Catecholamines in larvae and juveniles of the prosobranch gastropod, Crepidula fornicata

We investigated roles of catecholamines in metamorphosis of the prosobranch gastropod, Crepidula fornicata. Levels of DOPA, norepinephrine (NE) and dopamine (DA) were measured by high-pressure liquid chromatography (HPLC) in competent larvae and juvenile siblings that metamorphosed in response to the natural adult-derived cue or to elevated K+. Competent larvae contained 1.58 +/- 0.26 (S.E.M.) x 10(-2) pmol DOPA, 0.91 +/- 0.45 x 10(-2) pmol NE, and 0.290 +/- 0.087 pmol DA (mean values per microg total protein, n = 4 batches of larvae). Levels of DA per individual were not different between larvae and juvenile siblings; levels of NE were higher in juveniles. The tyrosine hydroxylase (TH) inhibitor alpha-methyl-DL-m-tyrosine (alpha-MMT) depleted DOPA and DA to approximately half of control values without affecting levels of NE. Depletion of DOPA and DA was accompanied by inhibition of metamorphosis in response to the natural cue but not to elevated K+. The dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC) induced high frequencies of metamorphosis at concentrations of 0.1-10 microM. In juveniles induced by 10 microM DDTC, levels of both NE and DA averaged approximately 80% of those in control larvae. Catecholamines may function as endogenous regulators of metamorphosis in C. fornicata.     

RADIOENZYMATIC ASSAY OF FEMTOMOLE CONCENTRATIONS OF DOPA IN TISSUES AND BODY FLUIDS

Abstract— A single isotope radioenzymatic procedure for the measurement of DOPA has been developed. The assay combines O -methylation of DOPA by purified COMT using [³H]SAM as the methyl donor and subsequent purification as the DNFB derivative of 3- O -[methyl-³H]DOPA. The present method is about 100 times more sensitive than currently available DOPA methods. This is due to decreased blank values and increased enzymatic conversion giving transmethylation values of 50% with tissue extracts and values of almost 100% with pure solutions. Although COMT methylates a wide variety of catechol compounds, specificity of the assay is achieved by selective extraction and purification of the final product by tlc.
The method has good inter-assay reliability, the coefficient of variation being about 3.5%.
This ultramicromethod was used to determine the steady-state concentrations of endogenous DOPA in minute samples of brain areas of the rat. In untreated rats brain DOPA levels varied with the mode of death; the highest levels were found in animals killed by microwave irradiation. Unconjugated DOPA was measured in microlitre aliquots of human body fluids.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Properties of 4-ene-5 alpha-reductase and studies on its solubilization from porcine testicular microsomes

The activity of 4-ene-5 alpha-reductase was assayed in porcine testis homogenates and subcellular fractions, using testosterone as substrate. 'Marker' enzyme activities were utilized to indicate the purity of the subcellular fractions. 4-Ene-5 alpha-reductase activity was associated with the microsomal fraction; there was no activity in the purified nuclear fraction. Enzyme activity was higher in the testes of 6 week old pigs than those of 3 and 17 week old animals, and a range of activity was found. The enzyme was unstable when stored at -20 degrees C but the addition of albumin (0.1%, w/v) or glycerol (20%, v/v) to the buffer and storage at -70 degrees C or in liquid nitrogen ensured that maximal activity was retained for at least 35 days. In addition to 5 alpha-DHT, other 5 alpha-reduced metabolites and 4-androstenedione were formed in this reaction; NADPH was the preferred cofactor, but 40% of the 4-ene-5 alpha-reductase activity was retained when NADH was used. Solubilization of the microsomal enzyme was achieved using sodium citrate (0.1 M); 4-ene-5 alpha-reductase activity was enhanced to greater than 120% and 60% of this activity was in the soluble fraction. The optimum pH and temperature for both soluble and membrane-bound 4-ene-5 alpha-reductase were 6.9 and 32 degrees C, respectively. The mean apparent Km and Vmax were 0.6 mumol/l and 158 pmol/min/mg microsomal protein for the microsomal enzyme and 1.42 mumol/l and 212.0 pmol/min/mg soluble protein for the solubilized 4-ene-5 alpha-reductase. The estimated sedimentation coefficient was 11.6.     

