Glycaemic variability in paediatric patients with type 1 diabetes on continuous subcutaneous insulin infusion (CSII) or multiple daily injections (MDI): a cross-sectional cohort study.

OBJECTIVE: This cross-sectional observational cohort study was designed to investigate i) whether glycaemic variability in paediatric patients with type 1 diabetes is lower in those using an insulin pump (CSII) compared with those using multiple daily insulin injections (MDI) and ii) whether urinary F2 -isoprostanes and/or urinary prostaglandin F2 excretion as surrogate marker of oxidative stress and cyclooxygenase activity are associated with glycaemic variability. METHODS: 48 paediatric patients with type 1 diabetes (22 using an insulin pump) underwent an ambulatory 3-day continuous glucose monitoring. All patients continued with normal daily activities and collected urine for two consecutive 24 h periods. The glucose pentagon was used to calculate the glycaemic risk parameter. RESULTS: Insulin requirements, HDL-cholesterol, the mean of glycaemic excursions (P < 0·01) and the standard deviation of mean glucose concentration (P < 0·05) were significantly lower in patients with CSII compared with those using MDI. By contrast, averaged HbA1c during the last twelve months as well as at the time of sensor insertion did not differ significantly between both groups. Summarizing characteristic parameter of acute and long-term metabolic control into the glucose pentagon revealed a significantly lower glycaemic risk parameter in CSI patients compared with both, healthy subjects and patients using MDI (P < 0·05). CONCLUSIONS: Paediatric patients with type 1 diabetes using an insulin pump presented with lower glycaemic variability and a concomitantly lower glycaemic risk parameter compared with those using MDII. Whether these findings translate into a lower risk of diabetes associated cardiovascular complications remains to be elucidated.

Effect of bacterial vaginosis on the pharmacokinetics of misoprostol in early pregnancy.

BACKGROUND: Misoprostol has been shown to be an effective agent for cervical ripening and termination of early pregnancy especially when administered vaginally. Our objective was to evaluate whether bacterial vaginosis (BV) affected the pharmacokinetics of vaginally administered misoprostol during early pregnancy. METHODS: Ten women with BV and 10 healthy women requesting medical abortion up to 9 weeks of pregnancy were administered 200 mg mifepristone followed 24-48 h later by a single dose of 800 µg misoprostol vaginally. Blood samples were taken before (0 h) and 0.5, 1, 2, 3 and 4 h after misoprostol administration. Misoprostol acid was determined in serum samples using liquid chromatography/tandem mass spectrometry. RESULTS: All women with BV had a vaginal pH > 4.7. The mean bioavailability measured as the area under the curve (AUC) and maximum concentration (C(max)) appeared higher in the control than in the BV group (1458.7 versus 878.1 pg h/ml) and (630.7 versus 342.5 pg/ml), respectively, but did not achieve statistical significance and there was no other significant difference in the pharmacokinetics between the two groups. However, if two women with vaginal pH > 4.7 were excluded from the control group the difference in AUC240 (1359 versus 878.1 pgh/ml) reached statistical significance (P = 0.048). CONCLUSIONS: BV had an effect on pharmacokinetics of vaginally administered misoprostol in early pregnancy. However, the results should be interpreted with caution due to the small sample size and marked individual variations.

[Tissue engineering of cartilage and bone : growth factors and signaling molecules]. / Wachstumsfaktoren und Signalmoleküle zur Anwendung im "Tissue Engineering" : Targeting und innovative Releasesysteme.

Modern tissue engineering concepts integrate cells, scaffolds, signaling molecules and growth factors. In tissue engineering of cartilage, the growth plate of the long bone represents an interesting, well-organized developmental structure, with a spatial distribution of chondrocytes in different proliferation and differentiation stages embedded in a scaffold of extracellular matrix components. The proliferation and differentiation of these chondrocytes is regulated by various hormonal and paracrine factors. This article discusses some important growth factors in the process of endochondral ossification and demonstrates how this information could be translated into a controlled release system for different tissue engineering strategies.

Effects of selective COX-2 inhibition on prostanoids and platelet physiology in young healthy volunteers.

