Different pharmacokinetics of tramadol in mothers treated for labour pain and in their neonates.

OBJECTIVE: The aim of the present study was to investigate the pharmacokinetic profile of tramadol hydrochloride in neonates, born from mothers who underwent analgesia with tramadol for the relief of labour pain. METHODS: Intramuscular tramadol (100--250 mg) was administered to 22 mothers giving birth who requested pain relief. At the time of birth (1.5--6.0 h after last tramadol dose), maternal and umbilical blood samples were taken. Another venous blood sample was drawn from each neonate, and at the same time from its mother, at 1, 2, 3, 6 or 12 h post-partum, providing the data for a population pharmacokinetic evaluation of tramadol and its metabolite M1. Routine APGAR scores and a standard neurological and adaptive capacity test were considered for evaluation of the effect of tramadol on the neonates. RESULTS: Serum tramadol concentrations at the time of birth (t(0)) were 243+/-102 ng/ml (mean+/-SD, umbilical vein), 258+/-103 ng/ml (umbilical artery) and 250+/-113 ng/ml (maternal vein). Serum M1 concentrations were 52+/-27 ng/ml (umbilical vein), 47+/-24 ng/ml (umbilical artery) and 56+/-21 ng/ml (maternal vein). The two-compartment type elimination profiles during the first 12 h post-partum for neonates (and mothers, respectively) were characterised by terminal t(1/2) (tramadol)=7.0 (7.2) h and t(1/2) (metabolite M1)=85.0 (5.5) h. CONCLUSION: The intramuscular application of tramadol in birth-giving mothers almost freely reaches the neonate, confirming a high degree of placental permeability. The neonates already possess the complete hepatic capacity for the metabolism of tramadol into its active metabolite. However, the renal elimination of the active tramadol metabolite M1 is delayed, in line with the slow maturation process of renal function in neonates. Despite this difference in pharmacokinetics between neonates and adults, the intramuscular application of tramadol at the recommended dosage range during delivery appears to effective in the relief of labour pain.

Effects of D-glucose upon D-fructose metabolism in rat hepatocytes: A 13C NMR study.

Isolated hepatocytes from fed rats were exposed for 120 min to D-glucose (10 mM) and either D-[1-13C]fructose, D-[2-13C]fructose or D-[6-13C]fructose (also 10 mM) in the presence of D2O. The identification and quantification of 13C-enriched D-fructose and its metabolites (D-glucose, L-lactate, L-alanine) in the incubation medium and the measurement of their deuterated isotopomers indicated, by comparison with a prior study conducted in the absence of exogenous D-glucose, that the major effects of the aldohexose were to increase the recovery of 13C-enriched D-fructose, decrease the production of 13C-enriched D-glucose, restrict the deuteration of the 13C-enriched isotopomers of D-glucose to those generated by cells exposed to D-[2-13C]fructose, and to accentuate the lesser deuteration of the C, (as compared to C5) of 13C-enriched D-glucose derived from D-[2-13C]fructose. The ratio between C2-deuterated and C2-hydrogenated L-lactate, as well as the relative amounts of the CH3-, CH2D-, CHD, and CD3- isotopomers of 13C-enriched L-lactate were not significantly different, however, in the absence or presence of exogenous D-glucose. These findings indicate that exogenous D-glucose suppressed the deuteration of the C1 of D-[I-13C]glucose generated by hepatocytes exposed to D-[1-13C]fructose or D-[6-13C]fructose, as otherwise attributable, in part at least, to gluconeogenesis from fructose-derived [3-13C]pyruvate, and apparently favoured the phosphorylation of D-fructose by hexokinase isoenzymes, probably through stimulation of D-fructose phosphorylation by glucokinase.

Metabolism of alpha-D-[1,2-13C]glucose pentaacetate and alpha-D-glucose penta[2-13C]acetate in rat hepatocytes.

