Reference range for thyrotropin. Post hoc assessment.

UNLABELLED: Setting the reference range for thyrotropin (TSH) remains a matter of ongoing controversy. PATIENTS, METHODS: We used an indirect method to determine the TSH reference range post hoc in a large sample. A total of 399 well characterised subjects showing no evidence of thyroid dysfunction were selected for definition of the TSH reference limits according to the method of Katayev et al.. To this end, the cumulative frequency was plotted against the individual logarithmic TSH values. Reference limits were calculated by extrapolating the middle linear part of the regression line to obtain the cut-offs for the 95% confidence interval. We also examined biological variation in a sample of 65 subjects with repeat measurements to establish reference change values (RCVs). RESULTS: Based on these, the reference interval obtained by the novel technique was in close agreement with the conventionally established limits, but differed significantly from earlier recommendations. DISCUSSION: Following unverified recommendations could result in a portion of patients with subclinical thyroid dysfunctions being missed, an important consideration in a setting with a high prevalence of thyroid autonomy. CONCLUSION: Indirect post hoc verification of reference intervals from a large retrospective sample is a modern approach that gives plausible results. The method seems particularly useful to assess the adequacy and performance of reference limits reported or established by others in a particular setting. The present data should encourage re-evaluation of reference systems on a broader scale.

Release of S(+) enantiomers in breath samples after anaesthesia with isoflurane racemate.

BACKGROUND AND OBJECTIVE: Isoflurane is a chiral volatile anaesthetic, routinely administered as racemate. It has a low metabolic rate and is mostly eliminated via respiration. In blood samples, S(+) enantiomers are found in greater proportion in the days immediately after administration of isoflurane racemate whereas the ratio in breath samples is unknown. METHODS: Breath and blood samples were drawn immediately after recovery and daily up to 19 days after operation from patients undergoing anaesthesia with isoflurane racemate. The percentage of isoflurane enantiomer was determined by gas chromatography mass spectrometry in blood and thermodesorption gas chromatography mass spectrometry in breath samples. RESULTS: In breath samples, there were significant differences in S(+) enantiomers at all time points compared to the racemate. During the early postoperative phase, the percentage of S(+) enantiomers were significantly enhanced whereas 5 days after surgery predominantly R(-) enantiomers (50.41%) were detected in the breath samples. Also in blood samples a statistical significant accumulation of the S(+) enantiomer was noted between days 1 and 5 compared to isoflurane racemate blood control. S(+) enantiomers were significantly higher in blood compared to breath samples and was most evident on the third day after surgery (51.43%). CONCLUSIONS: During the first days after application of isoflurane racemate, the percentage of S(+) enantiomers are higher in breath and blood samples of patients. We suggest that resorption and/or redistribution of enantiomers are responsible for the different kinetics of isoflurane enantiomers.

Glucose oversupply increases Delta9-desaturase expression and its metabolites in rat skeletal muscle.

AIM/HYPOTHESIS: Previous studies have shown that prolonged glucose infusion causes insulin resistance and triglyceride accumulation in rat skeletal muscle. In this study, we investigated a possible relationship between insulin resistance and the composition of different accumulated lipid fractions in rat skeletal muscle. METHODS: Continuous glucose infusion was carried out in rats for 7 days. Lipids were extracted from skeletal muscle, separated by thin layer chromatography and fatty acid composition of phospholipids, triglycerides, diglycerides, free fatty acids and cholesterol esters fractions was analysed by gas chromatography. Delta9-Desaturase mRNA was measured by real time polymerase chain reaction. The enzyme activity was measured in the microsomal fractions. RESULTS: Prolonged glucose infusion (5 days) increased the relative content of palmitoleic acid (16:1 N7) several-fold (2.3- to 5.8-fold) in four out of five lipid fractions and enhanced oleic acid (18:1 N9) two-fold in three lipid fractions suggesting increased Delta9-desaturase activity while the content of several polyunsaturated fatty acids was reduced. In parallel, Delta9-Desaturase mRNA contents and enzyme activities in skeletal muscle were increased 10-fold, 75-fold, 2.6-fold and 7.7-fold after 2 and 5 days of glucose infusion, respectively. CONCLUSION/INTERPRETATION: Our results suggest that long-term glucose oversupply induces a rapid increase in Delta9-desaturase expression and enzyme activity in skeletal muscle which leads to fast and specific changes in fatty acid metabolism possibly contributing to the insulin resistance in this animal model.

