Do intracellular, extracellular or urinary magnesium concentrations predict renal retention of magnesium in critically ill patients?

BACKGROUND AND OBJECTIVE: Magnesium disorders are common in hospitalized patients. In patients with low or normal magnesium, the intravenous magnesium loading test has been demonstrated to be a sensitive test to assess magnesium deficiency in critically ill patients. However, it is more time consuming and more difficult than the measurement of intracellular or extracellular magnesium concentrations. This study evaluated whether erythrocyte, plasma and urinary magnesium concentrations predict renal magnesium retention measured by th magnesium loading test. METHODS: One-hundred-and-three intensive care patients (36 females, 67 males) in a tertiary care centre and 41 healthy subjects (13 females, 28 males) took part in this prospective study. Intracellular, total plasma, ionize extracellular and urinary magnesium concentrations were measured and also magnesium retention by intravenous magnesium loading test. RESULTS: Total plasma magnesium concentration was poorly correlated with magnesium retention (r = 0.36 r2 = 0.13) and was the only parameter that significantly predicted magnesium retention in intensive care patients (P < 0.01). However, only 10% of the magnesium retention data were linked to the total plasma magnesium concentration. CONCLUSIONS: Total plasma magnesium concentration predicts magnesium retention in critically ill intensive care patients but not intracellular and urinary magnesium concentrations. Only a small proportion of the magnesium retention was due to the total plasma magnesium concentration.

CPAP-therapy effectively lowers serum homocysteine in obstructive sleep apnea syndrome.

Assessment of serum total homocysteine (tHcy) in patients with obstructive sleep apnea (OSA) syndrome is highly relevant since both are strongly associated with stroke and cognitive dysfunction. Seven of 16 untreated OSA patients showed tHcy levels exceeding 11.7 micromol/l. The circadian pattern of serum tHcy in untreated and treated patients (p < 0.001) implied a diagnostic impact of blood sampling time. Treatment with continuous positive airway pressure (CPAP) effectively lowered tHcy levels in patients by about 30% (p < 0.005) and thus probably the (hyper)homocysteinemia-related cognitive dysfunction and the risk for cardio-/cerebrovascular diseases.

Clozapine-associated elevation of plasma cholinesterase.

OBJECTIVE: The goal of this study was to identify adverse effects of the atypical neuroleptic clozapine on liver function and lipid metabolism. METHODS: Data which included serum levels of clozapine and its hepatic metabolite N-desmethyl clozapine were collected from medical records of patients treated with clozapine and controls. RESULTS: We identified a clozapine-associated marked elevation of plasma cholinesterase (ChE) with unchanged levels of AST, ALT or g-GT. ChE was correlated to the serum level of clozapine and even closer to N-desmethyl clozapine. For the total patient group we observed significant correlations of ChE with the body-mass index and body weight. However, clozapine-treated patients and controls did not differ with regard to body-mass index, triglycerides, and cholesterol. CONCLUSION: We report for the first time a clozapine-associated and dose-dependent elevation of plasma ChE, which may be related to clozapine-associated effects on hepatic lipid metabolism or ChE enzyme induction.

Determination of the acyl glucuronide metabolite of mycophenolic acid in human plasma by HPLC and Emit.

