Richard E. Mullins

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.

Clinical Utility of Monitoring Tacrolimus Blood Concentrations in Liver Transplant Patients

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme‐linked immunosorbent assay (ELISA) for monitoring whole‐blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7‐day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

Steady-State Serum Concentrations of Progesterone Following Continuous Intravenous Infusion in Patients With Acute Moderate to Severe Traumatic Brain Injury

Progesterone (PG) has been shown to provide substantial neuroprotection after traumatic brain injury (TBI) in multiple animal models. As a first step in assessing applicability to humans, the authors examined the effects of acute TBI and extracranial trauma on the pharmacokinetics of PG given by intravenous infusion. Multiple blood samples were obtained from 11 female and 21 male trauma patients receiving PG and 1 female and 3 male patients receiving placebo infusions for 72 hours. Values for CSS, CL, t1/2, and Vd were obtained using AUC(0–72) and postinfusion blood samples. CSS values were 337 ± 135 ng/mL, which were significantly lower than the target concentration of 450 ± 100 ng/mL. The lower CSS is attributed to the CL, which was higher than anticipated. In addition, t1/2 was longer and Vd was higher than anticipated. These results demonstrate that stable PG concentrations can be rapidly achieved following TBI.

Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887.

To determine the possible biochemical effects of prolonged weightlessness on liver function, samples of liver from rats that had flown aboard Cosmos 1887 were analyzed for protein, glycogen, and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. Among the parameters measured, the major differences were elevations in the glycogen content and hydroxymethylglutaryl‐CoA (HMG‐CoA) reductase activities for the rats flown on Cosmos 1887 and decreases in the amount of microsomal cytochrome P‐450 and the activities of aniline hydroxylase and ethylmorphine N‐demethylase, cytochrome P‐450‐dependent enzymes. These results support the earlier finding of differences in these parameters and suggest that altered hepatic function could be important during spaceflight and/or the postflight recovery period.—Merrill, A. H., Jr.; Hoel, M.; Wang, E.; Mullins, R. E.; Hargrove, J. L.; Jones, D. P.; Popova, I. A. Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887. FASEB J. 4: 95‐100; 1990.

Functional differences in human neutrophils isolated pre‐ and post‐prandially

Activated polymorphonuclear leukocytes have been associated with neoplasia, atherogenesis and reperfusion injury. Since some of these conditions are also correlated with dietary fat, we examined the functional characteristics of leukocytes isolated from subjects before and after consumption of a lipid‐rich meal. There was up to 2‐fold greater superoxide generation in response to agonists in leukocytes obtained post‐prandially; the maximum increase was observed about 4 h after eating and followed the peak (2–4 h) in serum triglycerides. Neutrophils isolated post‐prandially also exhibited impaired chemotaxis and defective bacterial killing, but normal phagocytosis. These findings provide a new variable that should be considered in studies of leukocytes.

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.

Analysis of whole blood tacrolimus concentrations in liver transplant patients exhibiting impaired liver function.

In transplant patients with impaired liver function, HPLC methodologies have been suggested for monitoring whole blood tacrolimus concentrations because of the reported inaccuracy of immunoassay for whole blood tacrolimus concentrations. One hundred fifty whole blood samples from 50 subjects enrolled in a multicenter liver transplant trial were chosen for HPLC/MS/MS analysis without consideration of the clinical status of the patient at the time of sampling. These samples were chosen to represent the sampling intervals during the 12-week posttransplantation period. Retrospectively, the authors identified a subset of 39 samples from 27 subjects exhibiting impaired liver function as demonstrated by bilirubin concentrations > 3.0 mg/dL (mean +/-SD = 7.5 +/- 5.6 mg/dL). The authors compared the agreement of concentrations obtained from the PRO-Trac II ELISA and HPLC/MS/MS by least squares linear regression analysis and Bland/Altman analysis, in this subset against the agreement of concentrations for 76 samples with normal bilirubin. In the samples obtained from patients with impaired liver function the resulting regression equation was: ELISA = 1.19(HPLC) + 0.7; r = 0.9. The mean difference (HPLC/MS/MS - ELISA) was -2.5 ng/mL +/- 2.9 ng/mL (mean +/- SD). While 71% of samples agreed within 3 ng/mL, 3% (n = 1) exhibited a difference >10 ng/ml. The corresponding evaluation of the samples with normal bilirubin concentrations resulted in the regression equation ELISA = 0.96(HPLC) + 0.9; r = 0.9, and a mean difference of -0.6 ng/mL +/- 2.3 ng/mL. The authors conclude that while a small subset of patients with cholestasis may require closer evaluation with a more specific methodology, the majority of the patients may be satisfactorily monitored with the PRO-Trac II ELISA.

