Alex A. Pappas

Quantification of Vitamin D Receptor mRNA by Competitive Polymerase Chain Reaction in PBMC: Lack of Correspondence with Common Allelic Variants

It has been recently claimed that polymorphism for the vitamin D receptor (VDR) influences several aspects of calcium and bone metabolism. To evaluate the physiologic plausibility of these claims, we compared the abundance of the VDR mRNA in peripheral blood mononuclear cells (PBMCs) between different VDR genotypes using a quantitative reverse transcribed polymerase chain reaction–based method. The method is based on the coamplification of VDR cDNA and an internal standard consisting of known concentrations of a human VDR CDNA mutated at a BglII restriction site; the interassay coefficient of variation is 11%. To validate the method, we made use of earlier receptor binding studies indicating that normal human monocytes and activated, but not resting, lymphocytes expressed the VDR. The concentration of the VDR mRNA was 10−8 to 10−7 g/g of total RNA in cell‐sorted monocytes and in in vitro activated lymphocytes, but only 10−12 g/g of total mRNA in resting lymphocytes, establishing that the VDR mRNA determined by our method in PBMCs is due to constitutive expression in monocytes. Following an initial genotype screening of 85 normal volunteers by polymerase chain reaction or restriction fragment length polymorphism analysis, 14 individuals with the Bb genotype, 12 with the bb genotype, and 12 with the BB genotype were selected. The concentration of the VDR mRNA, corrected for the number of monocytes, was similar among the three genotype groups, as were the other variables examined: serum calcitriol, serum osteocalcin, and vertebral and hip bone density. We conclude that VDR polymorphism does not affect the abundance of the VDR mRNA.

Ethylene Glycol Toxicity: The Role of Serum Glycolic Acid In Hemodialysis

Objective: To correlate serum glycolic acid levels with clinical severity and outcome in ethylene glycol poisoning and to determine if glycolic acid levels are predictive of renal failure and the need for hemodialysis. Methods: We measured serum ethylene glycol and glycolic acid levels by gas chromatography/mass spectrometry for 41 admissions (39 patients) for ethylene glycol ingestion and performed retrospective chart reviews. Results: Eight patients died, all of whom developed acute renal failure. Of the survivors, 15 also developed acute renal failure, whereas 18 did not. Of those with normal renal function, 8 had glycolic acid levels below detection limits (<0.13 mmol/L) despite ethylene glycol levels as high as 710 mg/dL; 7 of these patients coingested ethanol. Pertinent initial laboratory data for each group are as follows (mean; range): Deceased: pH 6.99 (6.82–7.22); bicarbonate, 4.8 mmol/L (2–9); anion gap, 28.6 mmol/L (24–40); glycolic acid, 23.5 mmol/L (13.8–38.0); ethylene glycol, 136.5 mg/dL (6–272). Survived/acute renal failure: pH 7.07 (6.75–7.32); bicarbonate, 5.6 mmol/L (1–12); anion gap, 28.7 mmol/L (18–41); glycolic acid, 20.2 mmol/L (10.0–30.0); ethylene glycol, 238.8 mg/dL (12–810). No acute renal failure with glycolic acid >1.0 mmol/L: pH 7.29 (7.12–7.46); bicarbonate, 14.7 mmol/L (4–23); anion gap, 16.5 mmol/L (10–26); glycolic acid, 6.8 mmol/L (2.6–17.0); ethylene glycol, 269.1 mg/dL (6–675). No acute renal failure with glycolic acid <1.0 mmol/L: pH 7.41 (7.38–7.47); bicarbonate, 23.4 mmol/L (17–25); anion gap, 11.8 mmol/L (8–18); glycolic acid, 0.1 mmol/L (0–0.66); ethylene glycol, 211 mg/dL (8–710). The mean time postingestion to admission generally correlated with severity as follows: deceased, ≥10.4 h; survived/acute renal failure, ≥9.9 h; no acute renal failure with glycolic acid >1.0 mmol/L, ≥6.2 h; no acute renal failure with glycolic acid <1.0 mmol/L, ≥3.7 h. Hematuria was more prevalent than oxaluria (86% and 41%, respectively), but neither was individually predictive of acute renal failure. Good correlations were found between glycolic acid levels and anion gap (r2 = 0.7724), pH (r2 = 0.7921), and bicarbonate (r2 = 0.6579); poor correlations (r2 <0.0023) occurred between ethylene glycol levels and glycolic acid, pH, anion gap, and bicarbonate. Measured ethylene glycol values were highly correlated with ethylene glycol values calculated from the osmolal gap (r2 = 0.9339), but the latter overestimates the true value by about 7%, on average. An initial glycolic acid level ≥10 mmol/L predicts acute renal failure with a sensitivity of 100%, a specificity of 94.4%, and an efficiency of 97.6%. Ethylene glycol levels are not predictive of acute renal failure or central nervous system manifestations of toxicity. If only ethylene glycol values are available (measured or calculated), an initial anion gap >20 mmol/L is 95.6% sensitive and 94.4% specific for acute renal failure when ethylene glycol is present. Likewise, initial pH <7.30 is 100% sensitive and 88.5% specific for acute renal failure. Conclusion: We propose glycolic acid <8 mmol/L as a criterion for the initiation of hemodialysis in ethylene glycol ingestion. Patients with glycolic acid <8 mmol/L probably do not need dialysis, regardless of the ethylene glycol concentration, when metabolism of ethylene glycol is therapeutically inhibited. In the absence of glycolic acid values, an anion gap >20 mmol/L or pH <7.30 predicts acute renal failure.

