Biomedical subjects

R E Mullins

Publications and source records attributed to R E Mullins.

At least 37 records · Page 2Linked to original sources

PubMed1988

Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.

Acyltransferases↗

PubMed1988

Quantitation of free sphingosine in liver by high-performance liquid chromatography.

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.

Animals↗

PubMed1988

Ion transport mediated by copolymers composed of polyoxyethylene and polyoxypropylene.

Block copolymers composed of polyoxyethylene and polyoxypropylene were found to increase the influx of Na+ and the efflux of K+ from human erythrocytes. They were, however, ineffective at promoting the transport of Ca2+. The size of the ion fluxes induced by the copolymers correlated with their efficacy in stimulating inflammation. These compounds were also found to induce conductance increases in planar lipid bilayers in a nonvoltage dependent and nonstepwise manner. In both experimental systems, ion transport was facilitated only under temperature and ionic-strength conditions in which the polymers form aggregates in aqueous solution. In neither system did the concentration dependence of transport activity exhibit a pronounced cooperativity. These observations are consistent with the view that aqueous monomers of these surface active agents partition into the membrane, where they facilitate the conductive movement of monovalent cations by means of a carrier type mechanism. As a novel class of ionophores, these substances are of practical interest because they can be water soluble and are potentially reversible.

Biological Transport↗

PubMed1987

Neuron-specific enolase. Assessment by ELISA in patients with small cell carcinoma of the lung.

Measurement of tissue-specific enolase isoenzymes may be of assistance in identifying small cell carcinomas of the lung and in distinguishing them from other pulmonary tumors. Enolase (E.C. 4.2.1.11) is a dimeric enzyme composed of various permutations of three immunologically distinct subunits alpha, beta, and gamma. Five isoenzymes alpha alpha, beta beta, gamma gamma, alpha beta, and alpha gamma have been identified. Immunohistochemical studies using antibodies to the gamma subunit have localized alpha gamma and gamma gamma specifically within neuronal and neuroendocrine tissues. Because of this limited distribution, neuron-specific enolase (NSE) can function as a biochemical marker for neuroendocrine tumors. The authors developed an enzyme-linked immunosorbent assay (ELISA) using the double antibody sandwich method. The sandwich is composed of rabbit antirat enolase that cross-reacts to the human gamma monomer, making the test specific for the gamma gamma isoenzyme. The avidin-biotin-peroxidase complex system is used to provide increased assay sensitivity. Serum samples from patients with histologically diagnosed small cell carcinoma have concentration of NSE 20- to 30-fold greater than that found in normal serum. Studies were conducted on patients with a variety of malignant pulmonary lesions and compared with controls to determine the value of NSE as a tumor marker.

Carcinoma, Small Cell↗

PubMed1987

Monitoring aminoglycoside therapy.

The aminoglycoside antibiotics are indispensible therapeutic agents; however, a high incidence of toxic effects mandates that they be used with caution. In many patients, careful monitoring of serum concentrations is required. Because of their chemical characteristics, aminoglycosides are given parenterally except in certain well-defined clinical situations such as bowel preparation prior to surgery. The drugs act by interfering with microbial protein synthesis and are uniformly bactericidal. Plasmid-mediated resistance remains a problem, particularly in the hospital environment. Aminoglycosides find their most frequent use in infections caused by aerobic gram-negative bacilli. The pharmacokinetics have been well characterized, and an appropriate dosage regimen can be selected to achieve peak bactericidal levels while maintaining trough concentrations low enough to minimize toxicity. Monitoring of serum aminoglycoside concentrations is essential in many types of patients, such as those having compromised renal function, those receiving a maximum dose of the drug, the obese, the elderly, and patients with cystic fibrosis. Close cooperation between the clinical laboratory and all groups involved in the patient's care is necessary to make maximum clinical use of drug monitoring information.

Aminoglycosides↗

PubMed1987

Electrophoretic mobility, concentration, and activity of alpha 1-antitrypsin in serum of patients undergoing bone-marrow transplantation.

