NMR-based metabolic profiling in healthy individuals overfed different types of fat: links to changes in liver fat accumulation and lean tissue mass.

BACKGROUND: Overeating different dietary fatty acids influence the amount of liver fat stored during weight gain, however, the mechanisms responsible are unclear. We aimed to identify non-lipid metabolites that may differentiate between saturated (SFA) and polyunsaturated fatty acid (PUFA) overfeeding using a non-targeted metabolomic approach. We also investigated the possible relationships between plasma metabolites and body fat accumulation. METHODS: In a randomized study (LIPOGAIN study), n=39 healthy individuals were overfed with muffins containing SFA or PUFA. Plasma samples were precipitated with cold acetonitrile and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Pattern recognition techniques were used to overview the data, identify variables contributing to group classification and to correlate metabolites with fat accumulation. RESULTS: We previously reported that SFA causes a greater accumulation of liver fat, visceral fat and total body fat, whereas lean tissue levels increases less compared with PUFA, despite comparable weight gain. In this study, lactate and acetate were identified as important contributors to group classification between SFA and PUFA (P<0.05). Furthermore, the fat depots (total body fat, visceral adipose tissue and liver fat) and lean tissue correlated (P(corr)>0.5) all with two or more metabolites (for example, branched amino acids, alanine, acetate and lactate). The metabolite composition differed in a manner that may indicate higher insulin sensitivity after a diet with PUFA compared with SFA, but this needs to be confirmed in future studies. CONCLUSION: A non-lipid metabolic profiling approach only identified a few metabolites that differentiated between SFA and PUFA overfeeding. Whether these metabolite changes are involved in depot-specific fat storage and increased lean tissue mass during overeating needs further investigation.

Feasibility studies. The use of NMR spectrometry as a possible substitute of or complement to several analytical tests in pharmacopoeia monographs.

NMR spectrometry has many analytical applications; for instance, the identification of known substances; the structure elucidation of unknown ones; the quantification of APIs, impurities, solvent and water; kinetic studies, stereochemistry determinations, and the analyses of complex mixtures as in metabonomics. NMR spectrometry has the potential to substitute or complement existing analyses that are performed on APIs. In this work, 4 different NMR analyses were done on 2 APIs: fluvastatin sodium and benzalkonium chloride with good results.

Quality control of metoprolol extended-release formulations in the presence of ethanol.

This paper presents in-vitro metoprolol release from four different extended-release (ER) formulations, i.e. Metoprolol GEA® Retard, Logimax® forte, Metoprolol Sandoz® and Seloken ZOC® in the presence of 10 to 40% (v/v%) ethanol at pH 1.2 and pH 6.8. The assay of metoprolol in the dissolution media was performed by reversed phase liquid chromatography (RP-LC) using a mixture of methanol and 100 mM phosphate buffer (pH 3.5) in 40:60 ratio as eluent. The dissolution data showed that the metoprolol contents of Metoprolol Sandoz® and Seloken ZOC® were released fast in the presence of 20% ethanol at the investigated conditions, while the other products demonstrated much more stability against ethanol. Unexpectedly it was discovered that the release of metoprolol from Metoprolol GEA® Retard and to some extent also from Logimax® forte decreased in the ethanol containing media.

Vitrification with DMSO protects embryo membrane integrity better than solutions without DMSO.

Vitrification has become common for cryopreservation of embryos. However, the most optimal protocol for vitrification is still to be found. Two vitrification protocols with similar osmolarities were compared: Protocol A, containing dimethyl sulphoxide (DMSO), propane-2-diol, and ethylene glycol, and Protocol B, containing propane-2-diol and ethylene glycol. Viability and the importance of specific incubation times for early embryo recovery, survival, and cleavage were studied. For assessment of cryodamage, embryos were labelled with Alexa Fluor 488-conjugated annexin V and propidium iodide. Vitrification studies on early mouse embryos were followed up with studies on human embryos. The two vitrification protocols did not differ in embryo survival rates and were equally efficient in both mouse and human embryo models. Morphological assessment of embryos directly after vitrification was not a useful tool for assessing survival in this study. Extended exposure of embryos with both vitrification protocols showed that the DMSO-containing vitrification solutions did not lead to cell membrane damage and death as quickly as the DMSO-free vitrification solutions. To assess embryo viability, the authors recommend that vitrification of early embryos should be combined with extended culture and assessment of normal blastocyst development before transferring to patients.