BACKGROUND: The acyl glucuronide (AcMPAG) of mycophenolic acid (MPA) has been found to possess pharmacologic and potentially proinflammatory activity in vitro. To establish its pharmacologic and toxicologic relevance in vivo, a reversed-phase HPLC method was modified to simultaneously determine MPA, the phenolic MPA-glucuronide (7-O-MPAG), and AcMPAG. In addition, cross-reactivity of AcMPAG in the Emit assay for MPA was investigated. METHODS: The procedure used simple sample preparation, separation with a Zorbax Eclipse-XDB-C8 column, and gradient elution. AcMPAG was quantified as 7-O-MPAG-equivalents. RESULTS: The assay was linear up to 50 mg/L for MPA, 250 mg/L for 7-O-MPAG, and 10 mg/L for AcMPAG (r >0.999). Detection limits were 0.01, 0.03, and 0.04 mg/L for MPA, 7-O-MPAG, and AcMPAG, respectively. The recoveries were 99-103% for MPA, 95-103% for 7-O-MPAG, and 104-107% for AcMPAG. The within-day imprecision was <5.0% for MPA (0.2-25 mg/L), <4.4% for 7-O-MPAG (10-250 mg/L), and < or =14% for AcMPAG (0.1-5 mg/L). The between-day imprecision was <6.2%, <4.5%, and < or =14% for MPA, 7-O-MPAG, and AcMPAG, respectively. When isolated from microsomes, purified AcMPAG (1-10 mg/L) revealed a concentration-dependent cross-reactivity in an Emit assay for the determination of MPA ranging from 135% to 185%. This is in accordance with the bias between HPLC and Emit calculated in 270 samples from kidney transplant recipients receiving mycophenolate mofetil therapy, which was greater (median, 151.2%) than the respective AcMPAG concentrations determined by HPLC. AcMPAG was found to undergo hydrolysis when samples were stored up to 24 h at room temperature or up to 30 days at 4 degrees C or -20 degrees C. Acidified samples (pH 2.5) were stable up to 30 days at -20 degrees C. CONCLUSIONS: The HPLC and Emit methods for AcMPAG described here may allow investigation of its relevance for the immunosuppression and side effects associated with mycophenolate mofetil therapy.

Induction of cytokine release by the acyl glucuronide of mycophenolic acid: a link to side effects?

OBJECTIVES: We have identified an acyl glucuronide (M-2) of the immunosuppressant mycophenolic acid (MPA). Acyl glucuronides have toxic potential and may contribute to drug toxicity. Whether acyl glucuronides are able to induce release of proinflammatory cytokines is unknown. Gastrointestinal disturbances have been observed during MPA therapy and may involve an inflammatory reaction. This study investigated whether M-2 can induce IL-6 and TNF-alpha release as well as gene expression of these cytokines in leukocytes. DESIGN AND METHODS: M-2 was produced by incubation of MPA with human liver microsomes. Human mononuclear leukocytes were incubated in the presence of M-2. Concentrations of IL-6 and TNF-alpha were measured by ELISA. Expression of mRNA was determined by quantitative RT-PCR. RESULTS: Incubation of 3 x 10(6) cells with M-2 resulted in a time and dose dependent release of cytokines, whereas MPA or its phenolic glucuronide MPAG were without effect. Cytokine liberation depended on mRNA induction. Response to M-2 showed much inter individual variability (30-fold for IL-6, 3-fold for TNF-alpha). CONCLUSIONS: If M-2 promotes release of cytokines in vivo, these may mediate some of the toxic actions of MPA.

Area under the plasma concentration-time curve for total, but not for free, mycophenolic acid increases in the stable phase after renal transplantation: a longitudinal study in pediatric patients. German Study Group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Mycophenolate mofetil, an ester prodrug of the immunosuppressant mycophenolic acid (MPA), is widely used for maintenance immunosuppressive therapy in pediatric renal transplant recipients. However, little is known about the pharmacokinetics of MPA in this patient population in the stable transplant phase, and dosage guidelines are preliminary. The authors therefore compared the pharmacokinetics of MPA, free MPA, and the renal metabolite MPA glucuronide (MPAG) in the initial (sampling at 1 and 3 weeks) and stable phases (sampling at 3 and 6 months) posttransplant in 17 children (age, 12.0 +/- 0.77 years; range, 5.9 to 15.8 years), receiving the currently recommended dose of 600 mg MMF/m2 body surface area (BSA) twice a day. Plasma concentrations of MPA and MPAG were measured by reverse phase HPLC. Because MPA is extensively bound to serum albumin and only the free drug is presumed to be pharmacologically active, the authors also analyzed the MPA free fraction by HPLC after separation by ultrafiltration. The intraindividual variability of the area under the concentration-time curves (AUC0-12) of MPA throughout the 12-hour dosing interval was high in the immediate posttransplant period, but declined in the stable phase, whereas the interindividual variability remained unchanged. The median MPA-AUC0-12 values increased 2-fold from 32.4 (range, 13.9 to 57.0) mg x h/L at 3 weeks to 65.1 (range, 32.6 to 114) mg x h/L at 3 months after transplantation, whereas the median AUC0-12 values of free MPA did not significantly change over time. This discrepancy can be attributed to a 35% decline of the MPA free fraction from 1.4% in the initial phase posttransplant to 0.9% (p < 0.01) in the stable phase. In conclusion, pediatric renal transplant recipients given a fixed MMF dose exhibit a 2-fold increase of the AUC0-12 of total MPA in the stable phase posttransplant and a 35% decrease of the MPA free fraction, whereas the AUC0-12 of free MPA remains unchanged over time. Because the latter pharmacokinetic variable is theoretically best predictive of the clinical immunosuppressive efficacy of MMF, these findings may have consequences for the dosing recommendations of MMF in renal transplant recipients.

