David W. Nierenberg

Determination of vitamins a and e in serum and plasma using a simplified clarification method and high-performance liquid chromatography

A method of sample clarification and high-performance liquid chromatography specifically developed to permit precise and rapid determination of vitamin A (retinol) and vitamin E (alpha-tocopherol) in serum and plasma is reported. Serum proteins were denatured by the addition of acetonitrile containing alpha-tocopherol acetate, the internal standard; the vitamins were subsequently extracted into an organic matrix consisting of ethyl acetate-butanol (1:1); no solvent evaporation step was required. The three vitamins of interest were eluted from a reversed-phase C18 column with an isocratic mobile phase methanol-water (95:5); detection was accomplished by measuring ultraviolet absorption at 280 nm. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from spiked aqueous samples averaged 100.0, 100.0 and 98.8%, respectively. Recoveries of retinol, alpha-tocopherol and alpha-tocopherol acetate from plasma and serum relative to water were 102.6, 96.9 and 96.5%, respectively. Retinol and alpha-tocopherol were stable in the extraction matrix for up to 3.5 h, and were stable in heparinized plasma stored at room temperature for two days. Oxalate, citrate and EDTA caused significant losses of retinol and alpha-tocopherol, while vitamin levels in serum and heparinized plasma were similar. Limits of detection for retinol and alpha-tocopherol were 60 ng/ml and 0.9 micrograms/ml, respectively. Each run required 12 min. Same-day coefficients of variation were 3.5 and 3.6% for retinol and alpha-tocopherol, respectively (n = 11). Between-day coefficients of variation for retinol and alpha-tocopherol were 4.8 and 5.5%, respectively (n = 5). This method permits simple, rapid, sensitive, selective and precise determination of retinol and alpha-tocopherol using 0.5 ml of serum or heparinized plasma.

Neuropathologic lesions and CSF morphine concentrations during chronic continuous intraspinal morphine infusion. A clinical and post-mortem study

&NA; Seven patients with chronic intractable pain due to cancer were given chronic intraspinal narcotic administration (CINA) and subsequently underwent postmortem examination. All deaths were unrelated to CINA. Two of these patients were found to have clinically unsuspected posterior column degeneration. Both patients had had epidural catheters placed and one had received prior radiotherapy to ports which included parts of the spinal cord. In retrospect, it is impossible to ascertain whether the degeneration occurred before or after infusion of morphine began. Review of the potential causes for posterior column degeneration suggests that neuropathy associated with malignant disease is more likely the cause of the degeneration rather than intraspinal infusion of morphine. However, continued vigilance at autopsy is recommended. In addition, utilizing a new method for measuring cerebrospinal fluid (CSF) concentrations of morphine via high‐pressure liquid chromatography, CSF morphine levels at steady state were measured in 5 patients. These levels were much lower than peak levels previously reported following bolus intraspinal administration. The ability of these measurements to contribute to knowledge of efficacy, toxicity. lumbar‐cisternal concentration gradients and differentiation of tolerance from drug delivery problems is discussed.

The central nervous system serotonin syndrome

Clinical Pharmacology and Therapeutics (1993) 53, 84–88; doi:10.1038/clpt.1993.12

Serum and plasma β-carotene levels measured with an improved method of high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of beta-carotene concentrations in serum and plasma is reported. Using a method modified from a previously published technique, serum and plasma proteins are denatured by exposure to perchloric acid, and beta-carotene is subsequently extracted into an organic matrix consisting of ethyl acetate-tetrahydrofuran (1:1); no evaporation step is required. Separation is achieved using isocratic elution from a reversed-phase C18 column with UV detection at 436 nm. Recovery of beta-carotene from water and plasma was greater than 98.1%; beta-carotene was stable in the extraction matrix for at least 4 h. Three anticoagulants (oxalate, citrate, and EDTA) caused losses of beta-carotene; perchloric acid and tetrahydrofuran could also destroy beta-carotene under certain conditions. Each run required less than 15 min; within-day coefficient of variation for identical samples averaged 2.3%, between-day coefficient of variation was 4.4% and sensitivity was better than 10 ng/ml. Stability of beta-carotene in plasma was also examined. This method permits a simple, rapid, sensitive, precise, and accurate determination of beta-carotene using 0.5 ml of serum or heparinized plasma.