The Youth Self-Report (YSR) and the Depression Self-Rating Scale (DSRS) as measures of depression and suicidality among adolescents.

Two hundred and thirty-seven adolescents from a junior high school in a small community outside Göteborg, Sweden, completed the Youth Self Report (YSR) and the Depression Self Rating Scale (DSRS). Self-reported suicidality and biographical data were also recorded. The school doctor and nurse assessed the adolescents' somatic, psychological and behavioural problems using school health-records. The convergent validity of the YSR total problems scale and syndrome scales were tested against the DSRS. Discriminant validity was assessed by the two measures' ability to predict suicidality and school health problems. The Internalising (r = 0.65**) and Anxious/Depressed (r= 0.61**) syndrome scales of the YSR had the highest correlations with the DSRS. However, all YSR syndrome scales were significantly, though more modestly, correlated with the DSRS. Using stepwise logistic regression analysis, four YSR sub-scales [Social Withdrawal, Anxious/Depressed, Attention problems and Delinquency] predicted mild-severe self-reported depression (DSRS scores 12 and above). The YSR syndrome scales Anxious/Depressed and Delinquency predicted suicide ideation whereas the Self-destructive/Identity problem and Social Withdrawal (low scores) scales predicted Suicide attempts. The YSR Anxious/Depressed sub-scale and the DSRS total score seem to measure a similar dimension. However, the Anxious/Depressed and Selfdestructive/Identity problem scales were superior in predicting suicidality.

Determination of sameridine in blood plasma by nitrogen-selective gas chromatography.

A gas chromatographic method was developed and validated for the determination of sameridine in human plasma. Sameridine is a new type of compound with both local anaesthetic and analgesic properties, when administered intrathecally. The method is based on liquid-liquid extraction of sameridine from 1.0 ml of plasma, followed by gas chromatography with nitrogen-phosphorus detection. Method validation results showed that this method is very sensitive, selective and robust. The limit of quantification was 1 nM for 1.0 ml of human plasma in the low-level range (1.00-75.0 nM) and the between-day accuracy and precision were measured at 99-104% of nominal values and 3.4-5.6% (RSD), respectively.

Evaluation of solid-phase microextraction for the study of protein binding in human plasma samples.

Solid-phase microextraction (SPME) in combination with capillary gas chromatography and a nitrogen-phosphorous detector is used to study protein binding in human plasma samples. Local anesthetics of the amide-type (ropivacaine, bupivacaine, mepivacaine, prilocaine, and lidocaine) are used as model compounds in this evaluation. Carbowax/divinylbenzene (CW/DVB), polyacrylate, and polydimethylsiloxane fibers are tested. Sampling on CW/DVB fibers give the highest recovery in plasma samples compared with other fibers. Ultrafiltrate spiked with each of the substances is used for the construction of calibration curves. The protein binding is investigated at four different total concentrations from 0.5 to 15.0 microM. The degree of protein binding increases when the solute concentration decreases. Protein binding of the five solutes is investigated at four pH levels (6.4, 7.4, 8.4, and 9.4). It is found that protein binding increased with increasing pH. The influence of temperature variation (from 32 degrees C to 40 degrees C) on protein binding is also investigated. The protein binding decreases when the temperature increases. The methodology is validated and good correlation and precision are obtained. Back-calculated quality control samples give accuracy within 20% of theoretical values for all five substances. This study shows that SPME as a sample-preparation method gives the same protein binding for the studied local anesthetics as that achieved using earlier presented methods.

High-performance liquid chromatography-tandem electrospray mass spectrometry for the determination of lidocaine and its metabolites in human plasma and urine.

A sensitive, selective and accurate high-performance liquid chromatographic-tandem mass spectrometric assay was developed and validated for the determination of lidocaine and its metabolites 2,6-dimethylaniline (2,6-xylidine), monoethylglycinexylidide and glycinexylidide in human plasma and urine. A simple sample preparation technique was used for plasma samples. The plasma samples were ultrafiltered after acidification with phosphoric acid and the ultrafiltrate was directly injected into the LC system. For urine samples, solid-phase extraction discs (C(18)) were used as sample preparation. The limit of quantification (LOQ) was improved by at least 10 times compared to the methods described in the literature. The LOQ was in the range 1.6-5 nmol/l for the studied compounds in plasma samples.