We have observed an electrophoretically abnormal, nonfunctional species of alpha 1-antitrypsin in serum from patients who were receiving bone-marrow transplants for treatment of leukemia or aplastic anemia. Three of four patients in whose serum this protein appeared died soon after; the fourth recovered, and the disappearance of the abnormal alpha 1-antitrypsin paralleled his recovery. This suggests that the inability to maintain functional activity of alpha 1-antitrypsin predisposes patients to life-threatening complications during recovery from bone-marrow transplants.

Anemia, Aplastic↗

PubMed1986

Stability of ranitidine admixtures frozen and refrigerated in minibags.

The stability of ranitidine hydrochloride stored frozen and refrigerated in polyvinyl chloride minibags was studied. Ranitidine hydrochloride was added to either 5% dextrose injection or 0.9% sodium chloride injection to yield concentrations of 0.5, 1.0, and 2.0 mg/mL. In phase 1 of the study, admixtures containing ranitidine hydrochloride 1 mg/mL were stored at 4 degrees C for 10 days. In phase 2, solutions were frozen for 30 days at -30 degrees C and were later refrigerated for 14 days. Ranitidine concentration was tested using a stability-indicating high-performance liquid chromatographic assay at time zero and at intervals during storage. Sterility tests were performed on some samples, and various admixtures were visually inspected and tested for pH. At least 90% of the initial concentration of ranitidine remained in all solutions at all storage conditions. No visual changes or changes in pH or sterility were observed. Ranitidine hydrochloride in concentrations of 0.5, 1.0, and 2.0 mg/mL in 5% dextrose injection or 0.9% sodium chloride injection may be stored in polyvinyl chloride minibags frozen for 30 days followed by refrigeration for an additional 14 days.

Drug Combinations↗

PubMed1986

Sensitivity of isoelectric focusing, ion exchange, and affinity chromatography to labile glycated hemoglobin.

We examined the effect of labile glycated hemoglobin on measurements of glycated hemoglobin by several commercial procedures. Erythrocytes from diabetic and nondiabetic patients were incubated in vitro with various concentrations of glucose, to generate labile glycated hemoglobin, and the species of glycated and nonglycated hemoglobin in each sample were identified by isoelectric focusing. Glycated hemoglobin was then assayed by the Bio-Rad A1 column method (I), the Bio-Rad A1c column method (II), and the Pierce affinity column method (III). I was sensitive to the labile (aldimine) fraction of glycated hemoglobin, and percentages of glycated hemoglobin so determined represented the sum of the labile fraction plus hemoglobin A1c and other stable glycated species. This spurious increase in glycated hemoglobin concentration by the aldimine could be obviated by any of three wash procedures, which eliminated the labile fraction from the samples: incubating the erythrocytes (a) in phosphate-buffered saline (pH 7.4) for 18 h at 22 degrees C, (b) in 100 mmol/L citrate (pH 5.0) for 30 min at 37 degrees C, or (c) in saline containing, per liter, 30 mmol of semicarbazide and 12 mmol of aniline hydrochloride (pH 5.0) for 30 min at 37 degrees C. Methods II and III did not detect the labile fraction. However, treatments a-c decreased the concentrations of stable glycated hemoglobin as determined by all three column-chromatographic methods as compared with unwashed sample. By isoelectric focusing we determined that blood with high glucose content had concentrations of aldimine roughly proportional to the blood glucose concentration. The kinetics of formation of labile glycated hemoglobin in these cells were consistent with the reported rate constants determined by using purified hemoglobin preparations in vitro.

Chromatography, Affinity↗

PubMed1985

Alpha 1-antitrypsin in cerebrospinal fluid of patients with neurologic diseases.