Accumulation of S(+) enantiomer in human beings after general anaesthesia with isoflurane racemate.

BACKGROUND AND OBJECTIVE: Isoflurane is a chiral, volatile anaesthetic with low metabolic rate (0.17%) that is routinely administered in its racemic form. Knowledge about the distribution of the enantiomers in human beings may give some important information about the understanding of the mechanisms of volatile anaesthetics. METHODS: Blood samples were drawn immediately after tracheal extubation and daily up to 8 days postoperatively from patients undergoing general anaesthesia with isoflurane racemate. The enantiomer enrichment of isoflurane was determined by headspace gas chromatography-mass spectrometry. RESULTS: At all time points, there was a statistically significant accumulation of the S(+) enantiomer in blood, especially at days 2 (52.01%) and 7 (52.1%). Separate analysis of obese patients or in a small group of patients with co-existing lung disease did not show any difference to the total population. In addition, duration of anaesthesia did not influence the enantiomer concentrations. CONCLUSIONS: We suggest that a slower association and dissociation rate is responsible for the S(+) enrichment.

Insulin sensitivity of glucose disposal and lipolysis: no influence of common genetic variants in IRS-1 and CAPN10.

AIMS/HYPOTHESIS: This study aimed to assess the physiologic relationships between insulin sensitivity of lipolysis and that of glucose disposal and whether two common genetic polymorphisms associated with insulin resistance partially explained the remaining variation. METHODS: We measured suppression of lipolysis (isotopically [primed-continuous infusion of d5 glycerol] measured glycerol rate of appearance) and glucose disposal during a three-step (n = 24) or standard (n = 57) hyperinsulinaemic euglycaemic clamp in 81 healthy subjects. We also compared insulin sensitivity of lipolysis in carriers (vs. wildtype controls) of the CAPN10 UCSNP43 G/A and IRS-1 Gly972Arg polymorphisms. RESULTS: We observed a significant correlation between insulin sensitivity of glucose disposal and insulin sensitivity of lipolysis ( r = -0.39, p < 0.001) which was retained, albeit weaker, after adjusting for BMI ( r = -0.27, p = 0.002). No significant difference in insulin sensitivity of lipolysis was found between Gly/Gly compared with X/Arg ( IRS-1) and G/G compared with G/A + A/A ( CAPN10) (all p values > 0.15). CONCLUSION/INTERPRETATION: Insulin sensitivity of lipolysis has a considerable variation in healthy human beings and independently explains about 10% of the variation in insulin sensitivity of glucose disposal (or vice versa). It is possible that mediated through NEFAs, insulin resistance of glucose disposal is secondary to that of lipolysis. Alternatively, the biological variation in insulin sensitivity, to some extent, affects both systems in parallel. Neither of the two putatively insulin resistance-related polymorphisms that were tested contributed measurably to the biological variation of insulin sensitivity of lipolysis.

Two novel prevalent polymorphisms in the hormone-sensitive lipase gene have no effect on insulin sensitivity of lipolysis and glucose disposal.

Free fatty acids released during triglyceride lipolysis play an important role in obesity-associated insulin resistance of glucose disposal. Individual sensitivity of lipolysis to the suppressive effect of insulin varies greatly among healthy subjects. It is possible that genetic factors contribute to this variation. Among the many proteins involved in the regulation of lipolysis, hormone-sensitive lipase (HSL) represents a prime candidate for genetic variants contributing to the biological variation of insulin sensitivity of lipolysis. We determined the insulin sensitivity of lipolysis (suppression of isotopically [primed-continuous infusion of d5 glycerol] measured glycerol rate of appearance) and of glucose disposal, using a three-step (n = 20) or standard (n = 53) hyperinsulinemic euglycemic clamp in 73 healthy, unrelated subjects. To assess the possible role of genetic polymorphisms, we directly sequenced the coding region of the HSL gene and the noncoding exon B from these subjects. We identified two silent mutations and three amino acid polymorphisms: Arg262Met (prevalence, 5%), Glu620Asp (prevalence, 31%) and Ser681Ile (prevalence, 22%). The latter two are located in the regulatory domain of HSL but neither had a significant impact on insulin sensitivity of lipolysis or glucose disposal (with and without adjustment for obesity and age as covariates; all P values > 0.20). We conclude that a number of genetic polymorphisms in HSL exist, some of which are highly prevalent. Neither of the polymorphisms we identified in the coding region, however, contributed measurably to the biological variation of insulin sensitivity in our lean, healthy population.

