Technical and biochemical factors affecting cerebrospinal fluid 5-MTHF, biopterin and neopterin concentrations.

BACKGROUND: The diagnosis of pediatric neurologic disorders with a deficiency in the biosynthesis of either the neurotransmitters serotonin and dopamine, or the co-factor tetrahydrobiopterin or a cerebral 5-methyltetrahydrofolate (5-MTHF) deficiency, strongly relies on a robust analysis of neurotransmitter metabolites, pterins and 5-MTHF in the cerebrospinal fluid (CSF). The aim of this study was to investigate which technical and biochemical factors affect the CSF concentration of 5-MTHF, neopterin and biopterin in a pediatric population. METHODS: We studied effects of the ventriculo-spinal gradient, total protein concentration, pretreatment with ascorbic acid (in case of 5-MTHF analysis), pretreatment of CSF with trichloro acetic acid (TCA)/dithiotreitol (DTE) and oxidation with either iodine or manganese oxide (in case of pterin analysis), storage time and age of the patients. We included CSF samples from children until the age of 18 years and analysed 5-MTHF, neopterin, biopterin, homovanillic acid (HVA), 5-hydroxy-indoleacetic acid (5-HIAA) and total protein. RESULTS: The major findings of our study are: (1) CSF 5-MTHF, neopterin and biopterin concentrations are not affected by the ventriculo-spinal gradient; (2) pretreatment of CSF with ascorbic acid has negligible effects on 5-MTHF concentrations; (3) pretreatment of CSF with TCA/DTE and oxidation with iodine results in the most accurate determination of neopterin and biopterin; (4) when adjusted for age and total protein, CSF 5-MTHF correlated with 5-HIAA, but not with HVA; (5) the reference value of 5-MTHF in CSF in childhood is age-dependent (r=-0.634; p0.001); (6) we did not observe an age-dependency for neopterin and biopterin in CSF. CONCLUSION: 5-MTHF, neopterin and biopterin can be analysed in any volume of CSF that is collected. For correct analysis of pterins, CSF will have to be pretreated to stabilize the concentrations and stored properly, whereas such pretreatment is not necessary for 5-MTHF.

Recovery and long-term renal excretion of propofol, its glucuronide, and two di-isopropylquinol glucuronides after propofol infusion during surgery.

BACKGROUND: The metabolism of the short-acting anaesthetic agent propofol has been described over the first 24 h. However, the long-term disposition of propofol and its metabolites is unclear. We describe the pharmacokinetics (renal excretion rates and renal clearance) of propofol and its metabolites over 60 h. METHODS: Ten patients undergoing lung surgery were included in the study. They received anaesthesia with continuous i.v. propofol at an average rate of 10 mg min(-1). During surgery and 60 h thereafter, we sampled blood and urine. Propofol and its metabolites were measured using gradient high performance liquid chromatography (HPLC). RESULTS: In nine patients, propofol and its glucuronides were found in the plasma over the first 15 h. In the urine, however, even after 60 h, propofol and its quinol glucuronides were still detectable. One patient had a markedly different pharmacokinetic profile, showing a limited renal excretion or absorption of 12% of the dose. CONCLUSIONS: After an infusion of propofol, patients excrete propofol and its metabolites in the urine over a period in excess of 60 h. We hypothesize that (re)absorption of propofol and its metabolites by the kidney is a major process in elimination and that the reabsorbed compounds are gradually conjugated in the kidney and excreted in the urine. One patient showed a different pharmacokinetic profile for which we currently have no explanation.

Similar motor block effects with different disposition kinetics between lidocaine and (+ or -) articaine in patients undergoing axillary brachial plexus block during day case surgery.