Proteases and their inhibitors have been implicated in the pathogenesis of neuroimmunologic diseases, particularly multiple sclerosis (MS). We measured the immunochemical level and functional activity of alpha 1-antitrypsin (AAT) in cerebrospinal fluid (CSF) and serum in patients with MS and other neurologic diseases. Increases in the immunochemical level of AAT in CSF correlated directly with disturbances in the blood-brain barrier, as reflected by the ratio of albumin in CSF to that in serum. The AAT activity in CSF directly correlated with the immunochemical level when all patients were compared. However, the AAT activity in patients with inflammatory diseases tended to be decreased relative to the immunochemical level, suggesting inactivation of AAT in these disorders. The AAT activity was not significantly altered in patients with MS, despite reports of increased protease activity in active MS.

Blood-Brain Barrier↗

PubMed1985

Stability of ranitidine hydrochloride and amino acids in parenteral nutrient solutions.

The stability of ranitidine hydrochloride in parenteral nutrient (PN) solutions and the effect of ranitidine hydrochloride on the amino acids in the PN solutions were studied. Six PN solutions (three each with amino acid contents of 2.125 and 4.25%) were prepared. Each PN solution also contained dextrose 25%, electrolytes, trace elements, vitamins, and heparin sodium. Ranitidine hydrochloride injection was added to four of the PN samples. Of the final samples, two contained no ranitidine, two contained ranitidine hydrochloride 50 micrograms/mL, and two contained ranitidine hydrochloride 100 micrograms/mL. Admixtures of ranitidine hydrochloride at the two concentrations in 0.9% sodium chloride injection were also prepared. Samples were observed for color change and tested for pH during storage at room temperature. Concentrations of amino acids were measured after 24 hours in samples without ranitidine and in samples containing ranitidine hydrochloride 100 micrograms/mL. Ranitidine hydrochloride content was determined by high-performance liquid chromatography at 12, 24, and 48 hours. No visual changes or pH changes occurred by 24 hours. All PN solutions became darker by 48 hours. The presence of ranitidine hydrochloride did not substantially affect amino acid concentrations. At 24 hours, at least 90% of the initial ranitidine concentrations remained in all samples. In three of the four PN samples at 48 hours, less than 90% of initial ranitidine concentrations remained. Ranitidine hydrochloride in concentrations of 50 and 100 micrograms/mL in parenteral nutrient solutions containing 4.25 and 2.125% crystalline amino acids is stable for 24 hours at room temperature. Under these conditions, concentrations of the amino acids contained in the PN solutions were not affected by the addition of ranitidine hydrochloride.

Amino Acids↗

PubMed1985

Assay of total and free propranolol in plasma by liquid chromatography with fluorometric detection.

We report a liquid-chromatographic method for assay of total and free propranolol and its metabolite, propranolol glycol, in plasma. An ultrafiltration device is used to separate the free drugs from the protein-bound drugs. The total and free drugs are isolated from plasma or ultrafiltrate by liquid-solid extraction on a Waters C18 SEP-PAK and the drugs are quantified on a C18 mu-Bondpak radial-compression column. The mobile phase consists of equal parts of methanol and a solution of potassium 50 mmol/L phosphate and 2.5 mmol/L 1-pentane-sulfonic acid, pH 5.0. Detection was by fluorescence, with excitation at 296 nm and emission at 338 nm. Time required for sample preparation for total-propranolol assay is 40 min per eight samples. Sample preparation for free-propranolol assay requires an additional 1 h per eight samples. Chromatography requires 15 min per sample. The extraction recovery is approximately 75% and the assay curve is linear from 5 to 400 micrograms/L. The run-to-run CV is 4% for total propranolol, 15% for free propranolol.

Binding, Competitive↗

PubMed1984

The effect of storage conditions on ion exchange and affinity chromatographic assays for glycated hemoglobin.

