Determination of the cephalosporin antibiotic cephradine in human plasma by high-performance liquid chromatography with ultraviolet detection.

A simple and sensitive high-performance liquid chromatographic method, for the determination of cephradine in human plasma samples has been developed and validated. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation. Aliquots of the extracts were analysed by reversed-phase high-performance liquid chromatography (HPLC) utilising a polymeric reversed-phase PLRP-S column, followed by ultraviolet detection at 260 nm. The method has a working dynamic range from 0.2 to 30.0 microg/ml from 200 microl human plasma. The precision of the method at 0.2 microg/ml was 4.9% (intra-assay) and negligible (inter-assay) as calculated by one-way analysis of variance and the accuracy of the method at 0.2 microg/ml was -4.1% in terms of percentage bias. This method has been successfully applied to clinical studies including an oral bioequivalence study comparing the pharmacokinetics of 500 mg tablets of Kefdrin with 500 mg tablets of Velosef in healthy human volunteers.

Rapid determination of the anti-cancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry.

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C18 sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4-800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 microl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran following single doses of oral tablet formulations.

Peptide histidine valine: its haemodynamic actions and pharmacokinetics in man differ from those of vasoactive intestinal peptide and peptide histidine methionine.

1. The effects of intravenous and intra-arterial infusion of the peptides derived from prepro-vasoactive intestinal peptide, vasoactive intestinal peptide, peptide histidine methionine and peptide histidine valine, were examined in six healthy volunteers. 2. Vasoactive intestinal peptide given intravenously caused a significant increase in heart rate and a decrease in diastolic, but not systolic, blood pressure, whereas peptide histidine valine caused an increase in heart rate alone, despite higher achieved circulating peptide concentrations. Peptide histidine methionine did not affect heart rate or blood pressure. Forearm blood flow was increased by vasoactive intestinal peptide and peptide histidine valine when infused locally intra-arterially, although vasoactive intestinal peptide was more potent than peptide histidine valine. 3. Plasma concentrations of cardiodilatin (the N-terminal peptide derived from pro-atrial natriuretic peptide) were increased by intravenous infusion of vasoactive intestinal peptide, but were unaffected by peptide histidine methionine or peptide histidine valine. Circulating plasma concentrations of adrenaline and noradrenaline did not change during infusion of vasoactive intestinal peptide, peptide histidine methionine or peptide histidine valine. 4. Peptide histidine valine had a long half-life when compared with peptide histidine methionine and vasoactive intestinal peptide. 5. We conclude that peptide histidine valine is active in the human cardiovascular system and has a similar, though less potent, vasodilating action to vasoactive intestinal peptide. The higher circulating levels of peptide histidine valine found in man suggest that it may be important in modulating vascular tone.

Leukotriene E4 release in cold urticaria.

Cold urticaria is a rare condition characterized by abnormal wealing following exposure to cold. It has been suggested that lipid-derived mediators may be involved in the pathogenesis of this condition. We have investigated whether the inflammatory reaction in cold urticaria is associated with the release of cysteinyl-leukotrienes. Leukotriene E4 (LTE4; a stable metabolic product of LTC4 and LTD4) and histamine were measured in the blood draining the site of a cold-challenge in five patients with clinical histories of cold urticaria. Three of the patients showed a typical clinical response to the challenge, and this was associated with an increase in the concentration of LTE4 and histamine. No increase in LTE4 or histamine levels were observed following cold challenge in the non-responding individuals.

Development of a solid-phase extraction technique for alpha-human atrial natriuretic peptide in human plasma.