Hepatocytes from fed rats were incubated for 120 min in the presence of alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM), both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM), alpha-D-glucose penta[2-13C]acetate (1.7 mM), or D-[1,2-13C]glucose (8.3 mM). The amounts of 13C-enriched L-lactate and D-glucose and those of acetate and beta-hydroxybutyrate recovered in the incubation medium were comparable under the first two experimental conditions. The vast majority of D-glucose isotopomers consisted of alpha- and beta-D[1,2-13C]glucose. The less abundant single-labeled isotopomers of D-glucose were equally labeled on each C atom. The output of 13C-labeled L-lactate, mainly L-[2-13C]lactate and L-[3-13C]lactate, was 1 order of magnitude lower than that found in hepatocytes exposed to 8.3 mM D-[1,2-13C]glucose, in which case the total production of the single-labeled species of D-glucose was also increased and that of the C3- or C4-labeled hexose was lower than that of the other 13C-labeled isotopomers. In cells exposed to alpha-D-glucose penta[2-13C]acetate, the large majority of 13C atoms was recovered as [2-13C]acetate and, to a much lesser extent, beta-hydroxybutyrate labeled in position 2 and/or 4. Nevertheless, L-[2-13C]lactate, L-[3-13C]lactate, and single-labeled D-glucose isotopomers were also produced in amounts higher or comparable to those found in cells exposed to alpha-D-[1,2-13C]glucose pentaacetate. However, a modest preferential labelling of the C6-C5-C4 moiety of D-glucose, relative to its C1-C2-C3 moiety, and a lesser isotopic enrichment of the C3 (or C4), relative to that of C1 (or C6) and C2 (or C5), were now observed. These findings indicate that, despite extensive hydrolysis of alpha-D-glucose pentaacetate (1.7 mM) in the hepatocytes, the catabolism of its D-glucose moiety is not more efficient than that of unesterified D-glucose, tested at the same molar concentration (1.7 mM) in the presence of the same molar concentration of unesterified acetate (8.5 mM), and much lower than that found at a physiological concentration of the hexose (8.3 mM). The present results also argue against any significant back-and-forth interconversion of D-glucose 6-phosphate and triose phosphates, under conditions in which sizeable amounts of D-glucose are formed de novo from 13C-enriched Krebs cycle intermediates generated from either D-[1,2-13C]glucose or [2-13C]acetate.

Metabolism of D-[1,2-13C]glucose and alpha-D-[1,2-13C]glucose pentaacetate in tumoral pancreatic islet cells.

Tumoral pancreatic islet cells of the RINm5F line were incubated, in groups of 25x106 cells each, for 120 min at 37 degrees C in media (5. 0 ml) containing either alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM) or both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM). In both cases, the amounts of 13C-enriched metabolites (D-glucose, L-lactate and acetate) and non-enriched metabolites (acetate) recovered in the incubation medium after incubation were close to the initial amount of esterified or non-esterified D-[1, 2-13C]glucose and acetate, respectively. The 13C-enriched metabolites corresponded mainly to double-labelled D-[1, 2-13C]glucose, L-[2,3-13C]lactate and [1,2-13C]acetate. The output of L-[2,3-13C]lactate and [1,2-13C]acetate was about 3-4 times lower in the cells exposed to alpha-D-[1,2-13C]glucose pentaacetate than in those incubated with unesterified D-[1,2-13C]glucose. These findings indicate that, despite extensive hydrolysis of alpha-D-[1, 2-13C]glucose pentaacetate in the RINm5F cells, the hexose moiety of the ester is less efficiently metabolized than unesterified D-[1, 2-13C]glucose tested at the same molar concentration (1.7 mM) in the presence of 8.5 mM acetate. Thus, a higher utilization of the hexose moiety of alpha-D-glucose pentaacetate than that of unesterified D-glucose, as previously documented in isolated pancreatic islets, represents a far-from-universal situation.

Metabolism of [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-13C]succinate) in rat hepatocytes.