Effect of the pattern of elevated free fatty acids on insulin sensitivity and insulin secretion in healthy humans.

In order to investigate whether the pattern of elevated free fatty acids (FFAs) has any effect on insulin sensitivity and insulin secretion in humans, we produced 2 distinct serum FFA patterns (PT 1 and 2) by infusing 6 healthy volunteers with 2 different lipid emulsions plus heparin for 24 hours. A hyperglycemic clamp (approx. 8 mM, 140 min) was performed before and 5 and 24 hours after both lipid infusions to determine insulin sensitivity and insulin secretion simultaneously. Total FFAs had increased comparably by 24 hours (2020+/-268 microM in PT 1) and (1812+/-154 microM in PT 2, p =0.24). Serum PT 1 contained 66% saturated FFAs plus monoenes and 34% polyenes, while PT 2 contained 80% saturated FFAs plus monoenes and 20% polyenes. Thus, the ratio of polyunsaturated to saturated plus monoenes was about 0.5 in PT 1 vs. 0.25 in PT 2. In PT 1, the insulin sensitivity index (ISI) decreased by 20 +/- 7% and 27 +/- 10% from basal after 5 and 24 hours, respectively. In PT 2, the ISI decreased significantly more after 5 (41+/-7%, p = 0.008) and 24 hours (52+/-6%, p = 0.005). In contrast, different phases of insulin secretion did not change during the lipid infusion and did not vary between the two FFA profiles. In conclusion, these findings provide preliminary evidence that the composition of elevated serum FFAs influenced insulin sensitivity in humans. The FFA pattern low in polyunsaturated FFAs reduced insulin sensitivity more than the pattern high in polyunsaturated FFAs. In contrast, no effect on insulin secretion was observed.

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC-MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1,000 microg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1 +/- 13.5 microg/l for 2-ethylhexanoic acid, 104.8 +/- 80.6 microg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2 +/- 389.5 microg/l for 2-ethyl-3-oxohexanoic acid.

A novel use of the hyperinsulinemic-euglycemic clamp technique to estimate insulin sensitivity of systemic lipolysis.

The aim of the present study was to assess whether a standard hyperinsulinemic-euglycemic clamp can provide an estimate for the antilipolytic insulin sensitivity. For this purpose, we infused 9 non-obese, healthy volunteers with [2H5]glycerol and used the glycerol rate of appearance (Ra) in plasma as an index for systemic lipolysis during a standard (1 mU/kg x min, 120 min) and a 3-step (0.1, 0.25, 1.0 mU/kg x min) hyperinsulinemic-euglycemic clamp. The insulin concentration, which half-maximally suppressed lipolysis (EC50) in the three-step clamp, was considered to be the gold standard for the antilipolytic insulin sensitivity. Glycerol Ra decreased from 1.53+/-0.11 micromol/kg x min to 0.60+/-0.09 micromol/kg x min (p <0.001) during the standard clamp. The decrease in Ra at most time points during the standard clamp significantly correlated with the EC50. The highest correlation for the % decrease of glycerol Ra from baseline was found at 60 min (r = 0.96, p < 0.001) making this parameter a useful index for the antilipoytic insulin sensitivity. Neither plasma glycerol nor plasma free fatty acid (FFA) concentrations were significantly correlated with the EC50. In conclusion, the standard hyperinsulinemic-euglycemic clamp in combination with isotopic determination of glycerol Ra provides a reasonable estimate for the antilipolytic insulin sensitivity. In healthy subjects, the parameter best suited to estimate the insulin EC50 (by linear correlation) was the percentage decrease of glycerol Ra at 60 min.

Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma2 gene is associated with increased antilipolytic insulin sensitivity.