Determination of polyamines in human tissues by precolumn derivatization with 9-fluorenylmethyl chloroformate and high-performance liquid chromatography

A high-performance liquid chromatography (HPLC) assay with fluorescence detection was developed for the determination of the polyamines putrescine, spermidine, spermine in samples of human spinal cord, cerebellum, cerebrospinal fluid (CSF), skeletal muscle, and muscle microdialysates without an extensive sample preparation. The precolumn derivatization was performed with 9-fluorenylmethyl chloroformate (FMOC), and the derivatizated polyamines were stable for at least 14 h at 4 degrees C. All polyamines were separated within 35 min. The method was checked for linearity, and mean correlation coefficient values of 0.995, 0.999, and 0.991 were achieved for putrescine, spermidine, and spermine, respectively. The within- and between-assay coefficient of variation percentages evaluated in standard solutions varied between 1.0 and 4.9% and between 1.3 and 6.9%, respectively. The corresponding values obtained in samples of human spinal cord were between 1.0 and 5.0% and between 0.6 and 5.8%. The values of the recovery, evaluated in spinal cord tissue, varied between 83.7 and 93.5%.     

The measurement of 11 beta-hydroxy-4-pregnene-3,20-dione (21-deoxycorticosterone) by radioimmunoassay in human plasma

A specific radioimmunoassay (RIA) method is described for the determination of 21-deoxycorticosterone (21 DB) in human plasma. 21-Deoxycorticosterone-3-(O-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate antisera in rabbits. Steroids which reacted significantly with the antisera were found to be progesterone, pregnenolone, corticosterone and 11-oxo progesterone. However, after extraction of plasma and column chromatography on Celite, all these steroids were separated from 21-deoxycorticosterone and consequently did not interfere with the radioimmunoassay. The intra- and interassays coefficients of variation were 8% and 11% respectively. Mean plasma 21-deoxycorticosterone level for healthy subjects was very low: 17.8 +/- 14.8 pmol/l (mean +/- SD) with no statistical difference between males and females. During the ACTH stimulation test, the 21-deoxycorticosterone levels of healthy subjects increased to 84.7 +/- 26.3 pmol/l (mean +/- SD) for males and 79.3 +/- 31.6 pmol/l (mean +/- SD) for females. Consequently high levels of plasma 21-deoxycorticosterone were found in treated patients suffering from congenital adrenal hyperplasia (CAH) with 21-hydroxylase deficiency, particularly in CAH salt-losers with high plasma renin activity (PRA), where the plasma level reached 40,545 pmol/l. Thus, 21-deoxycorticosterone may be a new marker for adrenal 21-hydroxylase deficiency.     

GH values after clonidine stimulation measured by immunofluorometric assay in normal prepubertal children and GH-deficient patients

OBJECTIVE: To establish the cut-off values of GH measured by immunofluorometric assay, a more sensitive and specific assay, in normal prepubertal children and compare their values with those of proven GH-deficient patients. METHODS: 30 normal children (20 males) and 26 patients with known causes of GH deficiency were submitted to the clonidine test and their GH values were compared. A powdered clonidine tablet (0.1 mg/m(2)) was given orally and blood samples for GH measurements were drawn at times -30, 0, 60, 90 and 120 min. RESULTS: GH peak values presented a wide variation ranging from 1.7 to 25 micro g/l (mean +/- SD = 12.87 +/- 5.8 micro g/l) in the normal group. The cut-off values for the 5th and 10th percentiles of the distribution curve were 3.3 and 5.5 micro g/l, respectively. In the GH deficiency group, maximum GH levels after clonidine stimulation ranged from <0.1 to 2.1 micro g/l (0.56 +/- 0.58 micro g/l). CONCLUSIONS: The cut-off values obtained with the immunofluorometric method are lower than the ones obtained by radioimmunoassay. We suggest a cut-off value of 3.3 micro g/l (5th percentile) that ensures 100% of sensitivity along with 93% of specificity to exclude the diagnosis of GH deficiency when using this immunofluorometric method.     