BACKGROUND: Selective inhibitors of cyclooxygenase-2 (COX-2) called coxibs, are effective anti-inflammatory and analgesic drugs. Recently, these drugs were associated with an increased risk for myocardial infarction and atherothrombotic events. The hypothesis of thromboxane-prostacyclin imbalance has been preferred to explain these unwanted effects. METHODS: We studied the effects of 14 days intake of rofecoxib (25 mg q.d.), celecoxib (200 mg b.i.d.), naproxen (500 mg b.i.d.) and placebo in a randomized, blinded, placebo-controlled study in young healthy volunteers (median age 25-30 years, each group n = 10). We assessed prostanoid metabolite excretion (PGE-M, TXB(2), 6-keto-PGF(1alpha), 11-dehydro-TXB(2), 2,3-dinor-TXB(2), and dinor-6-keto-PGF(1alpha)), the expression of platelet activation markers (CD62P, PAC-1, fibrinogen), platelet-leukocyte formation, the endogenous thrombin potential, platelet cAMP content and plasma thrombomodulin level. RESULTS: Naproxen suppressed biosynthesis of PGE-M, prostacyclin metabolites and thromboxane metabolites and thrombomodulin levels. In contrast, both coxibs had an inhibitory effect only on PGE-M, 6-keto-PGF(1alpha), and on dinor-6-keto-PGF(1alpha), whereas TXB(2), 2,3-dinor-TXB(2) and 11-dehydro-TXB(2) excretion were unaffected. None of the coxibs exerted significant effects on the expression of platelet activation markers, cAMP generation, platelet-leukocyte formation, or on thrombomodulin plasma levels. Interestingly, platelet TXB(2) release during aggregation was enhanced after coxib treatment following arachidonic acid or collagen stimulation. CONCLUSION: In young healthy volunteers coxibs inhibit systemic PGE(2) and PGI(2) synthesis. Platelet function and expression of platelet aggregation markers are not affected; however, coxibs can stimulate TXB(2) release from activated platelets. Combined decrease in vasodilatory PGE(2) and PGI(2) together with increased TXA(2) in proaggregatory conditions may contribute to coxib side effects.

Pharmacokinetic profiles up to 12 h after administration of vaginal, sublingual and slow-release oral misoprostol.

BACKGROUND: It has been shown that the route of administration of misoprostol has a strong impact on the pharmacokinetic profile and result in different clinical efficacy. No study has so far evaluated the pharmacokinetics beyond 6 hours. Furthermore a new slow-release misoprostol formulation was included in the study. METHODS: Pharmacokinetics of a novel slow-release (SR) oral misoprostol was compared during 12 h after administration to conventional misoprostol administered vaginally or sublingually. Thirty-three women requesting surgical abortion up to 12 weeks were randomly allocated to groups receiving a single dose of 400 microg conventional misoprostol administered vaginally or sublingually or 800 microg SR oral misoprostol. Blood samples were taken before (0 h) and 0.5, 1, 2, 3, 4, 6, 8, 10 and 12 h after misoprostol administration. Misoprostol acid (MPA) was determined in serum samples using liquid chromatography/tandem mass spectrometry. RESULTS: Three women did not complete the study. Serum concentrations reached their highest level following sublingual misoprostol (P<0.0001) and the time to peak concentration was shortest for this group (P=0.0094). The area under the curve (AUC) up to 12 h was greater following sublingual treatment than for the other alternatives (P<0.0001) and lowest for SR misoprostol. Cumulative serum levels of MPA did not increase beyond 6 h following sublingual and vaginal administration, while they continued to increase up to 12 h following SR misoprostol. CONCLUSIONS: The new SR form of misoprostol demonstrated lower peak levels and a lower AUC but longer lasting elevation in serum levels when compared to conventional misoprostol administered sublingually or vaginally. SR misoprostol may offer an alternative to repeated administration of conventional misoprostol.

Pharmacokinetics of a novel oral slow-release form of misoprostol.

BACKGROUND: The pharmacokinetics of a novel slow-release (SR) misoprostol was studied and compared to conventional misoprostol. METHODS: Thirty-one women, pregnant between 8 and 12 weeks, requesting surgical abortion were randomly allocated to receive orally 400 microg conventional misoprostol, 400 microg SR misoprostol or 800 microg SR misoprostol. Venous blood samples were taken at 0, 30, 60, 120, 240 and 360 min after the administration of misoprostol. Misoprostol acid (MPA) was determined in serum samples using liquid chromatography/tandem mass spectrometry. RESULTS: Serum peak concentration (Cmax) was highest for conventional oral misoprostol. The time to peak concentration (Tmax) was similar for all groups. The area under the curve up to 360 min was similar for conventional and for 800 microg SR misoprostol and significantly greater for these groups compared to 400 microg SR misoprostol (P = 0.013). CONCLUSION: The new SR form of misoprostol demonstrated lower peak levels but longer-lasting elevation in plasma levels compared to conventional oral misoprostol. The AUC for 800 microg SR misoprostol was similar to that of 400 microg of conventional oral misoprostol. SR misoprostol may offer an alternative to repeated administration of oral misoprostol or to vaginal administration.

Effect of preterm formula with and without long-chain polyunsaturated fatty acids on the urinary excretion of F2-isoprostanes and 8-epi-prostaglandin F2alpha.