The Pro12Ala polymorphism of the peroxisome proliferator-activated receptor (PPAR)-gamma2 is associated with reduced transcriptional activity in vitro and increased insulin sensitivity in humans in vivo. The mechanism by which this polymorphism influences insulin sensitivity in humans is unclear. PPAR-gamma2 is mainly expressed in adipocytes, and free fatty acids released from adipose tissue are key mediators of peripheral insulin resistance. Therefore, we examined insulin suppression of lipolysis in 51 subjects without (Pro/Pro) and 17 subjects with the polymorphism (X/Ala). Both groups were lean (BMI <27.0 kg/m2) and matched for age, BMI, waist-to-hip ratio, and sex. The isotopically (infusion of d5 glycerol) determined glycerol rate of appearance was used as an index of lipolysis. Insulin sensitivity of lipolysis was expressed as the insulin concentration resulting in half-maximal suppression (EC50). This was directly determined during a three-step hyperinsulinemic-euglycemic clamp (n = 21) or estimated indirectly during a standard hyperinsulinemic-euglycemic clamp (n = 47). The insulin sensitivity index (ISI) of glucose disposal was 0.095+/-0.006 micromol x kg(-1) x min(-1) x pmol(-1) x l(-1) in the control group and 0.129+/-0.008 micromol x kg(-1) x min(-1) x pmol(-1) x l(-1) in the X/Ala group (P = 0.003). The EC50 was 56+/-2 pmol/l in the control group and 44+/-3 pmol/l in the X/Ala group (P = 0.001). The EC50 of lipolysis and ISI was significantly correlated (r = 0.42, P = 0.002). In conclusion, in lean subjects, the Pro12Ala polymorphism is associated with increased insulin sensitivity of glucose disposal and suppression of lipolysis. This result suggests that an altered transcriptional activity of PPAR-gamma2 in X/Ala subjects either causes a more efficient suppression of lipolysis in adipose tissue, which in turn results in improved insulin-stimulated glucose disposal in muscle, or, alternatively, beneficially affects insulin signaling in both tissues independently of one another.

Suppression of systemic, intramuscular, and subcutaneous adipose tissue lipolysis by insulin in humans.

In addition to sc and visceral fat deposits, muscle has been shown to contain relevant amounts of lipids whose breakdown is subject to hormonal regulation. The aim of the present study was to determine insulin dose-response characteristics of systemic, sc adipose tissue and muscle lipolysis in humans. We used a combination of isotopic (primed continuous infusion of [d5]glycerol) and microdialysis techniques (catheters placed in the anterior tibial muscle and sc abdominal adipose tissue) during a three-step hyperinsulinemic-euglycemic clamp (insulin infusion, 0.1, 0.25, 1.0 mU/kg x min) in 13 lean, healthy volunteers. The glycerol rate of appearance was used as the index for systemic lipolysis; interstitial glycerol concentrations were used as the index for muscle and sc adipose tissue lipolysis. The insulin concentrations resulting in a half-maximal suppression (EC50) of systemic lipolysis, adipose tissue, and muscle lipolysis were 51, 68, and 44 pmol/L, respectively (between one another, P < 0.001). For each compartment there were significant correlations between the EC50 and the insulin sensitivity index for glucose disposal (r > 0.67; P < 0.05). However, lipolysis (as percent of baseline) was similar during the first two insulin infusion steps, but was significantly lower in adipose (22+/-2%) than in muscle (53+/-4%; P < 0.001) during step 3. Although we have no direct measurement of interstitial insulin concentrations, we conclude that based on the EC50 values, muscle is more sensitive with respect to the net effect of circulating insulin (transendothelial transport plus intracellular action) on lipolysis than sc adipose tissue in terms of exerting its full suppression within the physiological insulin range. This could be important in muscle for switching from preferential utilization of free fatty acids to glucose in the postprandial state. Inadequate suppression of im lipolysis resulting in excessive local availability of free fatty acids may represent a novel mechanism contributing to the pathogenesis of impaired glucose disposal, i.e. insulin resistance, in muscle.

Application of capillary electrophoresis in clinical chemistry: the clinical value of urinary modified nucleosides.