Determination of medrogestone in plasma by high-performance liquid chromatography

Interference with the UV absorbance of medrogestone by endogenous steroids in plasma was prevented by reacting plasma with oxalyl chloride. The reduction of interference was effective when oxalyl chloride was in the range 10–50 μl/ml plasma. Reaction of oxalyl chloride with plasma for 10 min could reduce interference approximately 5.5-fold, and there was no significant reduction after 30 min. The limit of quantitative concentration for medrogestone in HPLC was 1 ng/ml. The standard curves were linear with the correlation coefficient greater than 0.999 in the range of 1–30 ng/ml. The coefficients of variation of both intra- and inter-day mean values were <12% and <10% of the actual values, respectively. The developed method for plasma sample preparation and the evaluated HPLC condition were further applied to an in vivo pharmacokinetic study.     

The elimination rates of intact GIP as well as its primary metabolite, GIP 3-42, are similar in type 2 diabetic patients and healthy subjects

The incretin hormone, glucose-dependent insulinotropic polypeptide (GIP, previously known as gastric inhibitory polypeptide), is rapidly degraded to the biologically inactive metabolite GIP (3-42) in the circulation, but little is known about the kinetics of the intact hormone and the metabolite and whether differences exist between patients with type 2 diabetes mellitus and healthy subjects. We examined eight type 2 diabetic patients (six men, two women); mean (range) age: 59 (48-69) years; BMI: 31.6 (26.0-37.7) kg/m2; HbA1C: 9.0 (8.2-13.2) %; fasting plasma glucose (FPG): 10.0 (8.3-13.2) mmol/l and 8 healthy subjects matched for age, gender and BMI. An intravenous bolus injection of GIP (7.5 nmol) was given and venous blood samples were drawn the following 45 minutes. Peak concentrations of total GIP (intact+metabolite, mean+/-SEM) and intact GIP (in brackets) were 920+/-91 (442+/-52) pmol/l in the type 2 diabetic patients and 775+/-68 (424+/-30) pmol/l in the healthy subjects (NS). GIP was eliminated rapidly with the clearance rate for intact GIP being 2.3+/-0.2 l/min in the type 2 diabetic patients and 2.4+/-0.2 l/min in the healthy subjects (NS). The volumes of distributions were similar in the two groups and ranged from 8 to 21 l per subject. The primary metabolite, GIP 3-42, generated through the action of dipeptidyl peptidase IV (DPP-IV), was eliminated with a mean half-life of 17.5 and 20.5 min in patients and healthy subjects (NS). CONCLUSION: Elimination of GIP is similar in obese type 2 diabetic patients and matched healthy subjects. Differences in elimination of GIP and its primary metabolite, therefore, do not seem to contribute to the defective insulinotropic effect of GIP in type 2 diabetes.     

High-performance liquid chromatographic method for rapid and highly sensitive determination of histidine using postcolumn fluorescence detection with o-phthaldialdehyde

A high-performance liquid chromatographic method was developed for the rapid and sensitive determination of histidine. The method is based on separation by reversed-phase ion-pair chromatography followed by highly selective fluorescence derivatization of histidine with o-phthaldialdehyde. A linear calibration curve was obtained over the range of 0.25–200 pmol per injection (10 μl) with the coefficient of variation of 0.9% at 2 pmol (n=10) and with the detection limit (S/N=8) of 25 fmol. The method was applicable to the assay of histidine in human serum. Serum histidine values obtained by the present method were in good agreement with values obtained with an amino acid analyzer.     