BACKGROUND: The objective of this study was to evaluate the effect of conventional and long-chain polyunsaturated fatty acids (LCP)-enriched preterm formula on the endogenous formation of F2-isoprostanes and 8-epi-prostaglandin (PG) F2alpha as possible markers of lipid peroxidation in preterm infants during their first weeks of life. METHODS: In a prospective, randomized, double-blind study, infants received either formula enriched with LCP (n = 8), standard preterm formula (n = 7), or (expressed) breast milk (n = 8). Urine was sampled at study entry and after the study period of 3 weeks. The formation of F2-isoprostanes and 8-epi-PGF2alpha was evaluated by measuring the urinary excretion by gas chromatography-mass spectrometry. RESULTS: No differences in the urinary excretion of F2-isoprostanes and 8-epi-PGF2alpha were observed at the end of the study period. CONCLUSIONS: This result suggests that supplementation of a preterm formula with LCP for a period of 3 weeks does not stimulate lipid peroxidation in preterm infants.

Determination of free and glucuronide conjugated 20-hydroxyarachidonic acid (20-HETE) in urine by gas chromatography/negative ion chemical ionization mass spectrometry.

20-Hydroxy-arachidonic acid (20-HETE) was determined in urine by an isotope dilution assay using gas chromatography/mass spectrometry (GC/MS). After addition of 18O2-internal standard, 20-HETE was extracted from urine with hexane either directly or after treatment with glucuronidase. 20-HETE was derivatized to the pentafluorobenzylester and the sample was applied to thin layer chromatography with iso-octane/iso-propanol 9:1 (v/v) as the developing solvent. The corresponding zone was extracted and 20-HETE was hydrogenated. After derivatization to the trimethylsilylether, 20-HETE was determined by GC/MS using the [M-pentafluorobenzyl]- -ion in the negative ion chemical ionization mode. Excretion rates of free and glucuronide conjugated 20-HETE was determined in healthy children and in children with hyperprostaglandin-E-syndrome/antenatal Bartter syndrome (HPS/aBS) with or without indomethacin treatment. Compared to the controls, the HPS/aBS children showed higher excretion rates of 20-HETE, which were suppressed to normal values under indomethacin medication. Free and glucuronide conjugated 20-HETE do not correlate with PGE2 excluding any participation in HPS/aBS.

Neonatal urinary prostanoid excretion.

Urinary excretion of prostanoids prostaglandin E2 (PGE2), PGE-M (7alpha-hydroxy-5,11-diketo-2,3,4,5,20-penta-19-carboxyprostano ic acid), 6-keto-PGF1alpha, 2,3-dinor-6-keto-PGF1alpha, thromboxane B2 (TxB2) 2,3-dinor-TxB2 and 11-dehydro-TxB2 was determined by gas chromatography/mass spectrometry in preterm and term infants to show that there is an age-dependent excretion rate of the above prostanoids in infants this young. Group I included premature children with normal postnatal development, Groups II and III included term children who were admitted in the neonatal period for observation because of feeding problems but who were subsequently found to be completely healthy. We present normal data of three primary prostanoids and four prostanoid metabolites. In Group I, excretion rates of 2,3-dinor-TxB2 were significantly lower than in Group II (P = 0.04) and in Group III (P = 0.05). Furthermore, the excretion rate of 11-dehydro-TxB2 in group I was significantly lower than in Group II (P = 0.05). We found no significant age-dependent differences between the three groups in excretion rates of PGE2, PGE-M, 6-keto-PGF1alpha, 2,3-dinor-6-keto-PGF1alpha, and TxB2.

Prostanoid formation during feeding of a preterm formula with long-chain polyunsaturated fatty acids in healthy preterm infants during the first weeks of life.

The objective of this study was to evaluate the effect of conventional and long-chain polyunsaturated fatty acids (LCP)-enriched preterm formula on prostanoid formation in preterm infants during their first weeks of life. In a prospective, randomized, double-blind study, healthy infants received either formula enriched with LCP (n = 10), standard preterm formula (n = 10), or (expressed) breast milk (n = 10). Urine was sampled, and anthropometric measurements were taken at study entry and after the study period of 3 wk. In vivo formation of prostaglandin E2, thromboxane A2, and prostacyclin was evaluated by measuring the urinary excretion of the respective index metabolities by gas chromatography-mass spectrometry. There were no significant differences in urinary prostanoid excretion and anthropometric data between the groups at the end of the study period. We conclude that neither conventional formula nor supplementation of a preterm formula with LCP for a period of 3 wk substantially influence prostanoid formation in healthy preterm infants.