Urinary modified nucleosides were determined by capillary electrophoresis using a 300 mM SDS-25 mM sodium tetraborate-50 mM sodium dihydrogenphosphate buffer. The nucleosides were extracted from urine by phenylboronate affinity gel chromatography. In cancer patients the levels of the modified nucleosides are generally elevated. By an artificial neural network method breast cancer patients were differentiated from normal individuals, which indicates that the modified nucleosides could be of clinical value as tumor markers.

Leptin levels in humans are acutely suppressed by isoproterenol despite acipimox-induced inhibition of lipolysis, but not by free fatty acids.

Leptin secretion is complexly regulated in humans. Insulin has been shown to stimulate leptin secretion, whereas in vitro data suggest that catecholamines and free fatty acids (FFAs) inhibit leptin secretion. To dissect differential effects on leptin secretion, we performed two experimental protocols in 11 lean healthy subjects in addition to a saline infusion plus oral acipimox to suppress lipolysis (SAL + ACX) as a control experiment: (1) isoproterenol (approximately 30 ng/kg x min, to increase the heart rate by approximately 50 bpm) plus oral acipimox (ISO + ACX, 240 minutes) and (2) Intralipid (Pharmacia & Upjohn, Erlangen, Germany) plus heparin (LIP, 420 minutes). During SAL + ACX, FFAs decreased from 0.44 +/- 0.04 to 0.06 +/- 0.02 mmol/L (P = .001), while serum insulin and leptin remained unchanged. During ISO + ACX, FFAs decreased similarly from 0.41 +/- 0.13 to 0.09 +/- 0.02 mmol/L (P= .001), while insulin increased from 47 +/- 8 to a maximum of 116 +/- 15 pmol/L (P= .001) and serum leptin decreased acutely from 6.4 +/- 2.1 to a minimum of 5.4 +/- 1.8 ng/mL after 90 minutes (P = .003 vSAL + ACX). After 150 minutes, leptin returned to control levels. During LIP, the elevation of FFAs from 0.34 +/- 0.04 to 1.71 +/- 0.19 mmol/L (P = .001) had no effect on serum insulin or leptin concentrations (both P = nonsignificant). In conclusion, our results show that in humans, isoproterenol acutely suppresses leptin levels independently of increased FFAs, and elevated FFAs have no acute effect on leptin levels. The fact that an inhibition of leptin secretion occurred despite conditions that are known to suppress intracellular cyclic adenosine monophosphate (cAMP) levels, as demonstrated by suppressed lipolysis, suggests that signaling mechanisms other than those mediated by cAMP must be involved in modulating leptin secretion.

Lipolysis in skeletal muscle is rapidly regulated by low physiological doses of insulin.

AIMS/HYPOTHESIS: Both patients with Type II (non-insulin-dependent) diabetes mellitus and normoglycaemic, insulin resistant subjects were shown to have an increased lipid content in skeletal muscle, which correlates negatively with insulin sensitivity. Recently, it was shown that during a hyperinsulinaemic euglycaemic clamp interstitial glycerol was reduced not only in adipose tissue but also in skeletal muscle. To assess whether lipolysis of muscular lipids is also regulated by low physiological concentrations of insulin, we used the microdialysis technique in combination with a 3-step hyperinsulinaemic glucose clamp. METHODS: Nineteen lean, healthy subjects (12 m/7 f) underwent a glucose clamp with various doses of insulin (GC I = 0.1, GC II = 0.25 and GC III = 1.0 mU x kg(-1) x min(-1)). Two double lumen microdialysis catheters each were inserted in the paraumbilical subcutaneous adipose tissue and in skeletal muscle (tibialis anterior) to measure interstitial glycerol concentration (index of lipolysis) and ethanol outflow (index of tissue flow). RESULTS: During the different steps of the glucose clamp, glycerol in adipose tissue was reduced to 81 +/- 7 % (GC I), 55 +/- 8 % (GC II) and 25 +/- 5 % (GC III), respectively, of basal. In contrast, glycerol in skeletal muscle declined to 73 +/- 5 % (GC I) and to 57 +/- 6 % (GC II) but was not further reduced at GC III. Tissue flow was higher in the skeletal muscle and remained unchanged in both compartments throughout the experiment. CONCLUSION/INTERPRETATION: This study confirms the presence of glycerol release in skeletal muscle. Lipolysis in skeletal muscle and adipose tissue are suppressed similarly by minute and physiological increases in insulin but differently by supraphysiological increases. Inadequate suppression of intramuscular lipolysis resulting in increased availability of non-esterified fatty acids, could represent a potential mechanism involved in the pathogenesis of impaired glucose disposal, i. e. insulin resistance, in muscle. [Diabetologia (1999) 42: 1171-1174]

Identification of plasticizers in medical products by a combined direct thermodesorption--cooled injection system and gas chromatography--mass spectrometry.

