for 2 h, then every 1-2 h for 4 h. Blood was analyzed for busulfan concentration by high performance liquid chromatography and AUC calculated by the trapezoidal rule. Seventeen patients (25.8%) were not evaluable for AUC calculation due to slow absorption and/or elimination: 13 of 27 (48.1%) received the first dose between 18.00-24.00 vs four of 39 (10.2%) patients who received the first dose at 09.00 (P < 0.001). Eighteen of 51 (35.3%) evaluable patients had an AUC > 1500 mumol x min/l; 10 of whom received doses reduced proportionally to achieve an AUC = 1200 mumol x min/l starting with the 10th to 15th dose. Six of 18 (33.3%) patients with an initial AUC > 1500 mumol x min/l developed VOD vs one of 33 (3.0%) patients with an initial AUC < 1500 mumol x min/l (relative risk = 11.1; P = 0.0056). Other pharmacokinetic parameters, age, gender, type of BMT, previous therapy or pre-transplant liver function tests were not predictive of VOD. A higher incidence of VOD occurred in patients receiving BUCY (4 of 10) compared to those receiving BUCYAra-C (1 of 18) or BUCYVP16 (7 of 38), which could not be attributed to increased busulfan exposure in the BUCY patients. Routine pharmacotherapeutic monitoring of busulfan is recommended with further study to evaluate the impact of earlier and greater overall dose reduction in patients with high initial busulfan exposures.

Interaction of cyclosporin A with human lipoproteins.

The ultracentrifugal fractionation of human serum after previous incubation with cyclosporin A showed that, in healthy fasting individuals, 8% of cyclosporin A was found in the very low density lipoproteins (VLDL), 31% in the low density lipoproteins (LDL), 46% in the high density lipoproteins (HDL) and 15% in the non-lipoprotein protein fraction. In non-fasted, healthy and in non-fasted, lipaemic individuals, 7 and 6% of cyclosporin A was found in chylomicrons, 9 and 13% in VLDL, 28 and 30% in LDL, 39 and 37% in HDL, and 12 and 13% in the non-lipoprotein protein fraction, respectively. In patients receiving cyclosporin A the distribution varied from 12 to 19% in VLDL, 21 to 28% in LDL, 33 to 43% in HDL, and 13 to 20% in non-lipoprotein proteins. The interaction between cyclosporin A and isolated normal human lipoproteins was also studied by ultrafiltration. All lipoproteins exhibited a non-saturable, low affinity, high capacity uptake for cyclosporin A. Analysis of the uptake by phospholipid vesicles showed a similar uptake, suggesting that cyclosporin A dissolves in the lipophilic portion of the lipoprotein molecule rather than being associated with specific binding sites.

Improved ultrafiltration method for determining unbound cortisol in plasma.

We describe a modified ultrafiltration method for measuring unbound cortisol in diluted or undiluted plasma or serum. After equilibration at 37 degrees C with purified [3H]cortisol, plasma or serum with or without buffer was placed in the ultrafiltration cell and two successive 0.2-mL fractions of protein-free ultrafiltrate were obtained. Under our conditions, free ligand concentration was independent of flow rate. The second fraction (the first is discarded) was used for determining the proportion of unbound cortisol. The assay is rapid (less than 2 h), practical (no more than 1.5 mL of plasma or serum is necessary), and reproducible (CV: 4.5% within assay and 5.2% in different assays). Samples from normal men and women (blood taken at 0800 and 1600 hours), from pregnant women, and from patients with Cushing's disease and adrenal insufficiency gave results that agreed with those obtained by equilibrium dialysis.

An improved ultrafiltration method for free thyroxine and triiodothyronine in serum.

We describe an ultrafiltration technique for rapidly and directly determining free triiodothyronine or free thyroxine, or both. After equilibrating serum at 37 degrees C with purified tracer of high specific activity, we placed 0.15 mL of serum in 2.8 mL of phosphate buffer (0.1 mol/L, pH 7.4) in the ultrafiltration cell and obtained successive 0.2- and 0.6-mL fractions of protein-free ultrafiltrate. Under our conditions free ligand concentration was independent of flow rate. After purifying the second fraction with protein-coated charcoal, we could determine the proportion of free triiodothyronine or free thyroxine. Samples from normal adult men and women, including women who were taking oral contraceptives or were pregnant, and from hypo- and hyperthyroid patients gave results that agreed with those obtained by equilibrium dialysis. Speed is the main advantage of the method: one technologist can complete the procedure in 2 h and, using a multi-micro-ultrafiltration system, can process many samples in one day. For laboratories where index-type reactions are performed routinely and direct free triiodothyronine or free thyroxine is determined only on selected specimens, this method is superior to dialysis. It is also very convenient for rapidly purifying tracers, to at least 97% radiochemical purity, with 94% recovery and no dilution.

Lipid interference in steroid radioimmunoassay.

To study lipid interference in steroid radioimmunoassays in which dextran-coated charcoal is used as the separating agent, we tested triolein and phosphatidylcholine as model hydrophobic and amphipathic lipids, respectively. Addition of either caused distortion of the standard curve to an extent that was inversely related to the polarity of the steroid molecule. Both lipids form a dispersion that entraps steroid molecules. When we increased the charcoal concentration, the effect of phosphatidylcholine addition was eliminated for assays of both polar and nonpolar steroids. In contrast, the effect from triacylglycerol was not corrected, particularly in assays of nonpolar steroids. We also studied mixtures of lipids mimicking the mixture of lipids extracted from plasma of normolipemic and hyperlipemic indviduals. The degree of lipemia that can be tolerated differs from assay to assay, and primarily varies directly with the polarity of the steroid being assayed.

Mechanisms of interference of nonesterified fatty acids in radioimmunoassays of steroids.

Addition of nonesterified fatty acids caused an apparent increase in unbound steroid present in supernatants in radioimmunoassays for steroids utilizing dextran-coated charcoal. Nonesterified fatty acid interference occurred at the initial binding interaction between steroid and its antiserum, and also at the step separating bound from unbound steroid. It was determined that nonesterified fatty acids, which had been added to radioimmunoassays, formed micelles and trapped steroids. The extent of the entrapment was inversely related to the number of polar groups in the steroid molecule, that is, hydrophobic steroids more easily interacted with the nonesterified fatty acid micelles. However, if the charcoal concentration were increased, the nonesterified fatty acid effect was eliminated for assays of polar steroids and greatly reduced for non-polar steroid assays.