Hepatocytes prepared from overnight-fasted rats were incubated for 120 minutes in the presence of 2.5 mmol/L [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) or glycerol-1,2,3-tris(methyl[2,3-13C]succinate). The identification and quantification of 13C-enriched metabolites by a recently developed method for the deconvolution of nuclear magnetic resonance (NMR) spectra with multiplet structures and constraints documented a virtually complete recovery of [1,3-13C]glycerol-1,2,3-tris(methylsuccinate) in 13C-labeled glycerol, lactic acid, and glucose. In hepatocytes exposed to [1,3-13C]glycerol-1,2,3-tris(methylsuccinate), glucose was symmetrically labeled, with the vast majority of hexose molecules being enriched with 13C on both C1 and C3 and/or C6 and C4. The respective abundance of glucose isotopomers labeled either on both C3 and C4 or on only 1 of these 2 C atoms indicated that the triose phosphates generated from [1,3-13C]glycerol represented 44% +/- 1% of the total amount of triose phosphates incorporated into the hexose. In hepatocytes exposed to glycerol-1,2,3-tris(methyl[2,3-13C]succinate), the recovery of [2,3-13C]succinate, [2,3-13C]fumarate, and either double- or single-labeled malate, lactate, alanine, and glucose accounted for about half the initial 13C content of the ester. The majority of the glucose molecules were now labeled in both C, and C2 or C6 and C5, with a preferential labeling of C6-C5 relative to C1-C2, the paired C6/C1 and C5/C2 ratios averaging 1.33 +/-0.04. These findings show that glycerol-1,2,3-tris(methylsuccinate) is efficiently and extensively metabolized in hepatocytes. They reinforce the concept that the asymmetry of glucose 13C-labeling by triose phosphates generated from Krebs cycle intermediates is modulated by the availability of glycerol-derived triose phosphates. Lastly, the present study indicates that the latter triose esters, under the present experimental conditions which do not aim at duplicating the physiological in vivo situation, are largely directly channelled in the gluconeogenic pathway, with only a limited intrahepatic contribution of the "indirect" pathway involving their back-and-forth interconversion to and from pyruvate.

Proline metabolism in the wild-type and in a salt-tolerant mutant of nicotiana plumbaginifolia studied by (13)C-nuclear magnetic resonance imaging

To obtain insight into the link between proline (Pro) accumulation and the increase in osmotolerance in higher plants, we investigated the biochemical basis for the NaCl tolerance of a Nicotiana plumbaginifolia mutant (RNa) that accumulates Pro. Pro biosynthesis and catabolism were investigated in both wild-type and mutant lines. (13)C-Nuclear magnetic resonance with [5-(13)C]glutamate (Glu) as the Pro precursor was used to provide insight into the mechanism of Pro accumulation via the Glu pathway. After 24 h under 200 mM NaCl stress in the presence of [5-(13)C]Glu, a significant enrichment in [5-(13)C]Pro was observed compared with non-stress conditions in both the wild type (P2) and the mutant (RNa). Moreover, under the same conditions, [5-(13)C]Pro was clearly synthesized in higher amounts in RNa than in P2. On the other hand, measurements of enzyme activities indicate that neither the biosynthesis via the ornithine pathway, nor the catabolism via the Pro oxidation pathway were affected in the RNa mutant. Finally, the regulatory effect exerted by Pro on its biosynthesis was evaluated. In P2 plantlets, exogenous Pro markedly reduced the conversion of [5-(13)C]Glu into [5-(13)C]Pro, whereas Pro feedback inhibition was not detected in the RNa plantlets. It is proposed that the origin of tolerance in the RNa mutant is due to a mutation leading to a substantial reduction of the feedback inhibition normally exerted in a wild-type (P2) plant by Pro at the level of the Delta-pyrroline-5-carboxylate synthetase enzyme.

Metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C]succinate) in hepatocytes from Goto-Kakizaki rats.

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

Effects of starvation and diabetes on the metabolism of [2,3-13C]succinic acid dimethyl ester in rat hepatocytes.