High-throughput and simultaneous measurement of homocysteine and cysteine in human plasma and urine by liquid chromatography-electrospray tandem mass spectrometry

Total homocysteine (tHcy) and cysteine (tCys) concentrations in biological fluids are routinely used in the clinical diagnosis of genetic and metabolic diseases, and this necessitates the development of rapid and sensitive methods for quantification. Liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) was used to measure tHcy and tCys in 23 plasma and 21 urine samples from healthy adults and 14 urine samples from healthy children. The results were compared with a standard high-performance liquid chromatography (HPLC) method. The coefficient of variation (CV) for the LC-MS/MS method ranged from 2.9% to 6.1% for the intraassay and 4.8% to 6.4% for the interassay. Mean recoveries were close to 100% for both plasma and urinary tHcy and tCys. The mean plasma tHcy and tCys concentrations in healthy adults were 8.62 and 261.40 micromol/L, respectively. The mean urinary tHcy and tCys in adults were 0.98 and 22.60 micromol/mmol creatinine, respectively. The mean urinary tHcy and tCys in children were 1.17 and 27.43 micromol/mmol creatinine, respectively. Bland-Altman difference plots of method comparison between LC-MS/MS and HPLC showed good agreement in plasma and urinary tHcy and tCys concentrations. Our method is suitable for rapid measurements, and the reported urinary values in children will help to develop a pediatric reference range for clinical use.     

Effect of a test meal,duodenal acidification,and tetragastrin on the plasma concentration of β-endorphin-like immunoreactivity in man

The effects of various test materials on plasma β-endorphin-like immunoreactivity (β-EpLI) were investigated in man using a specific radioimmunoassay developed by the authors. Plasma β-EpLI was determined after extraction by the acid/acetone method (recovery 73±5%). The intraassay and interassay coefficients of variation were 5.0% and 7.6%, respectively. The plasma concentrations of human β-EpLI in normal subjects were 11.6±4.0 pmol/l for men (n=23) and 10.7±4.8 pmol/l for women (n=27). Ingestion of a test meal (150 g of Campbell's condensed meat soup) resulted in a biphasic rise in plasma β-EpLI from the basal level of 4.4±1.0 pmol/l to 29.2±1.9 pmol/l after 5 min and 24.8±6.7 pmol/l after 90 min. Intraduodenal infusion of 115 ml of 0.1 M HCl over 10 min increased the plasma β-EpLI level from 8.7±0.5 pmol/l to 15.5±0.4 pmol/l at 10 min after the start of infusion, but the level rapidly returned to the initial value after the end of the infusion. Intramuscular injection of 4 μg/kg body weight of tetragastrin markedly stimulated gastric acid output and β-EpLI release, but pretreatment with 10 mg of histamine H₂ receptor antagonist inhibited the gastric acid output and plasma β-EpLI release induced by tetragastrin.These results indicate that β-EpLI release is stimulated by ingestion of meat soup, duodenal acidification and tetragastrin administration. It is suggested that gastric acid participates, at least in part, in postprandial release of β-EpLI, probably from the gastrointestinal tract.     

Effect of near physiologic insulin therapy on hypoglycemia counterregulation in type-1 diabetes

OBJECTIVES: The aim of this study was to examine hormonal counterregulation during insulin-induced hypoglycemia in type-1 diabetic patients during long-term near normoglycemic insulin therapy and intensive clinical care. METHODS: Type-1 diabetic patients (age 35.3 +/- 2 years, body mass index 22.8 +/- 1 kg x m(-2), mean diabetes duration 13.6 (11-17 years), mean HbA1c during the last year 6.6 +/- 0.1%) and nondiabetic subjects were studied during (0-120 min) and after (120-240 min) hypoglycemic (3.05 mmol/l) hyperinsulinemic (approximately 330 pmol/l) clamp tests. RESULTS: During hypoglycemia peak plasma concentrations of glucagon (199 +/- 16 vs. 155 +/- 11 ng/l, p < 0.05), epinephrine (4,514 +/- 644 vs. 1,676 +/- 513 pmol/l, p < 0.001), norepinephrine (2.21 +/- 0.14 vs. 1.35 +/- 0.19 nmol/l, p < 0.01) and cortisol (532 +/- 44 vs. 334 +/- 61 nmol/l) were reduced in the diabetic patients. Plasma lactate did not change from baseline values (0.51 +/- 0.06 mmol/l) in diabetic but doubled in healthy subjects (1.13 +/- 0.111 mmol/l, p < 0.001 vs. control). During the posthypoglycemic recovery period plasma concentrations of free fatty acids were higher in diabetic patients at 240 min (1.34 +/- 0.12 vs. 2.01 +/- 0.23 mmol/l, p < 0.05). CONCLUSION: Despite long-term near physiologic insulin substitution and the low incidence of hypoglycemia, hormonal hypoglycemia counterregulation was impaired in type-1 diabetic patients after a diabetes duration of more than 10 years.     