Facilitating prompt diagnosis and treatment of the neuroleptic malignant syndrome

Clinical Pharmacology and Therapeutics (1991) 50, 580–586; doi:10.1038/clpt.1991.184

Two Cases of Rapid Onset Parkinson's Syndrome Following Toxic Ingestion of Ethylene Glycol and Methanol

Ethylene glycol and methanol are toxic alcohols commonly found in a variety of commercial products. We report two cases, one associated with ethylene glycol and one with methanol poisoning, which both led to acute hemorrhagic necrosis of the basal ganglia and resulted in acute Parkinsons syndrome. It is unlikely that oxalate crystal deposition is the only mechanism for such basal ganglia necrosis, because similar findings were seen following methanol intoxication. We discuss other possible mechanisms that may contribute towards this unusual neurotoxicity. Both of our patients survived their toxic ingestions, but then developed acute Parkinsons syndrome within 10 days of the ingestion. However, the patient who ingested methanol developed respiratory muscle stiffness/weakness, which responded poorly to anti‐Parkinsonian drug therapy. Treatment with carbidopa/levodopa improved cogwheel rigidity and bradykinesia in both patients. We conclude that acute Parkinsonism is one of the lesser‐recognized devastating complications of both ethylene glycol and methanol poisoning.

Determination of serum and plasma concentrations of retinol using high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of retinol concentrations in serum and plasma is reported. Retinol and retinol acetate (the internal standard) are extracted into butanol-ethyl acetate, with no subsequent evaporation step. Separation is achieved on a reversed-phase C-18 column, with a mobile phase consisting of acetonitrile-1% ammonium acetate (89:11), and UV detection at 313 nm. Recoveries of both retinol and the internal standard were 100%, and both compounds were stable in the extraction solvent for at least 2.5 h. Three anticoagulants (oxalate, citrate, EDTA) and perchloric acid (used in some methods to denature protein) all caused losses of retinol. Each run required 9 min; same-day coefficient of variation (C.V.) for identical samples averaged 2.5%; between-day C.V. was 6.4%; sensitivity was better than 10 ng/ml, while clinical concentrations were 400-1200 ng/ml. This method permits simple, rapid, sensitive, precise, and accurate determination of retinol using 0.5 ml serum or heparinized plasma.

The Challenge of “Teaching” Large Groups of Learners: Strategies to Increase Active Participation and Learning

While teaching in a tutorial, seminar, or problem-based learning group format may be the most fun and most active/interactive for both learner and faculty mentor, there are situations in medical student education in which various constraints require the use of the “lecture” format. Similar constraints may occur in the field of continuing medical education, or graduate medical education, as well. When this occurs, the faculty mentor can increase the active participation of the learners in the audience by continuously stressing seven key pedagogical (androgogical) principles. These include: 1) begin the learning exercise with a clinical example or anecdote to show the relevance of the material to the student; 2) frequently ask the students whether they have ever seen examples of what you describe in their previous experience with patients, personal experience, experience with relatives, etc.; 3) ask students frequently whether they have heard similar material presented differently in other courses; 4) recruit students to help solve “mystery cases”; 5) show examples of similar material from real life (e.g., patient descriptions, or even excerpts from favorite TV shows); 6) ask students to help summarize key points at the end of the session; and 7) allow, or even encourage, whispering during the class. Using some or all of these techniques can help turn a “lecture format” into a much more fun, interactive, and valuable session that emphasizes “learning” rather than “teaching.”

Acute short-term memory loss from trimethyltin exposure

We report a case of organic tin exposure in a graduate chemistry student. The inhalational and transcutaneous exposure occurred following a laboratory explosion. The patient initially presented with first and second degree burns of the face and chest, and developed an acute loss of short-term memory 72 hours after exposure. The memory loss gradually improved over the course of several months.

Determination of tocopherol and tocopherol acetate concentrations in human feces using high-performance liquid chromatography

A method of sample clarification and high-performance liquid chromatography (HPLC) specifically developed to permit simple and rapid determination of vitamin E (alpha-tocopherol, E) and vitamin E acetate (EA) in feces is reported. Retinol acetate (RA) was used as an internal standard. The vitamins of interest were extracted from an aqueous stool suspension into an organic phase (ethyl acetate-butanol), which was injected directly onto the reversed-phase HPLC system. An isocratic mobile phase of methanol-water (97:3) was employed, with ultraviolet detection at 275 and 285 nm (to permit simultaneous monitoring and absorbance ratio determination). Recoveries of exogenous RA, E, and EA from stool suspensions (relative to water) were 99.0 +/- 7.0, 100.9 +/- 7.0, and 101.2 +/- 13.3%, respectively (n = 10). The organic matrix could be stored at -35 degrees C overnight with no change in E or EA results. Sensitivities for E and EA were 80 and 102 micrograms/g of stool, respectively. Each analysis required nine min. The within-day coefficients of variation were 2.9, 3.6, and 3.0% (n = 7) for RA, E, and EA, respectively. Neither E nor EA were detected in baseline fecal samples from fourteen subjects, but both were present in high but varied concentrations after four weeks supplementation with oral d,l-EA. E but not EA was present in blood samples drawn during periods of oral supplementation with EA. There was poor correlation between fecal levels of E and EA, and the increase in serum levels of E. This method permits rapid, selective, and precise determination of E and EA in human fecal samples.