Elizabeth Sykes

Effects of Cremophor EL on distribution of Taxol to serum lipoproteins.

The clinical formulation of the anti-tumour agent Taxol involves the use of a mixture of ethanol and Cremophor EL. Gel electrophoresis and density-gradient ultracentrifugation were used to detect effects of Taxol infusions on serum lipoproteins. Use of the Cremophor vehicle results in a decrease in the electrophoretic mobility of serum lipoproteins along with the appearance of a lipoprotein dissociation product. These effects persist during a 24 h infusion and for at least 1.5 h afterwards, and can be reproduced in vitro using purified high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In control serum, Taxol binds to albumin > HDL, but after serum is exposed to Cremophor EL in vitro or in vivo substantial binding of Taxol to the lipoprotein dissociation product(s) was observed. The latter could represent an important factor in taxol biodistribution.

Analytical relationships among Biosite, Bayer, and Roche methods for BNP and NT-proBNP.

This study determined whether, for patient monitoring, it is feasible to convert B-type natriuretic peptide (BNP) results obtained using Triage (BNP, Biosite, San Diego, CA), Centaur (BNP, Bayer Diagnostics, Tarrytown, NY), and Elecsys 2010 (N-terminal proBNP; Roche, Indianapolis, IN) assays. Concordance between assays and effects of renal impairment also were assessed. Samples were primarily from emergency center patients. Biosite testing was performed immediately; Bayer and Roche testing was performed later on plasma stored frozen (–30°C). Logistic regression relationships were as follows: Bayer = 0.57 Biosite + 23.1, n = 121, R 2 = 0.85; Roche = 6.09 Biosite – 220.4 + 1,131.6 (if female), n = 131, R 2 = 0.57; and Roche = 15.34 Bayer + 2,400.8, n = 150, R 2 = 0.23. An increased serum creatinine level (≥2 mg/dL [≥177 μmol/L]) influenced the Roche results. We conclude the following from this preliminary study: (1) Results from one method cannot be converted reliably to another using regression relationships. (2) When using manufacturers’cutoff values, concordance between assays was acceptable. (3) Renal impairment affected Roche results.

Interactions of Solutol HS 15 and Cremophor EL with plasma lipoproteins

Two emulsifying agents, Solutol HS15 and Cremophor EL, were compared with regard to their effects on human plasma lipoproteins in vitro and on mouse plasma lipoproteins in vitro and in vivo. Both agents promoted binding of a hydrophobic photosensitizing agent (C8KC) to a circulating plasma species of low bouyant density. Persistence of this material was greater with Cremophor than with Solutol. Experiments carried out with labeled Solutol indicated that the vehicle itself is a component of this new species. High concentrations of either vehicle ( > or = 0.06%) led to decreased electrophoretic mobility of human LDL and HDL in vitro. In the mouse, a different effect was observed, resulting in complex changes in electrophoretic mobility of plasma lipoproteins. The plasma half-life of C8KC in the circulation of the mouse was correlated with the persistence of an altered electrophoretic lipoprotein pattern. Since Solutol and C8KC showed similar half-lives, this result suggests that the plasma half-life of the sensitizer is correlated with the persistence of the vehicle. While Solutol and Cremophor were designed to be vehicles for drug formulation, they also influence persistence of some drugs in the circulation.

Pharmacokinetics of Photofrin II distribution in man

Plasma samples were obtained from 4 patients at intervals after administration of Photofrin II (2 mg/kg). Total porphyrin in plasma and protein/lipoprotein fractions was estimated after hydrolysis, and levels of different Photofrin II components were measured by HPLC. Pharmacokinetic data are provided relating to rates of loss of total porphyrin from plasma and to pool size and half-lives of sensitizers bound to albumin and lipoprotein fractions. Preferential persistence of the most hydrophobic components of Photofrin II was observed.

Effect of S-adenosylhomocysteine on insulin-independent release of pyruvate dehydrogenase activator from rat adipocyte plasma membranes☆

The addition of insulin to adipocyte plasma membranes has been shown to release a low molecular weight, acid stable mediator which activates mitochondrial pyruvate dehydrogenase. The insulin-dependent release of this activator is dependent on the method used to prepare the plasma membranes. Adipocyte plasma membranes prepared in 0.25 M sucrose, 10 mM MOPS, pH 7.4 released an activator of pyruvate dehydrogenase in an insulin-independent manner. Insulin is required to stimulate phospholipid methylation in these membranes. The inhibition of insulin-stimulated phospholipid methylation with 1 mM S-adenosylhomocysteine resulted in a significant increase in amount and/or activity of the pyruvate dehydrogenase activator. The insulin-dependent dependent release of mediators of insulin action from adipocyte plasma may be regulated by phospholipid methylation.