AIM: The aim of this investigation was to compare the clinical effects and pharmacokinetics of lidocaine and articaine in two groups of 15 patients undergoing axillary brachial plexus anesthesia. METHOD: The study had a randomized design. Thirty patients were allocated to one of the two groups. Each patient received either lidocaine (600 mg = 2.561 mMol + 5 microg/ml adrenaline) or articaine (600 mg = 2.113 mMol + 5 microg/ml adrenaline), injected via the axilla of the brachial plexus over a period of 30 seconds. Onset of surgical analgesia was defined as the period from the end of the injection of the local anesthetic to the loss of pinprick sensation in the distribution of all three nerves. RESULTS: The mean onset time of sensory block of the median nerve of both lidocaine and articaine were approximately 10 min. Lidocaine is biexponentially eliminated with a t1/2alpha of 9.95 +/- 14.3 min and a t1/2beta of 2.86 +/- 1.55 h. Lidocaine is metabolized into MEGX (mono-ethyl-glycyl-xilidide) (t(max) 2.31 +/- 0.84 h; C(max) 0.32 +/- 0.13 mg/l; t1/2beta 2.36 +/- 2.35 h). Lidocaine total body clearance was 67.9 +/- 28.9 l/h. Articaine is rapidly and monoexponentially eliminated with a t1/2beta of 0.95 +/- 0.39 h. The total body clearance of articaine is higher than that of lidocaine, 1,133 +/- 582 l/h vs 67.9 +/- 28.9 l/h, respectively (p < 0.0001). The volume of distribution (V(d)), of articaine is a factor 16 higher times than that of lidocaine (p < 0.0001). CONCLUSION: For the axillary administration, lidocaine and articaine show similar pharmacodynamics with a different pharmacokinetic behavior and can therefore be used to the clinical preference for this regional anesthetic technique.

High-performance liquid-chromatographic-atmospheric-pressure chemical-ionization ion-trap mass-spectrometric identification of isomeric C6-hydroxy and C20-hydroxy metabolites of methylprednisolone in the urine of patients receiving high-dose pulse therapy.

Fourteen metabolites of methylprednisolone have been analysed by gradient-elution high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). The compounds were separated on a Cp Spherisorb 5 microm ODS column connected to a guard column packed with pellicular reversed phase. The mobile phase was an acetonitrile- 1.0% aqueous acetic acid gradient at a flow rate of 1.5 mL min(-1) The analysis gave a complete picture of parent drug, prodrugs and metabolites, and the alpha/beta stereochemistry was resolved. The short (1-2 h) elimination half-life of methylprednisolone is explained by extensive metabolism. The overall picture of the metabolic pathways of methylprednisolone is apparently simple-reduction of the C20 carbonyl group and further oxidation of the C20,C21 side chain (into C21COOH and C20COOH), in competition with or in addition to oxidation at the C6 position.

High-performance liquid chromatography analysis, preliminary pharmacokinetics, metabolism and renal excretion of methylprednisolone with its C6 and C20 hydroxy metabolites in multiple sclerosis patients receiving high-dose pulse therapy.

A gradient eluent HPLC analysis in human plasma and urine was developed and validated for methylprednisolone (MP), its prodrug methylprednisolone-21-hemisuccinate (MPS) with the metabolites 6beta-hydroxy-6alpha-methylprednisolone (MPA), 20-hydroxymethylprednisolone (MPC), 6beta-hydroxy-20alpha-hydroxymethylprednisolone (MPB), 6beta-hydroxy-20beta-hydroxymethylprednisolone (MPE), 20-carboxymethylprednisolone (MPD), methylprednisolone-glucuronide (MPF) and 21-carboxymethylprednisolone (MPX). The column was Cp Spherisorb C8 5 microm, 250 mm x 4.6 mm I.D. (Chrompack, Bergen op Zoom, The Netherlands) with a guard column 75 mm x 2.1 mm, packed with pellicular reversed-phase. The eluent was a mixture of acetonitrile and 0.067 M KH2PO4 buffer, pH 4.5. At t=O, the eluent consisted of 2% acetonitrile and 98% buffer (v/v). Over the following 35 min the eluent changed linearly until it attained a composition of 50% acetonitrile and 50% buffer (v/v). At 37 min (t=37) the eluent was changed over 5 min to the initial composition, followed by equilibration over 3 min. The flow-rate was 1.5 ml/min and UV detection was achieved at 248 nm. Preliminary pharmacokinetic data were obtained from one patient who showed illustrative plasma concentration-time curves and renal excretion-time profiles after a short-lasting infusion (0.5 h) of 1 g of methylprednisolone hemisuccinate. The half-life of prodrug methylprednisolone-21-hemisuccinate (MPS) was 0.3 h, that of metabolite MPX (21-carboxy MP) was 0.4 h and that of the parent drug methylprednisolone (MP) was 1.4 h. The half-lives of the metabolites are almost similar (4 h). The main compounds in the urine are methylprednisolone hemisuccinate (prodrug, 15.0%), methylprednisolone (parent drug, 14.6%), metabolite MPD (20-carboxy, 11.7%), and metabolite MPB (13.2%). The renal clearance values of metabolites MPB, MPC and MPD are approximately 500 ml/min, that of MP is 100 ml/min.