Absorption of intragastrically administered DDAVP in conscious dogs

Plasma concentrations of DDAVP were measured after intragastric administration and intravenous infusion in dogs. Oral ingestion of DDAVP led to a dose dependent increase in peak plasma concentrations as well as area under the curve (AUC). Intravenous infusion of DDAVP (0.13 pmol/l min) resulted in a mean steady-state level of 20.3 pmol/l. Elimination half-lives for oral DDAVP were 77.6 and 76.1 min for low and high doses respectively. T1/2 estimated from the ascending part of the i.v. infusion curve was 50 min. A metabolic clearance rate (MCR) of 3.9 ml/kg . min was assessed from the i.v. steady-state level.     

Demonstration of aromatic l-amino acid decarboxylase activity in human brain with l-dopa and l-5-hydroxytryptophan as substrates by high-performance liquid chromatography with electrochemical detection

The enzymatic decarboxylations of l-DOPA and l-5-hydroxytryptophan (l-5-HTP) by aromatic l-amino acid decarboxylase (AADC) were measured with homogenates from human brain regions, caduate nucleus and hypothalamus, using our new and highly sensitive methods for l-DOPA decarboxylase and l-5-HTP decarboxylase by high-performance liquid chromatography with electrochemical detection (HPLC-ED). Dopamine formed from l-DOPA as substrate was measured for DOPA decarboxylase activity using d-DOPA for the blank. For 5-HTP decarboxylase activity, serotonin (5-HT) formed from l-5-HTP was measured, and the blank value in presence of NSD-1055 was subtracted. NSD-1055 inhibited 5-HTP decarboxylase activity completely at a concentration of 0.2 mM. In this study, the properties of l-5-HTP decarboxylase activity in human caudate nucleus were first examined. AADC activities in human brains were found to be widely variable for both l-DOPA and l-5-HTP as substrates. The ratio of the activities for l-DOPA and l-5-HTP were found to be significantly higher in hypothalamus than in caudate nucleus. AADC activity for l-DOPA in the brain was found to be linear up to 40 min of incubation, while that for l-5-HTP was found to be linear up to 240 min of incubation. The optimum pyridoxal phosphate concentration was found to be similar for both substrates and was between 0.01 and 0.1 mM. The optimum pH values were found to be 7.2 and 8.2 for l-DOPA decarboxylase and l-5-HTP decarboxylase, respectively. K_m and V_max values for a human caudate nucleus l-DOPA decarboxylase were found to be 414 μM and 482 pmol/min/g wet weight, respectively, while those for l-5-HTP decarboxylase were found to be 90 μM and 71 pmol/min/g wet weight, respectively.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20 267) in plasma and of letrozole and its metabolite, CGP 44 645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C₈ columns using the ASPEC system. The separation was achieved on an ODS Hypersil C₁₈ column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44 645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.     

Measurement of plasma histamine by stable isotope dilution gas chromatography-mass spectrometry: methodology and normal values

A new method for the determination of histamine by stable isotope dilution mass fragmentography is described. The method is specific, sensitive, and accurate, resulting in a within-day coefficient of variation of 4.1% and a day-to-day variation of 7.9%. It was shown that the first blood sample after a venipuncture can contain an artificially elevated plasma histamine concentration. Platelets contain about 7 pmol histamine/10(9) cells. Serum histamine was elevated about four times in comparison with plasma histamine. This phenomenon was mainly ascribed to degranulation of basophilic leukocytes by complement activation during blood clotting. Normal values for plasma histamine were (n = 25) 2.07 +/- 0.75 nmol/liter (mean +/- 1 SD), which is one of the lowest values reported up to now.     