Identification of glucoside and carboxyl-linked glucuronide conjugates of mycophenolic acid in plasma of transplant recipients treated with mycophenolate mofetil.

1. Mycophenolic acid (MPA), is primarily metabolized in the liver to 7-O-MPA-beta-glucuronide (MPAG). Using RP-h.p.l.c. we observed three further MPA metabolites, M-1, M-2, M-3, in plasma of transplant recipients on MMF therapy. To obtain information on the structure and source of these metabolites: (A) h.p.l.c. fractions containing either metabolite or MPA were collected and analysed by tandem mass spectrometry; (B) the metabolism of MPA was studied in human liver microsomes in the presence of UDP-glucuronic acid, UDP-glucose or NADPH; (C) hydrolysis of metabolites was investigated using beta-glucosidase, beta-glucuronidase or NaOH; (D) cross-reactivity of each metabolite was tested in an immunoassay for MPA (EMIT). 2. Mass spectrometry of M-1, M-2, MPA and MPAG in the negative ion mode revealed molecular ions of m/z 481, m/z 495, m/z 319 and m/z 495 respectively. 3. Incubation of microsomes with MPA and UDP-glucose produced M-1, with MPA and UDP-glucuronic acid MPAG and M-2 were formed, while with MPA and NADPH, M-3 was observed. 4. Beta-Glucosidase hydrolysed M-1 completely. Beta-Glucuronidase treatment led to a complete disappearance of MPAG whereas the amount of M-2 was reduced by approximately 30%. Only M-2 was labile to alkaline treatment. 5. M-2 and MPA but not M-1 and MPAG cross-reacted in the EMIT assay. 6. These results suggest that: (i) M-1 is the 7-OH glucose conjugate of MPA; (ii) M-2 is the acyl glucuronide conjugate of MPA; (iii) M-3 is derived from the hepatic CYP450 system.

Bright basal ganglia in T1-weighted magnetic resonance images are frequent in patients with portal vein thrombosis without liver cirrhosis and not suggestive of hepatic encephalopathy.

BACKGROUND/AIMS: Deposition of paramagnetic substances in basal ganglia, resulting in increased signals in T1-weighted magnetic resonance images (bright basal ganglia), is frequently seen in liver cirrhosis. The present study describes the prevalence of bright basal ganglia and its clinical significance in patients with long-standing portal vein thrombosis in the absence of liver cirrhosis. METHODS: Six patients with angiographically proven complete portal vein thrombosis and cavernomatous transformation without signs of acute or chronic liver disease were studied by magnetic resonance imaging of the brain, neuropsychiatric evaluation, psychometric tests, electroencephalography, and determination of arterial ammonia levels and of serum manganese concentrations from peripheral venous blood. RESULTS: Five out of six patients demonstrated increased signal intensity in the basal ganglia. Overt portal-systemic encephalopathy was not noted prior to or at the time of evaluation. Normal EEG results were recorded in all patients. Only one of the six patients had pathological results in at least two out of four psychometric tests. This latter patient had had a large right-sided brain infarction. Arterial ammonia concentrations were normal in four of the six patients; one patient with increased ammonia levels had concomitant renal insufficiency with azotemia. The other four patients had no relevant concomitant diseases. Serum manganese levels were non-significantly increased compared with a control group (p=0.06), but they were significantly correlated to basal ganglia signal intensity (R=0.88; p=0.02). CONCLUSIONS: Our results demonstrate that bright basal ganglia primarily represent shunt-induced alterations. They are not directly associated with disturbed liver function nor with portal-systemic encephalopathy.