Isolation and identification of the C6-hydroxy and C20-hydroxy metabolites and glucuronide conjugate of methylprednisolone by preparative high-performance liquid chromatography from urine of patients receiving high-dose pulse therapy.

In the present study metabolites of methylprednisolone were detected using gradient elution high-performance liquid chromatography. Separation was performed by a Cp Spherisorb ODS 5 microm (250 mmx4.6 mm I.D.) column, connected to a guard column, packed with pellicular reversed phase. The mobile phase was a mixture of acetonitrile and 1% acetic acid in water. At t = 0, this phase consisted of 2% acetonitrile and 98% acetic acid 1% in water (v/v). During the following 35 min the phase changed linearly until it attained a composition of acetonitrile-buffer (50:50, v/v). At 40 min (t = 40) the mobile phase was changed over 5 min to the initial composition, followed by equilibration during 2 min. The flow-rate was 1.5 ml/min. UV detection was achieved at 248 nm. We have isolated the respective compounds with the most abundant concentration and suggested their chemical structure based on NMR, IR, UV, MS, retention behaviour and melting points. The c/, stereochemistry could not be solved in this study. The overall picture of the metabolic pathways of methylprednisolone is apparently simple: reduction of the C20 carbonyl group and further oxidation of the C20-C21 side chain (into C21-COOH and C20-COOH), in competition with or additional to the oxidation at the C6-position.

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man.

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 microg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 microg/ml, Q1G 1.02 microg/ml and Q4G 0.278 microg/ml. The corresponding values in urine are: Pgluc 0.264 microg/ml, Q1G 0.731 microg/ml and Q4G 0.199 microg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.

Comparison of the effects and disposition kinetics of lidocaine and (+/-)prilocaine in patients undergoing axillary brachial plexus block during day case surgery.

OBJECTIVE: The aim of this investigation was to compare the clinical effects and pharmacokinetics of lidocaine and prilocaine in two groups of 15 patients undergoing axillary brachial plexus anaesthesia. METHODS: The study had a randomised design. Patients were allocated to one of the two groups of 15. Each group received either lidocaine (600mg = 2.56 mmol/L + 5 mg/L adrenaline) or prilocaine (600mg = 2.72 mmol/L + 5 mg/L adrenaline), injected over a period of 30 seconds. Onset of the surgical analgesia was defined as the period from the end of the injection of the local anaesthetic to the loss of pinprick sensation in the distribution of all three nerves. RESULTS: The mean onset time of surgical analgesia of both lidocaine and prilocaine was 10 minutes. Lidocaine was biexponentially eliminated with a rapid elimination phase half-life (t((1/2)alpha)) of 9.95 +/- 14.3 minutes and a terminal elimination phase half-life (t((1/2)beta)) of 2.86 +/- 1.55 hours. Lidocaine was metabolised to MEGX (monoethylglycylxylidide); time to reach maximum plasma concentration (tmax) 2.3 +/- 0.8 hours; maximum plasma concentration (C(max)) 0.32 +/- 0.13 mg/L; t((1/2)beta) 2.4 +/- 2.4 hours. Lidocaine total body clearance was 67.8 +/- 28.8 L/h. Prilocaine was rapidly and biexponentially eliminated with a t((1/2)alpha) of 9.4 +/- 18.4 minutes and a t((1/2)beta) of 2.12 +/- 1.28 hours. The total body clearance of prilocaine (150 +/- 53 L/h) was higher than that of lidocaine (p = 0.0255). Both compounds demonstrated a comparable volume of distribution (Vd), while the volume of distribution at steady-state (V(ss)) and the volume of distribution in the second compartment (V(beta)) values of prilocaine were a factor of 1.6 higher than those of lidocaine (p < 0.001). Both compounds showed a comparable t((1/2)alpha) (p > 0.8) and a comparable t((1/2)beta) (p = 0.26). CONCLUSION: Following axillary administration, lidocaine and prilocaine demonstrated similar pharmacokinetic behaviour and could therefore be used as the clinical preference for this regional anaesthesia technique.