Prevention of chronic radiation enteropathy by dietary glutamine

AbstractBackground: Nearly 50% of all cancer patients receive therapeutic radiation during the course of their disease. The risk of late complications is the main dose-limiting factor in the delivery of radiation therapy. The small intestine, the major site of chronic radiation enteropathy, is also the principal organ of glutamine consumption. We therefore hypothesized that the provision of supplemental glutamine may have a protective effect on the development of chronic radiation enteropathy. Methods: This study evaluated the effects of supplemental oral glutamine on the development of chronic radiation (XRT) enteropathy. After scrotalization of a loop of small intestine, rats were randomized to receive 1 g/kg/day glutamine (GLN) or glycine (GLY) by gavage. After 2 days of prefeeding, rats were randomized to 1 of 4 groups: GLN + XRT (n=10), GLY + XRT (n=10), GLN only (n=10), GLY only (n=10). Twenty Gy was delivered to the scrotalized bowel in the GLN + XRT and GLY + XRT groups via a collimated beam. Gavage was continued for 10 days. Animals were then pair-fed chow. Rats were killed at 2 months postirradiation. Chronic radiation injury was assessed microscopically. Results: Injury scores in GLN + XRT were similar to those of unirradiated bowel and significantly different from GLY + XRT (1.89 ± 0.48 in XRT + GLN vs. 6.42 ± 1.55 in the XRT + GLY,p<0.01). Elevated Injury Scores in the XRT + GLY group correlated with gross thickening and fibrosis, a 10-fold decrease in gut GLN extraction (1.40 ± 4.3% in GLY + XRT vs. 16.0 ± 5.1% in GLN + XRT,p<0.05), and a 30% decrease in glutathione content (2.46 ± 0.19 and GLY + XRT vs. 3.17 ± 0.17 GLN + XRT,p<0.05). Conclusions: Provision of GLN during abdominal/pelvic XRT may prevent XRT injury and decrease the long-term complications of radiation enteropathy.

Vancomycin serum protein binding determination by ultrafiltration.

Sixty-two serum concentrations were obtained from 12 infected patients enrolled in a vancomycin pharmacokinetic study. Both unbound and total serum vancomycin concentrations were measured using ultrafiltration and a commercial fluorescent polarization immunoassay. Ultrafiltrates were obtained by centrifugation at 1000 × g for ten minutes at room temperature and their assay indicated a range in protein binding from 7.9 to 71 percent. The mean protein binding (mean ±SD) was 41.95 ± 14.15 percent. No measurable adsorption of vancomycin onto the ultrafiltration membrane was noted. Orthogonal regression of unbound versus total vancomycin concentrations was described by the equation y=0.597x-0.362 with a correlation coefficient of 0.948.

31P NMR of phospholipid metabolites in prostate cancer and benign prostatic hyperplasia

1H MRSI in vivo is increasingly being used to diagnose prostate cancer noninvasively by measurement of the resonance from choline‐containing phospholipid metabolites. Although 31P NMR in vivo or in vitro is potentially an excellent method for probing the phospholipid metabolites prominent in prostate cancer, it has been little used recently. Here, we report an in vitro 31P NMR comparison of prostate cancer and benign prostatic hyperplasia, focusing on the levels of the major phospholipid metabolites. Unlike phosphocholine and glycerophosphocholine, phosphoethanolamine and glycerophosphoethanolamine (and their ratio) were significantly different between cancer and benign prostatic hyperplasia. The high level of phosphoethanolamine+glycerophosphoethanolamine relative to phosphocholine+glycerophosphocholine suggests that the former may be significant contributors to the “total choline” resonance observed by 1H MRSI in vivo. Magn Reson Med, 2010.