Experiments were performed to evaluate the effect on glycated hemoglobin determinations when erythrocytes were stored under various conditions. We studied the influence of time, temperature, glucose concentration, and pH on results obtained by three commercially available column chromatographic procedures designed to quantify glycated hemoglobin. An affinity chromatographic procedure which measures total glycated hemoglobins (GHb) was compared with two ion-exchange column methods. One ion-exchange method measures total hemoglobin A1 and the other is purported to measure hemoglobin A1c. Stability studies were performed using specimens from nondiabetics and diabetics, supplemented in vitro with glucose at pH 7.4 and pH 5.0. Results obtained from the A1c and the GHb tests were unchanged over time for samples stored under the described conditions. Erythrocytes were incubated with glucose to produce labile glycated hemoglobin and assayed before and after overnight incubations in saline. These experiments showed that the A1c and GHb assays were unaffected by the presence of labile glycated hemoglobin. Column fractions were analyzed by isoelectric focusing and scanned with a laser densitometer for quantitative assessment of hemoglobin species found in each fraction. These experiments showed that the three column methods measure very different hemoglobin species. Focusing of eluates from the A1 and GHb tests revealed carryover of several hemoglobin species into the measured fractions, while the A1c test was found to be much more specific for HbA1c. Our data suggest that it may be possible to analyze glycated hemoglobins in samples transported to a reference laboratory without special treatment.

Chromatography, Affinity↗

PubMed1984

Standardized automated assay for functional alpha 1-antitrypsin.

We describe an enzymic assay for the functional activity of alpha 1-antitrypsin, in which tosyl-Gly-Pro-Lys-p-nitroanilide acetate (Chromozym PL) is the trypsin substrate. For the indicating reaction, the concentration of substrate is about double the Km of its reaction with trypsin. This assay is an improvement over reactions involving N-alpha-benzoyl-DL-arginine p-nitroanilide as substrate, the solubility of which in water is less than the Km of its reaction with trypsin. The assay is standardized in terms of active sites of trypsin inhibited per liter of serum, with p-nitrophenyl-p'-guanidinobenzoate as the active site titrant. The results agreed well with those by an immunonephelometric assay for alpha 1-antitrypsin (r = 0.953). The within-run and run-to-run precision was approximately 3%. The results of the assay varied linearly with alpha 1-antitrypsin concentration from 0 to 60 mumol/L of serum. The minimum sample size was 50 microL, and analysis of 24 samples took less than 20 min.

Aniline Compounds↗

PubMed1983

Correlation of standardized serum protein determinations with calculated and measured colloid osmotic pressure.

The colloid osmotic pressure (COP) was measured on the serum of 250 individuals (healthy persons and patients) using a commercial oncometer. The total serum protein concentrations were measured by five methods, and the serum albumin concentrations were measured by four methods. The protein determinations were standardized as recommended by the Study Group on Proteins of the AACC Committee on Standards. The measured COP (mCOP) was correlated with each of the nine analytically determined protein concentrations. The mCOP also was correlated with a COP calculated (cCOP) from the Landis-Pappenheimer equation: cCOP = 2.1 (total protein [TP]) + 0.16 (TP)2 + 0.009 (TP)3. The authors found excellent linear correlations between mCOP and total protein concentration, and between mCOP and albumin concentration, when the protein determinations were standardized properly. The correlation between mCOP and protein concentrations was as good as the correlation between mCOP and cCOP in all cases. The serum albumin as determined by the DuPont ACA and mCOP gave the best linear relationship (r = 0.940) of the methods tested. Using the linear regression equation of albumin (ACA) versus mCOP, we were able to predict a cCOP to within +/- 3 mmHg for more than 95% of all samples tested.

Autoanalysis↗

PubMed1983

Effects of Fluosol-DA (artificial blood) on clinical chemistry tests and instruments.