Clinical pharmacokinetics of R(+)- and S(-)-mepivacaine after high doses of racemic mepivacaine with epinephrine in the combined psoas compartment/sciatic nerve block.

The purpose of this study was to investigate the pharmacokinetics of R(+)- and S(-)-mepivacaine in 10 male patients after injection of a high dose (731 mg) of racemic R,S-mepivacaine for a combined psoas compartment/sciatic nerve block. Arterial blood samples were taken, and the plasma concentrations of the stereoisomers R(+)- and S(-)-mepivacaine were measured by means of high-performance liquid chromatography (HPLC) with a Chiral AGP column. The S(-) isomer reached higher plasma concentrations than the R(+) isomer. The maximal plasma concentration (Cmax) of R(+) was 1.54 +/- 0.34 micrograms/mL, whereas that of the S(-) isomer was 2.34 +/- 0.51 micrograms/mL (P = 0.00050). The time at which Cmax was reached (Tmax) was identical for both isomers. The area under the plasma concentration-time curve from t = 0 to infinity (AUC infinity) of S(-)-mepivacaine was almost double that of R(+)-mepivacaine. The elimination half-life (T1/2) was identical for both isomers (3 h), which means that the calculated total body clearance and the calculated steady-state volume of the distribution of R(+) are, respectively, 1.7 and 1.5 times larger than that of the S(-) isomer. We conclude that the plasma concentrations of the S(-)-mepivacaine isomer were higher than those of the R(+) isomer because of a smaller volume of distribution and a slower total body clearance.

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 micrograms/ml of succinylcholine. In a pilot study the plasma concentration-time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.

Plasma concentrations after high doses of mepivacaine with epinephrine in the combined psoas compartment/sciatic nerve block.

A combination of psoas compartment block and sciatic nerve block was performed with a total dose of 731.5 mg mepivacaine (55 ml, 1.33%) with epinephrine (1:600,000) in patients scheduled for orthopedic surgery on one leg. In 20 patients, arterial blood samples were collected at timed intervals over a 6-hour period to determine the mepivacaine plasma concentration. In all patients, good sensory and motor blocks were obtained and no analgesics were required during surgery. Despite the high dose of mepivacaine, the plasma concentrations stayed below 6.0 micrograms/ml, with one exception, although no clinical signs of local anesthetic toxicity were observed. Plasma pharmacokinetic variables of mepivacaine were as follows: Cmax: 4.22 mg.l-1 (SD, 1.28); Tmax: 0.99 hours (SD, 0.76); T1/2: 3.25 hours (SD, 1.12); CL 0.55 1.hour-1.kg-1.

Strain-related patterns of biliary excretion and hepatic distribution of copper in the rat.