Transport, localization, and phototoxicity of m-THPC

Since the photosensitizing agent mTHPC is insoluble in water, a formulation procedure, usually involving poly(ethylene glycol) and ethanol, is required for clinical and pre-clinical studies. Using this vehicle, we found that drug accumulation by murine leukemia L1210 cells in vitro was slow; only non-viable cells with damaged membranes showed rapid uptake of mTHPC. The drug ultimately localized in the cytosol, and subsequent irradiation led to mitochondrial > lysosomal >> membrane photodamage, and a rapid apoptotic response. The rate-limiting step in drug accumulation appears to involve dissociation of a transient albumin-bound aggregated species which exhibited a low fluorescence yield. Initial formation of this product may explain the unusual mTHPC pharmacokinetics in man recently reported. Dilution of mTHPC with human plasma led to a gradual increase in fluorescence yield as the drug became associated with lipoproteins and proteins. When a steady-state was reached, density-gradient ultracentrifugation indicated distribution of mTHPC to HDL > LDL >> albumin.

Association of high-molecular-mass and electrophoretically atypical alkaline phosphatases

Polyacrylamide gel electrophoresis of alkaline phosphatase may yield abnormally migrating fractions; these include high-molecular-mass alkaline phosphatase, which remains at the gel origin, and immunoglobulin-alkaline phosphatase complexes, which have a mobility approximately 1/3 that of liver isoenzyme. We performed a retrospective study of 19 patients whose sera exhibited atypical alkaline phosphatase fractions, defined as bands whose mobility was slower than bone, liver, or intestinal alkaline phosphatase; 17 had a mobility approximately 1/3 that of liver isoenzyme and 16 also exhibited gel origin enzyme activity or high-molecular-mass bands. The strong association of the atypical and high-molecular-mass alkaline phosphatases suggests that they may be structurally related, both consisting of either immunoglobulin-enzyme complexes or membrane-alkaline phosphatase complexes. This hypothesis is supported by (1) one serum available for investigation containing alkaline phosphatase-immunoglobulin complexes in both abnormally migrating fractions, but on detergent treatment showing no evidence of membrane-bound enzyme; (2) detergent treatment of serum from patients with only high-molecular-mass alkaline phosphatase creating bands with a mobility of approximately 1/3 that of the liver isoenzyme.

Effect of insulin, S-adenosylhomocysteine, phospholipase C, n-butanol and Triton X-114 on alkaline phosphatase from isolated rat adipocyte plasma membranes

Alkaline phosphatase activity has been described in aqueous extracts of rat adipose tissue [l], in rat adipocyte plasma membrane fractions [2] and more recently in human adipose tissue [3]. Studies with hepatocytes have shown this to be a membrane-bound enzyme covalently linked to the polar head group of phosphatidylinositol (PI) [4]; this interaction is supported by the finding that PI-specific phospholipase C releases alkaline phosphatase from hepatocyte plasma membranes [5,6]. It has also been proposed that the butanol-extraction of alkaline phosphatase from plasma membranes is caused by an autolytic process derived from activation of a plasma membrane phospholipase, phospholipase D [7] or PI-specific phospholipase C [8]. Insulin stimulates phospholipid methylation in rat adipocyte plasma membranes [9,10] and releases a low molecular weight, acid-stable mediator which activates mitochondrial pyruvate dehydrogenase [ll]. Inhibition of insulin-stimulated phospholipid methylation by S-adenosylhomocysteine results in a significant increase in activity of the pyruvate dehydrogenase activator [12]. Saltiel et al [13] have suggested that insulin modulates the activity of both pyruvate dehydrogenase and low k, CAMP phosphodiesterase by activation of a PI-specific phospholipase C in plasma membranes, hydrolysis of a novel PI-glycan and release of a modulator of insulin action. Using polyacrylamide gel electrophoresis (PAGE), we describe the effect of phospholipase C, n-butanol, Triton X-114, insulin and S-adenosylhomocysteine on