A reliable extraction method was developed for alpha-human atrial natriuretic peptide (alpha-hANP) using Bond Elut C8 columns in tandem. This involved activation of the columns using methanol followed by a water wash to remove the excess methanol. Plasma (1 ml) was then added and a vacuum applied until all was drawn through. Excess protein and other endogenous compounds were removed by washing the columns with water and elution of the alpha-hANP was achieved with 0.75 ml acetonitrile-water-trifluoroacetic acid (80:19.8:0.2, v/v/v). Samples were evaporated under nitrogen and reconstituted in radioimmunoassay buffer ready for analysis. The recovery of alpha-hANP from plasma using this method was found to be 90% +/- 0.6% [mean +/- standard error of the mean (S.E.M.); coefficient of variation (C.V.) = 1.5%] which will allow more precise measurement of the peptide than is presently available. With this high precision of analysis available, having a limit of detection of 0.4 fmol/ml and a range of 0 to 32 fmol/ml, a low-dose infusion of alpha-hANP was conducted and the changes in plasma concentration were followed.

Effects of two differing halothane concentrations on the metabolic and endocrine responses to surgery.

The effects of two differing concentrations of halothane, 2.1 MAC or 1.2 MAC, on the metabolic and endocrine responses to abdominal hysterectomy were investigated. The changes in blood glucose and lactate values, and plasma glycerol, cortisol, insulin and catecholamine concentrations were similar in both groups. We conclude that high concentrations of halothane do not suppress the responses to pelvic surgery, and that accurate quantification of the dose of halothane, within the concentration range of 1.2 to 2.1 MAC, is not essential in studies of metabolic changes associated with surgery.

The effect of atrial natriuretic peptide on plasma renin activity, plasma aldosterone, and urinary dopamine in man.

The acute natriuretic effect of human atrial natriuretic peptide (ANP) has been well described in man. We have now studied possible hormonal mediators of this effect. We studied six healthy volunteers on two occasions when they received either an infusion of ANP of 1.5 pmol X kg-1 X min-1 for 30 min followed by 15 pmol X kg-1 X min-1 for a further 30 min, or matching vehicle infusions in a randomized single-blind fashion. On the placebo day, plasma renin activity (PRA) rose from 1.26 +/- 0.08 to 1.57 +/- 0.14 ng A1 X ml-1 X h-1, while on the ANP study day PRA fell from 1.45 +/- 0.15 to 1.28 +/- 0.05 ng A1 X ml-1 X h-1 (p less than 0.01). No significant changes were found in plasma aldosterone concentrations or in urinary dopamine excretion. These results provide evidence that ANP suppresses renin release in man.

Adrenaline and noradrenaline excretion in placebo treated patients in the Medical Research Council Hypertension Trial and in matched normotensive controls.

The 24-h excretion of adrenaline and noradrenaline was measured in 268 untreated essential hypertensives and in 268 age- and sex-matched controls. No significant difference was found between the groups. However, in the control group alone, a multiple regression analysis showed a highly significant correlation between systolic blood pressure and both noradrenaline (P = 0.0006) and adrenaline (P = 0.0001). These results suggest that any role for elevated sympatho-adrenal activity in long-term blood pressure regulation is limited to the time before hypertension is established.

The effect of human atrial natriuretic peptide on urinary sodium and urinary dopamine excretion in man.

Atrial extracts have long been known to produce natriuresis but it is only recently that atrial natriuretic peptide (ANP) itself has been isolated, purified and sequenced. We have now developed a specific radio-immunoassay for ANP and assessed its biological activity by infusing ANP in normal volunteers. Low dose ANP infusion (1.5 pmol/kg per min) produced no haemodynamic or natriuretic effects. High dose ANP infusion (15 pmol/kg per min) produced an increase in plasma immunoreactive ANP levels of 203 +/- 78 pmol/l and caused the urinary sodium excretion to increase from 3.5 +/- 1.6 to 11.0 +/- 7.4 mmol/30 min period (P < 0.05). No haemodynamic effects were seen with this high dose infusion. No changes in urinary dopamine were seen with either ANP infusion. This study shows that ANP is capable of producing a natriuresis in man in the absence of any generalized haemodynamic effects and that ANP may be an important modulator of salt and water balance in man.