Biliary copper excretion was studied in male, bile-cannulated rats of the inbred strains Fischer, Brown Norway, WAG/Rij and Lewis. After intravenous injection of 10, 30 and 50 micrograms copper per 100 gm body weight, two patterns of copper excretion were observed; their profiles varied with the copper dose and the strain of the rats used. The lowest amounts of copper were excreted by Fischer rats, the highest by WAG/Rij rats; this was related to the effect of the copper dose on both patterns. The subcellular distribution of copper in the liver was studied in Fischer and Brown Norway rats after doses of 50, 100, and 200 micrograms per 100 gm body weight. Brown Norway rats accumulated more copper in the liver, although the copper concentration was the same in both strains 1 hr after injection of all doses. Fischer rats accumulated proportionally more copper in lysosomal and nuclear mitochondrial fractions whereas Brown Norway rats accumulated proportionally more copper in the cytosol. Gel filtration of liver supernatants revealed that the amount of copper accumulating in the protein presumed to be metallothionein was 2 to 3 times higher in Brown Norway rats, whereas in the Fischer rats more copper eluted in the void volume fraction. We conclude that both biliary copper excretion and copper distribution in the liver are under genetic control. Because of its low copper excretion and reduced binding of copper to metallothionein the Fischer rat, compared to other strains, may be a suitable model for studying the involvement of the liver in copper intoxication.

Biochemical polymorphism in the rat: genetics of three electrophoretic variants and characterization of inbred strains.

Nine inbred strains of the rat (Rattus norvegicus) were screened for differences in electrophoretically detectable proteins. Interstrain variation was observed for 7 of 26 proteins. Three of these variants have not been described previously: leucine aminopeptidase (Lap-1), major urinary protein (Mup-1), and seminal vesicle protein (Svp-2). Genetic analysis revealed two autosomal alleles for each of these polymorphisms. The loci Lap=1, Mup-1, and Svp-2 are linked neither to one another nor to the previously described Svp-1 and Es-4 loci. Each of the nine strains can be identified now by a specific set of monogenic markers.

Genic heterogeneity and genetic monitoring of mouse outbred stocks.

An outbred stock of Swiss:SE mice has been surveyed for genic heterogeneity at 12 loci, encoding biochemical polymorphisms in mice. Using horizontal starch-gel electrophoresis, 6 loci (Es-1, Es-2, Es-3, Es-5, Trf, Dip-1) revealed no genic heterogeneity within the total sample of 289 male mice examined. The other 6 loci (Mpi-1, Mup-1, Hbb, Gpi-1, Pgm-1, Ldr-1) each showed a 2-allelic variation within each of the 3 stock units (pavillions) involved. A pavillion effect on the observed genotype numbers was found for Pgm-1 and Gpi-1 (P less than 0.10). Inadequate genotyping may have occurred at Gpi-1 and Ldr-1, as suggested by a 'week effect' on the observed genotype numbers (P less than 0.05). For studying long-term genetic changes within outbred stocks, a routine monitoring procedure using biochemical polymorphisms is recommended.

Genetically determined electrophoretic variants of the major urinary protein (Mup) complex in mouse urine.

The major urinary protein (Mup-complex) excreted in mouse urine, has been studied electrophoretically both on starch gel and on cellogel. On stargel six anodally migrating protein bands were observed. These bands are designated component 3, 2', 1, and 4 (i.e. two bands) in the order of decreasing mobility toward the anode. The slower protein band of component 4 on starch gel was not observed on cellogel. By testing mouse inbred strains, we were able to dinstinguish five male and four female Mup phenotypes. Test crosses suggested a four-allelic (a, b, c, d,) variation with regard to components 2', 2 and 1: 'group A' strains showed component 1, 'group B' strains components 1 and 2, 'group C' and 'group F' strains none, and 'group D' strains showed components 1 and 2'. Component 3 may be encoded by another Mup locus, although no crossing-over has been observed: presence (A, B, D, and F strains), absence (C strains). Insufficiently reproducible demonstration of the variation with regard to component 4, forced us to exclude this component for strain distinction. The Mup phenotypes described, can be useful for the detection of certain strain contaminations, especially if F1 hybrid Mup phenotypes are distinguishable.