The metabolism of [2,3-13C]succinic acid dimethyl ester ([2,3-13C]-SAD) 10 mmol/L was examined in hepatocytes from overnight-fasted normal rats, 3-day starved rats, and overnight-fasted hereditarily diabetic Goto-Kakizaki (GK) rats. The amount of 13C-labeled succinate, fumarate, malate, lactate, alanine, and aspartate released by the hepatocytes was much higher in fasted normal rats than in starved or diabetic animals. Although the integrated areas of the 13C2 and 13C3 signals assigned to double-labeled malate, lactate, or alanine were not significantly different, the amount of single-labeled malate, lactate, alanine, and aspartate was higher in C3- versus C2-labeled isotopomers. The release of 13C-labeled glucose by the hepatocytes was lower in fasted versus starved or diabetic rats. Virtually all hexose molecules double-labeled in the C1-C2-C3 and/or C6-C5-C4 moieties corresponded to the [1,2-13C] and/or [5,6-13C] isotopomers. However, in the case of the single-labeled species, 13C-labeling of C1 (or C6) exceeded that of C2 (or C5). Both the single- and double-labeled molecules enriched with 13C in the C1-C2-C3 moiety were less abundant than those labeled in the C6-C5-C4 moiety, with such asymmetry being most marked in overnight-fasted normal rats, less pronounced in diabetic animals, and virtually absent in starved rats. These findings document that SAD is efficiently metabolized in hepatocytes, with its use as a gluconeogenic precursor being influenced by the nutritional and hormonal status of the animals. The present experiments also reinforce the view that asymmetrical labeling of glucose by 13C-labeled precursors is modulated by the relative contribution of exogenous and endogenous nutrients to the production of triose phosphates incorporated into the hexose.

Metabolism of the dimethyl ester of [2,3-(13)C]succinic acid in rat hepatocytes.

Hepatocytes prepared from overnight fasted rats were incubated for 120 min in the presence of the dimethyl ester of [2,3-(13)C]succinic acid (10 mM). The identification and quantification of 13C-enriched metabolites in the incubation medium were performed by a novel computational strategy for the deconvolution of NMR spectra with multiplet structures and constraints. The generation of 13C-labelled metabolites, including succinate, fumarate, malate, lactate, alanine, aspartate and glucose accounted for about half of the initial amount of the ester present in the incubation medium. A fair correlation was observed between the experimental abundance of each 13C-labelled glucose isotopomer and the corresponding values derived from a model for the metabolism of [2,3-(13)C]succinate. Newly formed glucose was more efficiently labelled in the carbon C5 than C2, as well as the carbon C6 than C1, supporting the concept that D-glyceraldehyde-3-phosphate may undergo enzyme-to-enzyme channelling between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.

Asymmetrical labeling of D-glucose generated from [3(-13)C]pyruvate in rat hepatocytes.

The generation of 13C-labeled D-glucose isotopomers by rat hepatocytes incubated for 30 or 120 min in the presence of 10 mM [3-(13)C]pyruvate was assessed by 13C NMR. The amount of C1-labeled D-glucose exceeded that of C2-labeled hexose, which was itself higher than that of C3-labeled D-glucose. A comparable hierarchy was observed in the C6-C5-C4 moiety of the hexose. The latter moiety of D-glucose was more efficiently labeled, however, than the C3-C2-C1 moiety. This finding is similar to that both previously reported and again observed in the present study when hepatocytes were exposed to [2(-13)C]pyruvate. These converging observations thus support the concept of enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phospho-fructoaldolase.

Enzyme-to-enzyme channelling of Krebs cycle metabolic intermediates in Caco-2 cells exposed to [2-13c]propionate.

The generation of 13C-labelled lactate by colon carcinoma cells of the Caco-2 line incubated for 120 min in the presence of [2-13C]propionate (10 mM) was assessed by 13C NMR. About 10% of the total amount of 13C-labelled lactate was recovered in the cell pellet and displayed a [2-13C]lactate/[3-13C]lactate isotopomer ratio of 1.18 +/- 0.01. An even higher isotopomer ratio of 1.53 +/- 0.14 was observed in the case of 13C-labelled lactate released by the cells into the incubation medium. These findings indicate that, in the Caco-2 cells, metabolic intermediates of the Krebs cycle undergo enzyme-to-enzyme channelling in the sequence of reactions catalysed by succinyl-CoA synthetase, succinate dehydrogenase and fumarase.

D-glucose generation from [2-13C]pyruvate in rat hepatocytes: implications in terms of enzyme-to-enzyme channelling.