Urinary catecholamines in essential hypertension: results of 24-hour urine catecholamine analyses from patients in the Medical Research Council trial for mild hypertension and from matched controls.

Four consecutive 24-h urine samples were collected from 134 male and 134 female placebo-treated patients in the Medical Research Council Trial for Mild Hypertension. Similar samples were collected from age and sex-matched normotensive controls. On the fourth day noradrenaline excretion was 22.05 +/- 1.01 nmol/mmol creatinine in the hypertensives compared with 22.22 +/- 1.16 nmol/mmol creatinine in the controls. Adrenaline excretion on the same day was 6.13 +/- 0.33 nmol/mmol creatinine in the hypertensive subjects compared with 6.32 +/- 0.38 nmol/mmol creatinine in the controls. There was no significant difference for either catecholamine between the two groups. However, in the control group there was a highly significant correlation between excretion of adrenaline and systolic blood pressure (r = 0.218, p = 0.0004) and between noradrenaline excretion and systolic blood pressure (r = 0.200, p = 0.001). Catecholamine excretion and blood pressure were not significantly correlated in the hypertensive patients. There were no significant correlations in either group between catecholamine excretion and heart rate, caffeine intake, nicotine consumption or the Bortner self-assessment score of personality type. This study has found no evidence of elevated sympathoadrenal activity in mild hypertensives. The correlations in the control group may reflect the role of sympathoadrenal activity in acute fluctuations in blood pressure or may suggest that the level of blood pressure within the 'normal' range depends in part on the level of sympathoadrenal activity.

Circadian rhythms of epinephrine and norepinephrine in man.

The diurnal rhythms of plasma epinephrine and norepinephrine were investigated in a group of normal young men. Sleep, posture, illumination, and food intake were monitored. Plasma epinephrine demonstrated a statistically significant diurnal rhythm, with a mean amplitude of 14 +/- 1.6 (+/- SE) pg/ml superimposed on a mean level of 43 +/- 5.3 pg/ml. The trough occurred at 03.20 h +/- 35 min. Plasma norepinephrine had a significant diurnal rhythm, with a mean amplitude of 111 +/- 19 pg/ml superimposed on a mean level of 413 +/- 25 pg/ml, with the trough occurring at 02.20 h +/- 30 min. There was a significant correlation between the two rhythms at zero phase shift, with a pooled value for the group of r = 0.49. Epinephrine levels had no direct relationship to sleep or posture, whereas norepinephrine levels were significantly higher with upright posture and higher when the men were awake than when asleep. Our results indicate that circadian variations in the sympathetic-adrenal medullary system are not explained by a single controlling influence and that the norepinephrine rhythm can be accounted for as a direct response to changes in posture and sleep, whereas the epinephrine rhythm is probably controlled by a circadian oscillator.

Reversed-phase high-performance liquid chromatography and amperometric detection of 3-O-methyl isoprenaline sulphate: application to studies on the presystemic metabolism of d-isoprenaline in man.

A selective method for the determination of 3-O-methyl isoprenaline sulphate in human urine and blood plasma has been developed using reversed-phase high-performance liquid chromatography with amperometric detection. The sulphoconjugate was subjected to acidic hydrolysis and the liberated 3-O-methyl isoprenaline was isolated by organic extraction and conventional cation exchange. An internal standard of 3-O-methyl isoetharine was synthesized from commercially available isoetharine and used to correct for recovery losses. The assay was shown to be linear over the range 5 ng/ml to 20 micrograms/ml with a limit of detection of 2 ng/ml. The reliability of the analytical method was examined together with its applicability to in-vivo studies in man.

Can the human neonate mount an endocrine and metabolic response to surgery?