Increases in serum sphingomyelin by 17β-estradiol

The effects of estrogens on plasma sphingomyelin and the hepatic activity of the initial enzyme of sphingomyelin synthesis were examined using immature chicks. After three days of 17β-estradiol administration, serum sphingomyelin, total phospholipids, and cholesterol doubled, and triacylglycerol levels increased 7.5 fold. The sphingomyelin content and percentage of total phospholipids of liver were unaffected by estrogen treatment. The specific activity of serine palmitoyltransferase (EC 2.3.1.50) was unchanged, but the total activity appeared slightly higher due to increased liver weights. The higher spingomyelin may, therefore, be due less to increased levels of biosynthetic enzymes than to factors such as the substrate (i.e., fatty acid) supply or decreased clearance of plasma sphingomyelin. These results are similar to earlier findings with key enzymes of cholesterol and glycerolipid biosynthesis and suggest that the three lipid pathways may be coordinated during estrogen treatment and enhanced very-low density lipoprotein (VLDL) synthesis.

Gentamicin solution for mediastinal irrigation: Systemic absorption, bactericidal activity, and toxicity

Local irrigation with gentamicin sulfate represents a possible substitute for neomycin sulfate, used for many years but now no longer available for use as an irrigation fluid. In this investigation, mediastinal irrigation with gentamicin was used in 12 patients who had experienced problems after a heart operation. The regimen employed for mediastinal irrigation with gentamicin was equipotent with that using neomycin. We sought to determine the degree of absorption and risk of either inadequate or toxic blood levels that might follow gentamicin absorption. Irrigation periods were short, ranging from one to four days and determined by measurements of plasma gentamicin concentration using radioimmunoassay evaluation. Systemic gentamicin absorption occurred in all patients. Toxic levels of higher than 8.0 micrograms/mL occurred and were size related, ie, correlated with smaller body weight and surface area, and sex related, ie, female sex. Larger-sized patients often had inadequate levels. Despite the potential risk from toxic blood levels, major increases in serum creatinine levels were not seen. These findings suggest that monitoring of plasma gentamicin levels during mediastinal irrigation with gentamicin is mandatory to avoid both inadequate treatment and toxicity.

Comparison of whole-blood cyclosporine levels measured by radioimmunoassay and fluorescence polarization in patients post orthotopic liver transplant

Summary: This study compared the analysis of whole blood cyclosporine concentrations measured by fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA) polyclonal and monoclonal procedures. Fifteen orthotopic liver transplant patients with a mean age of 39 \pm 11.06 years were included in the study. One hundred thirteen levels were analyzed using FPIA, RIA polyclonal, and RIA monoclonal procedures. There was no difference statistically in comparing FPIA and RIA polyclonal results (p > 0.05). There was a statistical difference between FPIA and RIA monoclonal results (p = 0.0001). With use of least squares simple linear regression analysis, FPIA results showed good correlation with RIA polyclonal results (R2 = 0.87). Poor correlation was shown between FPIA and RIA monoclonal results (R2 = 0.51). In this study population, FPIA produced results 2.5% higher than the RIA polyclonal procedure.