The combination of a new thermodesorption module with a cooled injection system now provides a powerful system for direct analysis of volatile trace compounds in gaseous, liquid and solid samples by gas chromatography-mass spectrometry (GC-MS). As a cooled injection system is used for the cryofocusing of the desorbed volatiles the GC-MC system still can be used for the regular analysis of liquid samples. Although plasticizers usually are analyzed by GC-MS after solvent extraction, contaminated solvents and glassware are very well known problems. Analysis of plasticizers in plastic materials by direct thermodesorption instead saves time and avoids cross contaminations. Many medical products are made of plasticized polyvinyl chloride. Extraction of the common plasticizer di(2-ethylhexyl) phthalate (DEHP) into blood will occur, and harmful effects of DEHP in the human body have been suggested. We therefore analyzed 21 different plastic devices which are used for various invasive techniques in medicine by direct thermodesorption GC-MS. In some of the plastics up to 30 different components were identified. By far the most common plasticizer found was DEHP, followed by diethyl and dibutyl phthalates.

Analysis of volatile organic compounds in human urine by headspace gas chromatography-mass spectrometry with a multipurpose sampler.

A multipurpose sampler (Gerstel MPS), designed for liquid large volume, gaseous and headspace samples was used for the GC-MS analysis of organic volatiles in human urine. Headspace sampling with a volume-, temperature- and speed-controlled gas-tight syringe was combined with a temperature-controlled cold injection system (CIS) for cold trapping, enrichment and focusing of analytes. Regular 2-ml GC vials filled with 1 ml acidified urine were used as headspace sampling vials. A 100-vial autosampler tray was equipped with an additional temperature and heating time controlled "preheating station" for five vials. Profiles of organic volatiles in human urine were determined and 34 components identified. Trimethylamine (TMA) and 4-heptanone as two metabolites of medical interest were quantified. Calibration curves and intra assay imprecision for 4-heptanone concentrations in the range of 40 to 800 ng/ml showed a correlation coefficient of r = 0.9980 and a relative standard deviation (RSD) between 3.0 and 3.4%. Calibration curves and intra-assay imprecision for TMA concentrations in the range of medical interest from 0.5 to 20 micrograms/ml showed a correlation coefficient of r = 0.9968 and a RSD between 4.1 and 6.8%. The high practicability of the multipurpose sampler for both gaseous and liquid samples together with the here shown good reproducibility and sensitivity make this single CIS-GC-MS system very attractive for routine clinical use in metabolic profiling of organic volatiles (headspace) and non-volatiles (liquid).

Headspace gas chromatography-mass spectrometry analysis of isoflurane enantiomers in blood samples after anesthesia with the racemic mixture.

Several in vivo and in vitro studies on the stereoselective potency of isoflurane enantiomers suggest beneficial effects of the (+)-(S)-enantiomer. In order to detect possible differences in the pharmacokinetics of isoflurane enantiomers, a clinical study of 41 patients undergoing general anesthesia maintained with racemic isoflurane was performed. The isoflurane enantiomers were analyzed in blood samples drawn before induction, at cessation (day 0), and up to eight days after isoflurane anesthesia (day 1-8). A multipurpose sampler (Gerstel MPS) was used for the headspace gas chromatography-mass spectrometry (GC/MS) analysis, and it was combined with a cold injection system (Gerstel CIS 3) for coldtrapping, enrichment, and focusing of the analyte. The enantiomer separation was achieved by using a capillary column coated with octakis(3-O-butanoyl-2,6-di-O-pentyl)-gamma-cyclodextrin (Lipodex E) dissolved in the polysiloxane PS 255. Detection was done in the selected ion monitoring mode with ions m/z 117 and m/z 149. An enrichment of (+)-(S)-isoflurane in all blood samples drawn after anesthesia was found. The highest enantiomer bias, up to 52-54% (+)-(S)-isoflurane as compared to 50% for the racemate, was observed on day 2 for most of the patients. Furthermore, quantification of isoflurane in blood samples of five patients was done by enantiomer labeling, employing enantiomerically pure (+)-(S)-isoflurane as internal standard. The isoflurane concentration decreased rapidly from 383 nmol/ml to 0.6 nmol/ml (mean values) eight days after anesthesia. The present study shows differences in the pharmacokinetics of isoflurane enantiomers in man. However, it is not possible to distinguish between enantioselective distribution and enantioselective metabolism, if any.