Little is known of the ability of the human newborn infant to mount an endocrine and metabolic response to surgical trauma. Blood concentrations of glucose, lactate, pyruvate, alanine, hydroxybutyrate, acetoacetate, and glycerol together with plasma concentrations of insulin, glucagon, adrenaline, and nonadrenaline were measured in 33 infants (26 term, 7 preterm) subjected to surgery during the neonatal period. The results show that newborn infants can indeed mount a substantial endocrine and metabolic stress response, the main features of which are hyperglycemia and hyperlactatemia associated with the release of catecholamines and the inhibition of insulin secretion. There are specific differences between preterm and term neonates and between neonates anesthetised by different anesthetic techniques in the pattern of this response.

Circulating catecholamines in acute asthma.

Plasma catecholamine concentrations were measured in 15 patients (six male) aged 14-63 years attending the casualty department with acute severe asthma (peak expiratory flow 27% (SEM 3%) of predicted). Nine patients were admitted and six were not. The plasma noradrenaline concentration, reflecting sympathetic nervous discharge, was two to three times normal in all patients and was significantly higher in those who required admission compared with those discharged home (mean 7.7 (SEM 0.6) v 4.7 (0.5) nmol/l (1.3 (SEM 0.1) v 0.8 (0.08) ng/ml); p less than 0.001). Plasma adrenaline concentration, however, was not increased in any patient. This surprising failure of the plasma adrenaline concentration to increase during the stress of an acute attack of asthma was unexplained and contrasts with the pronounced rise in plasma adrenaline and noradrenaline concentrations in acute myocardial infarction, heart failure, and septicaemia. The failure of plasma adrenaline concentration to increase in acute asthma is unlikely to be explained by adrenal exhaustion, but it may be another example of impaired adrenaline secretion in asthma.

High-performance liquid chromatography with amperometric determination of plasma tyramine.

A method for the measurement of tyramine in human plasma is described. It is based on tetraphenylboron ion-pair extraction and reversed-phase ion-pairing liquid chromatography with amperometric detection. Tyramine can be reliably measured in the range 5-200 ng/ml with an absolute limit of detection of 0.50 ng/ml at a signal-to-noise ratio of 2.0. Correction for variable recovery is made by using a tritiated tyramine internal standard. This assay is suitable for studies on the bioavailability of ingested tyramine and should thus have a role in the development of safer monoamine oxidase inhibitor drugs.

Measurement of tyramine in human plasma, utilising ion-pair extraction and high-performance liquid chromatography with amperometric detection.

An assay for plasma tyramine has been developed which uses ion-pair extraction, reversed-phase ion-pair high-performance liquid chromatography and amperometric detection. Tritiated tyramine is used as the internal standard. The method can measure down to 0.5 ng/ml of tyramine in 1 ml of human plasma and is thus suitable for monoamine oxidase inhibitor studies involving oral dosing with tyramine.

Ascending brain-stem noradrenergic pathways modulate the renin response to haemorrhage.

Ventral noradrenergic projections (VNAB) from lateral tegmental (A1, A2) and dorsal noradrenergic projections (DNAB) from the coeruleal (A6) as well as A1 cell groups in the brain-stem of the rat were lesioned by intracerebral injection of 6-hydroxydopamine. A marked increase in plasma renin activity and adrenaline concentrations followed haemorrhage (0.5 ml/100 g body weight) in the sham-operated rats. VNAB-lesioned rats showed a similar response to the sham-operated controls, but in DNAB-lesioned animals the rise of plasma renin was markedly attenuated.

Determination of d-isoproterenol sulphate by high-performance liquid chromatography with amperometric detection.

The application of reversed-phase high-performance liquid chromatography (HPLC) to the determination of isoproterenol sulphate in human plasma and urine was investigated. Sulphoconjugation of the inactive isomer of isoproterenol was chosen as an experimental model to study individual variations in the rate of sulphation of phenols in man. This approach allowed the ingestion of relatively large amounts of drug and detection of the conjugated material after acid hydrolysis, using alumina clean-up and HPLC with amperometric detection. This method was found to be rapid, sensitive, precise and suited to pharmacokinetic studies in man.