Cyclosporin whole blood immunoassays (AxSYM, CEDIA, and Emit): a critical overview of performance characteristics and comparison with HPLC.

Assays with different specificity are used for cyclosporin monitoring in clinical transplantation. A recent survey of 35 centers showed that 86% used immunoassays for cyclosporin A (CsA). In consensus documents the following performance criteria were recommended: (a) imprecision < or = 10% at 50 microg/L and < or = 5% at 300 microg/L; and (b) comparison with the reference method (HPLC) should yield a slope of 0.9-1.1, an intercept of -15 to 15 microg/L, and S(y/x) < or = 15 microg/L. The newly developed CsA assays for the AxSYM (Abbott) and the CEDIA (Boehringer Mannheim) as well as the Emit assay (Behring Diagnostica) were evaluated. Results from samples of heart, kidney, and liver recipients (100 specimens each) were compared with a validated HPLC-ultraviolet detection method. Between-series imprecision (CV) with commercial controls was 5.8% and 1.7% for AxSYM (70 and 300 microg/L), 11% and 5.5% for CEDIA (90 and 200 microg/L), and 8.1% and 4.5% for Emit (63 and 172 microg/L). In the presence of 300 microg/L parent CsA, cross-reactivities were (for AxSYM, CEDIA, and Emit, respectively) 7%, 4%, and none for AM1 (1 mg/L) and 12.6%, 25%, and 6% for AM9 (0.5 mg/L). Comparison with HPLC showed in heart and kidney recipients an average overestimation with the Emit and the CEDIA of approximately 22%, with overestimation in the AxSYM of 32%. In liver recipients, the most challenging patient group, the CEDIA and the AxSYM showed a mean overestimation of 43% and 47%, respectively, and the Emit differed by 31% compared with HPLC. None of the immunoassays fully satisfied the performance criteria recommended in the consensus documents. In terms of specificity, Emit ranks before CEDIA, which ranks before AxSYM. Regarding imprecision, the ranking is AxSYM < Emit < CEDIA. These limitations must be considered when using these assays for therapeutic drug monitoring of CsA in clinical transplantation.

Pharmacokinetics of mycophenolic acid (MPA) and determinants of MPA free fraction in pediatric and adult renal transplant recipients. German Study group on Mycophenolate Mofetil Therapy in Pediatric Renal Transplant Recipients.