Liquid chromatographic bioanalytical determination of ropivacaine, bupivacaine and major metabolites.

Bioanalytical methods for the determination of ropivacaine, bupivacaine and their major metabolites in urine and blood plasma are presented. Ropivacaine is a new local anaesthetic drug mainly used for surgery and for postoperative pain relief. The samples are hydrolysed and cleaned using solid-phase extraction and analysed using ion-pair reversed-phase liquid chromatography with gradient elution. The analytes are detected using UV at 210 nm. The methods are highly selective and the limits of quantification were 1 microM in urine and 0.1 microM in plasma, respectively. The between-day variance was generally below 3% (RSD).

Determination of ropivacaine and [2H3]ropivacaine in biological samples by gas chromatography with nitrogen-phosphorus detection or mass spectrometry.

Bioanalytical methods for determining the total concentration of the new local anaesthetic drug ropivacaine in blood plasma, urine and tissues are presented. Ropivacaine is a drug mainly used in connection with surgery and for post-operative pain relief. The biological samples were prepared using liquid-liquid extraction and analysed using capillary gas chromatography with nitrogen-phosphorus detection or mass spectrometry. The methods are highly selective and reliable with a between-day precision, given as the relative standard deviation, generally below 6%. More than 20000 samples have been analysed using the methods described.

Determination of free concentration of sameridine in blood plasma by ultrafiltration and coupled-column liquid chromatography.

Sameridine is a new candidate drug with both local anaesthetic and analgesic properties. The free concentration of sameridine in blood plasma was determined by coupled-column liquid chromatography. Following adjustment of the pH and the temperature of the plasma samples, the free fraction was prepared by ultrafiltration. The coupled-column liquid chromatographic system consisted of a reversed-phase column, a cation-exchange extraction column and a cation-exchange analytical column. Sameridine was detected by UV determination at 205 nm and the system showed high selectivity. The limit of quantification was 1 nM and the within-day precision was 4.6% (R.S.D., n = 10).

Toxicity of bupivacaine and ropivacaine in relation to free plasma concentrations in pregnant rats: a comparative study.

The relationship between free drug concentration and toxicity of bupivacaine and ropivacaine, a new local anaesthetic agent, was studied in a pregnant rat model. The compounds were given subcutaneously to rats in late pregnancy. Dose levels (bupivacaine 5.5 to 24 mg/kg and ropivacaine 5.3 to 26 mg/kg) were selected based upon the proposed human dosage and the known pharmacological activity of the compounds. Chewing, spasm, dyspnoea, drowsiness, salivation and convulsions were observed in a dose-dependent manner in the animals given 14 to 24 mg/kg of bupivacaine, while only a few animals receiving 26 mg/kg of ropivacaine showed less severe symptoms. Deaths from clonic convulsions were occasionally seen in animals receiving 14 mg/kg or more of bupivacaine. No animals receiving ropivacaine died. No effects on litter size offspring loss or weight of the offspring at birth were observed with one exception. After 24 mg/kg of bupivacaine an increased postnatal loss of the offsprings were noticed, most likely due to impaired maternal care. Protein binding, at expected Cmax, were significantly lower for ropivacaine (around 49%) compared with bupivacaine (around 69%) at dose levels. The results suggest an increased safety margin before onset of toxic side effects after treatment with ropivacaine, compared to bupivacaine, in pregnant rase.

Metabolism and excretion of ropivacaine in humans.