In rat hepatocytes exposed to [2-13C]pyruvate, newly formed glucose was more efficiently labeled in the carbon C5 than C2, as well as in the carbon C6 than C1, suggesting enzyme-to-enzyme channeling of D-glyceraldehyde 3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase. Likewise the C1/C2 and C6/C5 ratios for 13C abundance in newly formed glucose, which largely exceeded the C3/C2 ratio of lactate or alanine and could reflect reversibility in the fumarase reaction, were compatible with the enzyme-to-enzyme tunneling of symmetrical Krebs cycle intermediates in the sequence of reactions catalyzed by succinyl-CoA synthetase, succinate dehydrogenase, and fumarase. This study further indicates that the major fraction of pyruvate is metabolized via pyruvate carboxylase rather than pyruvate dehydrogenase.

Enzyme-to-enzyme channelling of symmetric Krebs cycle intermediates in pancreatic islet cells.

Tumoural islet cells of the RINm5F line were incubated for 120 min in the presence of [2-13C]propionate (10 mmol/l), and the 13C enrichment of lactate released in the incubation medium was monitored by 13C nuclear magnetic resonance. The C3/C2 ratio of resonance areas was much lower than that found with naturally 13C-enriched lactate. This reveals that symmetric Krebs cycle intermediates undergo oriented transfer in the sequence of reactions catalysed by succinate thiokinase, succinate dehydrogenate and fumarase in the mitochondria of islet cells.

Reflectance pulse oximetry in fetal lambs: subcutaneous vessels and vasoconstriction affect its reliability.

OBJECTIVE: Reflectance pulse oximetry (RPOX) has been introduced for intrapartum fetal surveillance. The purpose of this study was to describe two possible effects on the reliability of RPOX, namely the effect of the presence of a subcutaneous vein and the effect of vasoconstriction by adrenaline, both at fetal SaO2 levels. METHODS: In four anesthetized fetal lambs, a prototype 660/890 nm reflectance sensor (Nellcor Inc.) was placed on the fetal head, with the photodiode of the sensor precisely over a superficial subcutaneous vein. Measurements were made before and after coagulation of the vein. In five anesthetized fetal lambs, one or two reflectance sensors were placed on the fetal head and/or neck and adrenaline was administered in doses of 0.02 to 0.04 mg via a brachial artery. Pulse oximeter saturation readings (SpO2) were compared with continuous arterial oxygen saturation (SaO2) values obtained using a fiberoptic catheter (Opticath, Abbott) in the carotid artery. RESULTS: When the sensor was placed over the vein, the pulse oximeter read 18% to 24% too low at a SaO2 level of 20% to 50%. After coagulation of the vein, SpO2 readings were in agreement with fiberoptic SaO2 values. Administration of adrenaline resulted in a large overestimation of the SaO2 in 6 of the 7 measurements. CONCLUSIONS: Subcutaneous veins and vasoconstriction can affect the reliability of reflectance pulse oximetry. As comparable situations may occur during labor, SpO2 readings should be interpreted with caution when this kind or comparable types of RPOX sensors are used at low SaO2 levels.

Lysine overproducer mutants with an altered dihydrodipicolinate synthase from protoplast culture of Nicotiana sylvestris (Spegazzini and Comes).

Two S-(2-aminoethyl)L-cysteine (AEC) resistant lines were isolated by screening mutagenized protoplasts from diploid N. sylvestris plants. Both lines accumulated free lysine at levels 10 to 20-fold higher than in controls. Lysine overproduction and AEC-resistance were also expressed in plants regenerated from the variant cultures. A feedback insensitive form of dihydrodipicolinate synthase (DHPS), the pathway specific control enzyme for lysine synthesis, was detected in callus cultures and leaf extracts from the resistant lines. Aspartate kinase (AK), the other key enzyme in the regulation of lysine biosynthesis, was unaltered in the mutants. Crosses with wild type plants indicated that the mutation conferring insensitivity to feedback in DHPS, with as result overproduction of lysine and resistance to AEC, was inherited as a single dominant nuclear gene.