Dosage guidelines for mycophenolate mofetil (MMF), an ester prodrug of the immunosuppressant mycophenolic acid (MPA), are still preliminary in children. This study compares the pharmacokinetics of MPA and its major metabolite MPA glucuronide (MPAG) in pediatric renal transplant recipients receiving 600 mg MMF/m2 body surface area twice a day to those of adults on the currently recommended oral dose of 1 g of MMF twice a day. Concentration-time profiles of 18 children (age, 10.7+/-0.72 yr; range, 5.9 to 15.3 yr) and 10 adults were investigated 1 and 3 wk after transplantation. Plasma concentrations of MPA and MPAG were measured by reverse-phase HPLC. Because MPA is extensively bound to serum albumin and only the free fraction is presumed to be pharmacologically active, the MPA free fraction was also analyzed by HPLC after separation through ultrafiltration. The areas under the concentration-time curves (AUC0-12) of total and free MPA throughout the 12-h dosing interval in children were, in general, comparable to the corresponding data in adult patients. The mean AUC0-12 of MPA and free MPA did not change significantly over the first 3 wk after transplantation, but there was substantial intra- and interindividual variation. MPAG-AUC0-12 values in children with primary renal transplant dysfunction were threefold higher than in those with functioning transplants. Renal impairment had no consistent effect on total MPA-AUC0-12 values, but the MPA free fraction in children (median, 1.65%; range, 0.40 to 13.8%) was significantly (r2=0.46) modulated by renal transplant function and serum albumin levels. In conclusion, concentration-time profiles of pediatric renal transplant recipients administered 600 mg MMF/m2 body surface area twice a day are comparable to those in adults on 1 g MMF twice a day in the first 3 wk after transplantation. Renal impairment and decreased serum albumin levels led to an increase in the free fraction of MPA and the free MPA-AUC0-12 values. Because the pharmacologic activity of MPA is a function of unbound drug concentration, these findings might be relevant for the pharmacodynamic effects of MPA.

Simultaneous determination of mycophenolic acid and its glucuronide in human plasma using a simple high-performance liquid chromatography procedure.

We describe a reversed-phase HPLC method for determination of total mycophenolic acid (MPA), its free concentration (MPAf), and the glucuronide metabolite (MPAG), based on simple sample preparation and gradient elution chromatography. The compounds were quantified in parallel by absorbance at 254 nm and 215 nm in the internal standard mode. Linearity was verified up to 50 mg/L for MPA and up to 500 mg/L for MPAG (r >0.999). Detection limits at 215 and 254 nm were, respectively, 0.01 and 0.03 mg/L for MPA, and 0.03 and 0.1 mg/L for MPAG. The recovery of MPA was 95-106%; recovery of MPAG was 96-106%. The imprecision (CV) for MPA (0.2-25 mg/L) was <8.4% (254 nm) and <4.4% (215 nm) within day (n = 12) and <9.2% (254 nm) and <6.2% (215 nm) between days (n = 12). The imprecision for MPAG (10-250 mg/L) was <4.9% (254 nm) and <3.4% (215 nm) within day, and <6.1% (254 nm) and <5.9% (215 nm) between days. For quantification of MPAf, 100 microL of ultrafiltrate was applied directly to the column. The detection limit was 0.005 mg/L at 215 nm and 0.015 mg/L at 254 nm. In the range between 18-210 microg/L, the within-day CVs were <11.8% (n = 12) and the between-day CVs were <15.8% (n = 12).

Determination of monoethylglycinexylidide by fluorescence polarization immunoassay in highly icteric serum samples: modified precipitation procedure and HPLC compared.

Hyperbilirubinemia, which frequently occurs in severe liver disease, interferes with the fluorescence polarization immunoassay (FPIA) monoethylglycinexylidide (MEGX) assay manufactured by Abbott Diagnostics. Because the MEGX test is particularly helpful in this clinical situation, strategies have been developed to overcome this problem. Precipitation of serum with the Abbott Digoxin II precipitation reagent eliminates bilirubin. Therefore, we compared FPIA results after precipitation of 81 icteric samples from 27 MEGX tests to results obtained using a validated HPLC method. The precipitation did not substantially alter the performance characteristics of FPIA: detection limit, 8 microg/L; between-days imprecision, 5.3-6.2%; recovery, 102-104% (50-200 microg/L). This pretreatment of serum did not eliminate all interference, and only a poor correlation was observed between serum MEGX concentrations measured with HPLC or modified FPIA (r2 = 0.46; S(y/x) = 20.0 microg/L). In contrast, MEGX formation values calculated by subtraction of the prelidocaine MEGX concentration were in close agreement (r2 = 0.98; S(y/x) = 2.3 microg/L). Because only MEGX formation is clinically relevant, this modified FPIA procedure offers a simple and rapid alternative to HPLC.