The pharmacokinetics, biotransformation, and urinary excretion of ropivacaine (Naropin), a new local anesthetic agent, have been studied in six healthy male volunteers after a 15-min iv infusion of 152 mumol (50 mg) of [14C]ropivacaine, with a specific radioactivity of 22.5 kBq/mumol (8.8 kBq/mg). Blood, urine, and feces were collected for up to 96 hr after administration. The plasma and urine samples were analyzed for unchanged ropivacaine and for four of its metabolites, i.e. 3-OH-2',6'-pipecoloxylidide (3-OH-PPX), 4-OH-ropivacaine, 3-OH-ropivacaine, and the N-dealkylated metabolite PPX, using GC and HPLC methods. The presence of 2,6-xylidine in plasma was also analyzed. The metabolites were quantified after acidic hydrolysis. The radioactivity could be followed in plasma for up to 14 hr after administration, with ropivacaine being the predominant compound in the early samples. The concentrations of the aforementioned metabolites in plasma were below or just above the lower limit of quantification, and no 2,6-xylidine was detected. The maximum plasma concentration of ropivacaine was 5.9 +/- 2.6 microM (1.6 +/- 0.7 mg/liter), with an elimination half-life of 2.0 +/- 0.3 hr and a total plasma clearance of 397 +/- 127 ml/min. The maximum plasma concentration value for the total radioactivity was 5.5 +/- 2.4 microM (1.5 +/- 0.7 mg/liter) and the elimination half-life was 5.4 +/- 2.9 hr. [14C]Ropivacaine and its metabolites were mainly excreted in the urine, with a total recovery of 86 +/- 3% in the urine and 9 +/- 1% in the feces after 96 hr. Most of the radioactivity (about 68%) was excreted within 12 hr. Ropivacaine was extensively metabolized, and only 1 +/- 0.6% of the dose was excreted unchanged in the urine. The major metabolite identified in the urine was conjugated 3-OH-ropivacaine, which was excreted to an extent of 37 +/- 3% of the dose. The urinary excretion of 4-OH-ropivacaine was < 1%, whereas the N-dealkylated metabolites PPX and 3-OH-PPX accounted for 2 and 3% of the dose, respectively. An additional hydroxylated metabolite, 2-OH-methyl-ropivacaine, was tentatively identified in the urine of some volunteers, accounting for about 4-15% of the dose.

Pharmacokinetics and analgesic effect of ropivacaine during continuous epidural infusion for postoperative pain relief.

BACKGROUND: The pharmacokinetics and clinical efficacy of ropivacaine (2.5 mg/ml) during a 24-h continuous epidural infusion for postoperative pain relief in 20 patients scheduled for abdominal hysterectomy were characterized using an open-label, increasing-dose design. METHODS: Through an epidural catheter inserted at T10-T12, a test dose of 7.5 mg ropivacaine was given 3 min before a bolus dose of 42.5 mg and immediately followed by a 24-h continuous epidural infusion with either 10 or 20 mg/h. Peripheral venous plasma samples were collected up to 48 h after infusion, and urinary excretion was followed up to the end of infusion. Postoperative pain at rest, on coughing, and at mobilization was assessed by means of a visual analog scale 2,4,6,8,12, and 24 h after the end of surgery. Sensory (pinprick) and motor block (modified Bromage scale) were assessed at the same intervals. RESULTS: The total plasma concentrations of ropivacaine increased markedly and consistently during the 24-h epidural infusion, in contrast to stable unbound concentrations. Both total and unbound plasma concentrations at the end of infusion were proportional to the total dose, although only the latter was proportional to the infusion rate. The total and unbound plasma clearance was independent of dose. Total mean clearance decreased on average by 21% (P < 0.001) during the last 12 h of epidural infusion, i.e., from 539 +/- 191 ml/min to 418 +/- 138 ml/min, indicating time-dependent kinetics. The unbound clearance also varied between estimates after 8 h of infusion and the end of treatment, i.e., a 5.3% decrease from 10.4 +/- 5.3 l/min to 9.5 +/- 3.9 l/min (P < 0.05). The unbound fraction of ropivacaine in plasma decreased during treatment, and this was related to the increase in alpha1-acid glycoprotein concentration. Pain was generally well controlled, and median visual analog scale scores during mobilization were less than 30 mm in patients receiving ropivacaine at 20 mg/h. CONCLUSIONS: The pharmacokinetics of ropivacaine were independent of dose, but total clearance decreased with time over 24 h. The consistent increase in total plasma concentration during the postoperative epidural infusion contrasted to much less variation in the unbound plasma concentrations of ropivacaine.

Determination of free concentration of ropivacaine and bupivacaine in blood plasma by ultrafiltration and coupled-column liquid chromatography.

A coupled-column liquid chromatographic method for determining the free concentration of ropivacaine and bupivacaine in blood plasma was developed. Following adjustment of the temperature and pH, the plasma samples were ultrafiltrated. Ropivacaine or bupivacaine in the ultrafiltrate was determined by direct injection into a coupled-column liquid chromatographic system, consisting of one reversed-phase and one ion-exchange column. The system was highly selective. Ropivacaine and bupivacaine were detected by UV at 210 nm. The limit of determination was 10 nM and the inter-assay precision at a concentration level of about 100 nM was 6% (R.S.D., n = 30) for ropivacaine and 7% (R.S.D., n = 30) for bupivacaine.