Discrepant intact parathyroid hormone result by immunoassay

a To convert to pg/ml, multiply by 1.0. b Range secondary to multiple separate assessments over a 6 month period. c Performed at ARUP Laboratories, Salt Lake City, UT. d Performed at Mayo Medical Laboratories, Rochester, MN. Intact parathyroid hormone (iPTH) measurement plays an essential role in thework-up of hypercalcemia and hypocalcemia.We recently encountered a 59-year-old Caucasian female with serum calcium of 2.8 mmol/l (reference interval: 2.1–2.6 mmol/l) and ionized calcium of 1.46 mmol/l (1.12–1.32 mmol/l, pH 7.34). Her serum iPTH concentration was 195 ng/l (9–69 ng/l) on the Siemens Immulite® 2000. Her serum creatinine on multiple separate occasions over the previous year ranged from 68.07 to 79.56 μmol/l (53.04–123.76 μmol/l) with an eGFR of 0.63–0.72 ml/s/m (0.58–0.86 ml/s/m). While she was mildly 25-hydroxy vitamin D deficient and taking oral supplementation, the 1,25-dihydroxy vitamin D concentration was normal. Serum alkaline phosphatase and phosphate were within the mid-portion of the reference interval and PTH-related peptide concentration was below the lower limit of detection. A parathyroid SPECT (Single Proton Emission Computerized Tomography) scan showed a lesion consistent with a parathyroid adenoma. On the day of surgery, however, the initial pre-operative plasma iPTH on the Siemens ADVIA Centaur® was 11.9 ng/l with a subsequent redrawn specimen being 12.2 ng/l, causing her surgery to be canceled. At our institution, the Centaur® is used for intra-operative iPTH testing because of its shorter assay turnaround time (18 min) compared to that of the Immulite® (60 min) which is used for routine testing. Additional serum samples were obtained from the patient. Dilution studies showed no evidence of high-dose hook effect on the Centaur® iPTH assay. Treatment of serum with heterophile antibody blocking reagent (Scantibodies Laboratory, Santee, CA) suggested lack of antibody interference. Subsequent outpatient testing continued to yield an elevated iPTH by Immulite® but a low-normal concentration by the Centaur® iPTH assay. In addition, serum samples were tested by three other methods, all yielding elevated iPTH concentrations (Table 1). Since the Centaurs iPTH assay product insert indicated high dose biotin therapy could potentially falsely depress results [1], we inquired whether the patient was taking biotin supplements, which she denied. Serum biotin concentration (4.9 nmol/l; adult 0.9–12.3 nmol/l) was not increased (Cambridge Biomedical Research, Boston, MA). We have not been able to determinewhy this patients iPTH concentration on the Centaur® is so discrepant when compared to the iPTH concentration on the Immulite® and other methods. The low iPTH value obtained on the Centaur® platform indicates that somehow one or both of the antibodies used in the immunoassay may not have been able to bind to their respective epitopes. Manufacturers use different antibodies to recognize epitopes on the iPTH molecule. The iPTH assay on the Centaur® uses a 2-site sandwich immunoassay using direct chemiluminometric technology [1]. A biotinylated polyclonal goat anti-human PTH recognizing amino acids 39–84 is used as the capture antibody [1]. An acridinium ester labeled polyclonal goat anti-human PTH recognizing amino

Effects of the bile acid UDCA on PDT efficacy in vitro and in vivo

The phototoxicity of PDT in cell culture can be promoted by the relatively hydrophilic bile acid UDCA (ursodeoxycholic acid). This was attributed to a conformational change in the anti-apoptotic protein Bcl-2, leading to an enhanced sensitivity to photodamage by sensitizers that target sites of Bcl-2 localization. UDCA also promoted the binding and inactivation of Bcl-2 by the non-peptidic antagonist HA14- 1, suggesting that UDCA may also be useful for promoting chemotherapy designed to target Bcl-2. In tumor-bearing animals, addition of UDCA to a PDT protocol involving the tin etiopurpurin SnET2 resulted in enhanced cancer control, but there was no effect on the extent of PDT-induced vascular shut-down. These results are consistent with the propo proposal that UDCA only promotes direct tumor cell kill. In this report, we have sal summarized recent research relating to mode of action of UDCA as it effects the on the efficacy of photodynamic therapy where Bcl-2 is among the PDT targets, and discuss the implications of the results.