[Stability of iodinated contrast media in UV-laser irradiation and toxicity of the photoproducts]. / Stabilität jodhaltiger Röntgenkontrastmittel unter UV-Laser-Bestrahlung und Toxizität der Photoprodukte.

PURPOSE: In XeCl-Excimer laser angioplasty, unintended and possibly harmful interaction of the UV-laser light and the contrast media may occur due to the high concentration of contrast medium proximal to the occlusion or subtotal stenosis. METHODS: One ml of three nonionic monomeric contrast agents (iopromide, iomeprol, iopamidol), one nonionic dimeric (jotrolane), and one ionic monomeric (amidotrizoate) X-ray contrast agent were irradiated with a XeCl excimer laser (lambda = 308 nm, pulse duration 120 ns, 50 Hz) using a 9 French multifiber catheter (12 sectors). Up to 20,000 pulses (106 J) were applied. Using high performance liquid chromatography the amount of liberated iodide as well as the fraction of unchanged contrast media were measured. Cytotoxicity of the photoproducts was tested in a colony formation assay of human skin fibroblasts. The contrast agents were irradiated with 2000 pulses/ml (5.3 mJ/pulse; 10.6 J) and then added to the cell cultures for a period of three hours in a concentration of 10%. RESULTS: Excimer laser irradiation induced iodide liberation of up to 3.3 mg iodide/ml. Up to 19% of the contrast agents changed their original molecular structure. Incubation of irradiated contrast agents resulted in a significantly decreased potential for colony formation (p values ranging from 0.0044 to 0.0102) with significantly higher toxicity of amidotrizoate and iomeprol in comparison to iopromide, iotrolan, and iopamidol. DISCUSSION: Due to the cytotoxic photoproducts and the high level of liberated iodide, it is recommended to flush the artery with physiological saline solution before applying a pulsed excimer laser in human arterial obstructions in order to reduce the contrast agent concentration at the site of irradiation.

Evidence for inhibition of leptin secretion by catecholamines in man.

The regulation of leptin secretion is complex and not entirely understood in humans. Insulin has been shown to stimulate leptin secretion in humans, whereas in vitro data suggest that catecholamines inhibit leptin secretion. The present studies were therefore undertaken to examine the leptin response to hyperinsulinemia in the presence and absence of elevated plasma levels of endogenous catecholamines in humans. Leptin concentrations were determined during both a euglycemic and hypoglycemic hyperinsulinemic clamp study in 10 normal and 10 type I diabetic subjects. Serum leptin increased during the hyperinsulinemic euglycemic clamp in normal (from 6.1 +/- 0.9 to 7.2 +/- 1.1 ng/dl, p = 0.003) and diabetic subjects (from 6.2 +/- 1.4 to 7.8 +/- 1.8 ng/dl, p = 0.001). During hyperinsulinemic hypoglycemia leptin concentrations increased significantly in type 1 diabetic patients (from 5.6 +/- 1.1 to 7.6 +/- 1.7 ng/dl, p = 0.003) but remained unaltered in normals (from 5.5 +/- 0.7 to 5.7 +/- 0.9 ng/dl, p = 0.7). During hypoglycemia in all subjects the increase in leptin was negatively correlated with the increase in epinephrine (r = 0.60, p = 0.005) and positively with the decrease in free fatty acids (r = 0.71, p = 0.003). In conclusion our results indicate that catecholamines play a suppressive role in the regulation of leptin secretion.