Lack of metabolic racemisation of ropivacaine, determined by liquid chromatography using a chiral AGP column.

Ropivacaine hydrochloride monohydrate (ropivacaine) is a new local anaesthetic agent which is administered exclusively as the (-)-(S)-form. The aim of the study was to determine whether metabolic racemisation of (-)-(S)-ropivacaine occurs. This was tested in man, rat, dog, and sheep after different routes of administration. The enantiomers of ropivacaine and two of the major metabolites, 3-hydroxy-ropivacaine and 2',6'-pipecoloxylidide (PPX), were determined in urine samples by liquid chromatography on a Chiral AGP column after liquid-liquid extraction. It was possible to detect < 1% of the (+)-(R)-enantiomer of both ropivacaine and the two major metabolites. In the samples examined, no trace of metabolic racemisation was observed. In pharmacokinetic, pharmacodynamic, toxicological, and metabolic studies, therefore, nonchiral assays are considered to be adequate.

Peak distortion in the column liquid chromatographic determination of omeprazole dissolved in borax buffer.

Injection of a sample containing omeprazole dissolved in borax buffer (pH 9.2) into a reversed-phase liquid chromatographic system consisting of a mixture of acetonitrile and phosphate buffer (pH 7.6) as the mobile phase and a C18 surface-modified silica as the solid phase resulted under special conditions in split peaks of omeprazole. The degree of peak split and the retention time of omeprazole varied with the concentration of borax in the sample solution and the ionic strength of the mobile phase buffer as well as with the column used. Borax is eluted from the column in a broad zone starting from the void volume of the column. The retention is probably due to the presence of polyborate ions. The size of the zone varies with the concentration of borax in the sample injected. In the borax zone the pH is increased compared with the pH of the mobile phase, and when omeprazole (a weak acid) is co-eluting in the borax zone its retention is affected. In the front part and in the back part of the borax zone, pH gradients are formed, and these gradients can induce the peak splitting. When the dissolving medium is changed to a phosphate buffer or an ammonium buffer at pH 9 no peak distortion of omeprazole is observed.

Determination of binding affinity of enantiomers to albumin by liquid chromatography.

The principles of the determination of the binding affinity constants of small molecules to albumin by liquid chromatography, using albumin as a mobile phase additive, are outlined. Chromatographic conditions for determinations of constants are presented and applied to enantiomers of tryptophan and omeprazole. The influence of albumin on the retaining properties of LiChrosorb RP-8, Phenyl Hypersil and LiChrosorb Diol was studied.

Effects of drug-protein binding on trace enrichment of drugs in blood plasma on short precolumns.

A reversed-phase liquid chromatographic precolumn venting plug technique was used for the determination of drugs by direct injection of large volume (500 microliters) blood plasma samples. Enrichment of the drugs from large plasma samples, as well as clean-up from less retained plasma components, such as proteins, was obtained on a short precolumn. Strong drug-protein binding resulted in losses of the drug during the enrichment step. The recovery was found to be inversely proportional to the degree of drug-protein binding in the sample solution. Techniques to increase the recovery were studied. These include methods to decrease the degree of drug-protein binding in the sample solution by dilution and changes of the pH, as well as methods to increase the residence time of the drug on the precolumn by increasing the precolumn length and precolumn hydrophobicity.

Effects of system peaks in a column-switching liquid chromatographic system for bioanalysis.

A reversed-phase liquid chromatographic column-switching system was used for the determination of drugs and metabolites in blood plasma with a direct injection procedure. A short precolumn was used for enrichment of the drugs and clean-up from weekly retained plasma components using an aqueous solution. In a second step the drugs were eluted, with a suitable eluent, from the precolumn to the separation column for the final separation. When the aqueous plug on the precolumn, created by the clean-up procedure, was transferred to the separation column a disturbance of the established equilibria in the separation column occurred. This resulted in migrating zones, one of each mobile phase component. If one mobile phase compound gives a detectable signal the zone will be visualized as an extra peak (system peak) in the chromatogram. Deformed peaks of the drug were obtained when the drug was co-eluted with a non-visible system peak. Changes in the retention and selectivity of weakly retained solutes on the separation column were obtained when the pH of the aqueous plug in the precolumn deviated from the pH of the mobile phase. This is explained by the formation of a pH gradient in the separation column.