Phenytoin and plasmapheresis: importance of sampling times and impact of obesity.

The effect of plasmapheresis (PP) on total and free phenytoin clearance is reported. An obese patient with the diagnosis of thrombotic thrombocytopenic purpura (TTP) was treated with PP. Twelve episodes of PP, having exchange volumes of 1.5-2.25 times the plasma volume with a mean +/- SD 7.7 +/- 0.8 L of plasma removed, were studied. A significant (p < 0.05) difference was observed with a mean change in plasma phenytoin concentrations from pre- to end-PP of 7.32 +/- 2.5 mg/L compared to 1.98 +/- 0.7 mg/L observed pre-PP to 1 h post-PP. These values corresponded to 48.4 +/- 11.6 and 15.0 +/- 6.7% decreases in phenytoin concentrations at the two aforementioned time periods. To prevent misinterpretation of plasma phenytoin concentrations, samples should not be obtained for at least 2 h after PP.

Measurement of serum isopropanol and the acetone metabolite by proton nuclear magnetic resonance: application to pharmacokinetic evaluation in a simulated overdose model.

The purpose of this investigation was to 1) compare the performance of proton nuclear magnetic resonance spectroscopy to gas chromatography head-space analysis in the measurement of serum isopropanol and its metabolite, acetone, obtained during a simulated overdose, and 2) compare pharmacokinetic parameters obtained using the two analytical techniques. Three healthy volunteers ingested 0.6 mL/kg of 70% isopropanol and blood samples were obtained at baseline, 0.16, 0.33, 0.66, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, and 24.0 hours post-ingestion. Resulting sera were analyzed by gas chromatography head-space analysis and proton nuclear magnetic resonance spectroscopy for determination of isopropanol and acetone concentrations. A correlation between concentrations quantitated by gas chromatography head-space analysis versus proton nuclear magnetic resonance spectroscopy was determined using linear regression. Pharmacokinetic disposition parameters were determined from serum concentration-time data and compared using analysis of variance. For isopropanol, the linear regression equation which describes the relationship between gas chromatography head-space analysis and proton nuclear magnetic resonance spectroscopy was y = 1.041x - 2.180 (r2 = 0.995, p < 0.0001); for acetone, y = 1.022x - 0.946 (r2 = 0.984, p < 0.0001). Pharmacokinetic disposition parameters derived from the two analytical methods were comparable. Proton nuclear magnetic resonance spectroscopy can be used to rapidly quantitate serum isopropanol and acetone concentrations in the same sample when gas chromatography head-space analysis is unavailable. Also, proton nuclear magnetic resonance spectroscopy can be used to follow serial serum concentrations during an ingestion for the purpose of pharmacokinetic analysis.

Carbamazepine and erythroid arrest

A patient who developed anemia with an isolated erythroid toxicity following chronic carbamazepine administration is reported. The anemia quickly resolved with discontinuation of this drug. Other hematologic toxicities of carbamazepine are well described; however, an isolated erythroid toxicity is unusual. In addition, the onset of this drug-induced toxicity developed later than is expected for carbamazepine-associated hematological toxicities. This case demonstrates the suddenness with which hematological toxicities can occur with carbamazepine, and affirms the need for regular monitoring of patients. Any significant decrease in the patients hemoglobin or hematocrit level requires close monitoring for the sudden development of serious anemia.

Nuclear magnetic resonance in pathology: I. Principles and general aspects

Nuclear magnetic resonance (NMR) has become established as a powerful diagnostic imaging technique in medicine. The power of NMR as a tool for characterizing molecular structure and for quantitative analysis of complex mixtures in the clinical laboratory or even in vivo is beginning to be exploited. In the clinical laboratory, NMR can analyze the complex mixtures of bodily fluids, intact cells, tissues, and their extracts. Phosphorus-31 or 1H NMR spectroscopy in vivo can provide a noninvasive probe of high-energy compounds, amino acids, and compounds of phospholipid metabolism. The basic principles of NMR spectroscopy are presented here, with emphasis on the various NMR parameters and the information they provide. A companion article presents a survey of applications in pathology.