Improved quantification of 8-epi-prostaglandin F2 alpha and F2-isoprostanes by gas chromatography/triple-stage quadrupole mass spectrometry: partial cyclooxygenase-dependent formation of 8-epi-prostaglandin F2 alpha in humans.

F2-isoprostanes are considered to be novel markers of lipid peroxidation. To study the in vivo formation of F2-isoprostanes, an improved method was developed for isotope dilution assays involving gas chromatography/triple-stage quadrupole mass spectrometry (GC/MS/MS) including thin-layer chromatography (TLC) (sum of all F2-isoprostanes) and high-performance liquid chromatographic (HPLC) purification (prostaglandin F2 alpha (PGF2 alpha) and 8-epi-PGF2 alpha). Following the addition of isotopically labeled prostaglandins to urine, the sample was acidified and applied to a C18 cartridge. After elution, prostaglandins were derivatized to pentafluorobenzyl esters and subjected to TLC. A broad zone was scratched off, isoprostanes were eluted and after formation of their trimethylsilyl ether derivatives the sum of F2-isoprostanes was determined by GC/MS/MS. For the determination of PGE2 alpha and 8-epi-PGF2 alpha prior to trimethylsilylation an additional HPLC step was performed and the fractions containing PGF2 alpha and 8-epi-PGF2 alpha were analyzed by GC/MS/MS. Using this technique, 8-epi-PGF2 alpha concentrations in urine samples as low as 5 pg ml-1 could be determined with high accuracy. The excretion rates of isoprostanes were studied in comparison with the classical prostaglandins in three different groups: healthy adults, healthy children and children with hyper-PGE syndrome (HPS), a pathological situation associated with a stimulated PGE2 synthesis. F2-isoprostanes represented the main arachidonic acid metabolites in these groups and 8-epi-PGF2 alpha excretion was comparable in its amount to the classical prostanoids. To delineate the cyclooxygenase-catalyzed contribution, the influence of indomethacin, an inhibitor of cyclooxygenases, on F2-isoprostane formation in healthy adults and in HPS children was analyzed. Significantly decreased excretion rates were observed 2 days after indomethacin administration for all prostanoids, including F2-isoprostanes and 8-epi-PGF2 alpha. However, the suppression of F2-isoprostanes and 8-epi-PGF2 alpha excretion rates was less pronounced in comparison with the classical prostanoids. An improved and reliable method for the determination of F2-isoprostanes and especially 8-epi-PGF2 alpha has been developed. The data obtained on human urine samples indicates a contribution of the cyclooxygenase pathway to the formation of isoprostanes.

Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.

A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated.

Determination of prostaglandin E1 and its main plasma metabolites 15-keto-prostaglandin E0 and prostaglandin E0 by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry.

Prostaglandin E1 (PGE1), 15-keto-PGE0 and PGE0 in plasma were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of deuterated internal standards, the prostaglandins were extracted by a solid-phase cartridge and derivatized to the pentafluorobenzyl ester methoxime. The samples were purified by thin-layer chromatography, converted to the trimethylsilyl ethers and quantified by gas chromatography/triple-stage quadrupole mass spectrometry. The parent ions in the negative ion chemical ionization mode were [M-pentafluorobenzyl]- ([P]-), the daughter ions used for quantification were [P-(CH3)3SiOH]- (PGE0 and 15-keto-PGE0) and [P-2(CH3)3SiOH]- (PGE1), respectively. Plasma concentrations in healthy subjects were at about 1-3 pg ml-1 for PGE1 and PGE0 and 2-15 pg ml-1 for 15-keto-PGE0. After infusion of 60 micrograms PGE1 in 2 h, the concentrations in plasma were 3-10 pg ml-1 for PGE1, 8-17 pg ml-1 for PGE0 and 115-205 pg ml-1 for 15-keto-PGE0.

Determination of seven prostanoids in 1 ml of urine by gas chromatography-negative ion chemical ionization triple stage quadrupole mass spectrometry.

In an isotope dilution assay, prostaglandin (PG) E2, 6-keto-PGF1 alpha, thromboxane (Tx) B2 and their metabolites PGE-M (11 alpha-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostano ic acid), 2,3-dinor-6-keto-PGF1 alpha, 2,3-dinor-TxB2 and 11-dehydro-TxB2 were determined in urine by gas chromatography-triple stage quadrupole mass spectrometry (GC-MS-MS). After addition of deuterated internal standards, the prostaglandins were derivatized to their methoximes and extracted with ethyl acetate-hexane. The sample was further derivatized to the pentafluorobenzylesters and purified by thin-layer chromatography (TLC). Three zones were scraped from the TLC plate. The prostanoid derivatives were converted to their trimethylsilyl ethers and the products were quantified by GC-MS-MS. In each run, two or three prostanoids were determined.