Artificial blood must be added to the list of therapeutic agents that produce interference with diagnostic laboratory tests. Fluosol-DA (Alpha Therapeutic Corp., Los Angeles, CA), a stable 20% emulsion of perfluorocarbons in aqueous medium, is being evaluated in clinical trials as a blood substitute in the United States. We investigated its effects in blood and serum samples on test results and instruments in the clinical chemistry laboratory. The 20% emulsion was added to blood or serum specimens in amounts corresponding to the replacement of in-vivo plasma volumes of 10-50%, concentrations that would be expected in blood samples obtained from patients who have received Fluosol. Observed interferences mimicked those caused by high triglyceride concentrations in serum specimens: interference with chemical reactions and generation of spurious absorbance readings because of turbidity. These types of errors are often additive, and the cumulative effect may cause either erroneously high or low values for the analytes concerned. Because Fluosol may be used widely, although infrequently, for patients refusing blood transfusions on religious grounds and for patients with rare antibodies to red blood cells who require transfusion, laboratories analyzing specimens containing Fluosol should be aware of the potential errors.

Alkaline Phosphatase↗

PubMed1982

Improved one-dimensional thin-layer chromatography of phospholipids in amniotic fluid.

With this extraction procedure and chromatographic method, six amniotic fluid phospholipids [phosphatidylcholine (lecithin), sphingomyelin, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and phosphatidylglycerol] are completely resolved in less than 70 min from the time the specimen is received. The mobile phase (chloroform/petroleum ether/methanol/acetic acid, 5/3/1.6/1, by vol) is used with commercially available 7.5 X 10 cm silica-gel plates. The phospholipids are made visible by immersing the plate in cupric acetate/phosphoric acid. Lecithin/sphingomyelin (L/S) ratios are determined densitometrically. Rf values are well reproducible for both samples and standards, as are L/S ratios, and the method is simple and inexpensive. The relation between L/S ratios (for 100 specimens of amniotic fluid measured by this method) and fetal status is consistent with other reports. We confirm phosphatidylglycerol to be a sensitive indicator of fetal lung maturity: when it was detectable in amniotic fluid, the newborn invariably showed no symptoms of the respiratory distress syndrome.

Amniotic Fluid↗

PubMed1982

Sample preparation and liquid-chromatographic analysis for tricyclic antidepressants in serum.

We report two separate sample-preparation procedures and a "high-pressure" liquid-chromatographic method for quantitating nine tricyclic antidepressant drugs: trimipramine, doxepin, amitriptyline, imipramine, desmethyldoxepin, nortriptyline, desipramine, maprotiline, and protriptyline. The drugs are extracted from 1 mL of serum by a single manual hexane extraction or by using DuPont's PREP I Automated Sample Processor. The drugs from either process are chromatographed on a mu-Bondapak-CN column with a mobile phase of acetonitrile/methanol/Na2HPO4, 5 mmol/L (60/15/25 by vol), pH 7.0, at a flow rate of 2.0 mL/min. Absorbance at 254 nm is linear with drug concentration from 25 to 1000 mg/L for each drug. With the manual extraction procedure the detection limit was 10 mg/L, absolute recovery 20-44%, within-run precision (CV) 10.1%, and run-to-run precision 11.1%. The performance of automated sample extraction was better, with a detection limit of 5 mg/l, absolute recovery 72-97%, within-run precision 6.1%, and run-to-run precision 6.6%.

Antidepressive Agents, Tricyclic↗

PubMed1980

Maltosyl isothiocyanate: an affinity label for the glucose transporter of the human erythrocyte membrane. 1. Inhibition of glucose transport.

Maltosyl isothiocyanate (MITC) has been synthesized from maltose with an overall yield of 88%. It has been found to be a potent irreversible inhibitor of zero trans influx of glucose with human erythrocytes. Kinetic analysis of glucose transport after treatment of erythrocytes with MITC revealed that VT was diminished while KT was unchanged. Transportable sugars and competitive inhibitors of monosaccharide transport protected against MITC inhibition, while carbohydrates which do not interact with the transporter gave no protection. Covalent inhibitors of anion transport were without effect on glucose transport. MITC fulfilled the kinetic requirements for an affinity label of the glucose transporter of human erythrocytes [Groman, E. V., Schultz, R. M., & Engel, L. L. (1977) Methods Enzymol. 46, 54].

Affinity Labels↗