Both GLP-1 and GIP are insulinotropic at basal and postprandial glucose levels and contribute nearly equally to the incretin effect of a meal in healthy subjects

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are both incretin hormones regulating postprandial insulin secretion. Their relative importance in this respect under normal physiological conditions is unclear, however, and the aim of the present investigation was to evaluate this. Eight healthy male volunteers (mean age: 23 (range 20-25) years; mean body mass index: 22.2 (range 19.3-25.4) kg/m2) participated in studies involving stepwise glucose clamping at fasting plasma glucose levels and at 6 and 7 mmol/l. Physiological amounts of either GIP (1.5 pmol/kg/min), GLP-1(7-36)amide (0.33 pmol/kg/min) or saline were infused for three periods of 30 min at each glucose level, with 1 h "washout" between the infusions. On a separate day, a standard meal test (566 kcal) was performed. During the meal test, peak insulin concentrations were observed after 30 min and amounted to 223+/-27 pmol/l. Glucose+saline infusions induced only minor increases in insulin concentrations. GLP-1 and GIP infusions induced significant and similar increases at fasting glucose levels and at 6 mmol/l. At 7 mmol/l, further increases were seen, with GLP-1 effects exceeding those of GIP. Insulin concentrations at the end of the three infusion periods (60, 150 and 240 min) during the GIP clamp amounted to 53+/-5, 79+/-8 and 113+/-15 pmol/l, respectively. Corresponding results were 47+/-7, 95+/-10 and 171+/-21 pmol/l, respectively, during the GLP-1 clamp. C-peptide responses were similar. Total and intact incretin hormone concentrations during the clamp studies were higher compared to the meal test, but within physiological limits. Glucose infusion alone significantly inhibited glucagon secretion, which was further inhibited by GLP-1 but not by GIP infusion. We conclude that during normal physiological plasma glucose levels, glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide contribute nearly equally to the incretin effect in humans, because their differences in concentration and potency outweigh each other.     

Determination of midazolam and its major metabolite 1'-hydroxymidazolam by high-performance liquid chromatography-electrospray mass spectrometry in plasma from children

We have developed a sensitive, selective and reproducible reversed-phase high-performance liquid chromatography method coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantification of midazolam (MDZ) and its major metabolite, 1'-hydroxymidazolam (1'-OHM) in a small volume (200 microl) of human plasma. Midazolam, 1'-OHM and 1'-chlordiazepoxide (internal standard) were extracted from alkalinised (pH 9.5) spiked and clinical plasma samples using a single step liquid-liquid extraction with 1-chlorobutane. The chromatographic separation was performed on a reversed-phase HyPURITY Elite C18 (5 microm particle size; 100 mm x 2.1mm i.d.) analytical column using an acidic (pH 2.8) mobile phase (water-acetonitrile; 75:25% (v/v) containing formic acid (0.1%, v/v)) delivered at a flow-rate of 200 microl/min. The mass spectrometer was operated in the positive ion mode at the protonated-molecular ions [M+l]+ of parent drug and metabolite. Calibration curves in spiked plasma were linear (r2 > or = 0.99) from 15 to 600 ng/ml (MDZ) and 5-200 ng/ml (1'-OHM). The limits of detection and quantification were 2 and 5 ng/ml, respectively, for both MDZ and 1'-OHM. The mean relative recoveries at 40 and 600 ng/ml (MDZ) were 79.4+/-3.1% (n = 6) and 84.2+/-4.7% (n = 8), respectively; for 1'-OHM at 30 and 200 ng/ml the values were 89.9+/-7.2% (n = 6) and 86.9+/-5.6% (n = 8), respectively. The intra-assay and inter-assay coefficients of variation (CVs) for MDZ were less than 8%, and for 1'-OHM were less than 13%. There was no interference from other commonly used antimalarials, antipyretic drugs and antibiotics. The method was successfully applied to a pharmacokinetic study of MDZ and 1'-OHM in children with severe malaria and convulsions following administration of MDZ either intravenously (i.v.) or intramuscularly (i.m.).