Influence of a single oral dose of vitamin D(2) on serum 25-hydroxyvitamin D concentrations in tuberculosis patients.

SETTING: Newham Chest Clinic, London, UK. OBJECTIVE: To determine the safety and efficacy of the administration of bolus-dose vitamin D(2) in elevating serum 25-hydroxyvitamin D (25[OH]D) concentrations in tuberculosis (TB) patients. DESIGN: A multi-ethnic cohort of TB patients was randomised to receive a single oral dose of 2.5 mg vitamin D(2) (n = 11) or placebo (n = 14). Serum 25(OH)D and corrected calcium concentrations were determined at baseline and 1 week and 8 weeks post-dose, and compared to those of a multi-ethnic cohort of 56 healthy adults receiving an identical dose of vitamin D(2). RESULTS: Hypovitaminosis D (serum 25[OH]D < 75 nmol/l) was present in all patients at baseline. A single oral dose of 2.5 mg vitamin D2 corrected hypovitaminosis D in all patients in the intervention arm of the study at 1 week post-dose, and induced a 109.5 nmol/l mean increase in their serum 25(OH)D concentration. Hypovitaminosis D recurred in 10/11 patients at 8 weeks post-dose. No patient receiving vitamin D(2) experienced hypercalcaemia. Patients receiving 2.5 mg vitamin D(2) experienced a greater mean increase in serum 25(OH)D at 1 week post-dose than healthy adults receiving 2.5 mg vitamin D(2). CONCLUSION: A single oral dose of 2.5 mg vitamin D(2) corrects hypovitaminosis D at 1 week but not at 8 weeks post-dose in TB patients.

Non-extraction HPLC method for the simultaneous measurement of theophylline and caffeine in human serum.

A non extraction HPLC method is described for the simultaneous measurement of theophylline and caffeine in human serum using a Pinkerton ISRP column and u.v. detection at 275 nm. The method is suitable for therapeutic monitoring of theophylline levels in adults and, in particular, quantitation of both theophylline and caffeine in premature neonates where as little as 10 microL of sample can be used. Comparison of theophylline levels obtained by this method with EMIT analysis show a correlation coefficient of 0.97 (n = 37) in adults and 0.79 (n = 16) in premature neonates. There was no correlation between serum theophylline and caffeine levels in premature neonates receiving theophylline therapy. No interference was encountered from endogenous plasma components or other drugs in the 53 patients studied. Precision of the assays compares well with reported values for extraction HPLC and immunochemical analyses.

Use of a non-extraction HPLC technique for measuring angiotensin-converting enzyme under optimum conditions.

A non-extraction high performance liquid chromatography assisted method for the measurement of angiotensin-converting enzyme in serum has been developed. Serum containing the enzyme was incubated with the synthetic substrate, furylacryloylphenylalanylglycylglycine at a concentration approaching 10 times the apparent Km, prior to injection onto a shielded hydrophobic reverse phase high performance liquid chromatography column. This allowed the product to be separated and measured directly without the need for a time consuming protein extraction step. The method is enzymatically sound and avoids the methodological problems associated with automated kinetic assays.

A non-extraction HPLC method for the simultaneous determination of serum paracetamol and salicylate.

A direct injection HPLC method for the sumultaneous measurement of serum paracetamol and salicylate is described using a Pinkerton internal surface reversed-phase column with benzoic acid as internal standard. The method is linear to at least 1000 mg/L for both drugs and shows good precision at levels of 62-500 mg/L. None of the drugs tested for interference affected the quantitation of either drug. In patient samples, the values obtained with this method correlated well with those from enzymatic paracetamol and Trinder salicylate methods.

Detection of synthetic glucocorticoids by liquid chromatography-tandem mass spectrometry in patients being investigated for Cushing's syndrome.

BACKGROUND: We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection of four commonly prescribed steroid drugs (prednisolone, dexamethasone, betamethasone and beclomethasone dipropionate) while simultaneously measuring 24-h urine free cortisol and cortisone in patients. METHODS: Two hundred and fifty microlitre aliquots of urine were spiked with internal standard and extracted with dichloromethane. The MS instrument was operated with positive electrospray and multiple reaction monitoring. Two transitions were monitored for each analyte of interest and the ratio of the intensities of the product ion fragments was established. RESULTS: The LC-MS/MS method for the measurement of urine free cortisol and cortisone was established. The assay was linear up to 788 nmol/L for cortisol and 777 nmol/L for cortisone, with a limit of quantitation of 5.0 nmol/L for both. Analysis time per sample was seven minutes. Transitions for four synthetic glucocorticoids were included, and they were identified based on the ratio of the intensities of product ion fragments. Analysis of 219 samples collected from 154 patients (55 male and 99 female) revealed the presence of prednisolone in five samples from three patients. Dexamethasone was detected in samples from four patients, and betamethasone was detected in one sample. CONCLUSION: This is the first LC-MS/MS method in routine use to combine quantification of urinary cortisol and cortisone and detection of synthetic glucocorticoids in patients being investigated for Cushing's syndrome. Since the most common quoted cause of Cushing's syndrome is steroid treatment, this is a valuable diagnostic tool.

Direct serum injection high-performance liquid chromatographic method for the simultaneous determination of phenobarbital, carbamazepine and phenytoin.

A method is described for the simultaneous measurement of serum levels of three antiepileptic drugs, phenobarbital, phenytoin and carbamazepine, by direct injection high-performance liquid chromatography on a 25-cm Pinkerton internal surface reversed-phase (ISRP) column. Several commonly available compounds were tested and found not to co-chromatograph with the three drugs of interest or the internal standard, 5-(p-methylphenyl)-5-phenylhydantoin. Results obtained on patients' samples with this method compared well with those from enzyme-multiplied immunoassay technique (EMIT).

Measurement of human proinsulin by an indirect two-site immunoradiometric assay.

An indirect two-site immunoradiometric assay is described for the measurement of human proinsulin in plasma. Polyethylene tubes coated with purified guinea-pig antibodies to insulin were used to extract proinsulin and insulin from plasma. Rabbit antibody to human C peptide was then added to react with the C-peptide moiety of the bound proinsulin. The uptake of this antibody was measured by the subsequent binding of 125I-sheep antibody to rabbit IgG. The binding of radioactivity to the tubes was a function of the proinsulin concentration in the sample. The sensitivity of the assay was 0.006 pmol/ml. Only 200 microliters of plasma was required in the assay and the 125I-labelled antibody was produced from readily available reagents. The polyethylene tubes remained stable for at least 5 months after coating. The mean fasting proinsulin level was 0.009 pmol/ml in sixteen normal subjects and 0.025 pmol/ml in twelve maturity onset diabetics. Oral glucose produced an 8 fold increase in proinsulin concentration but a decline in the plasma proinsulin/insulin molar ratio. Four patients with insulinoma had extremely elevated proinsulin levels and proinsulin/insulin ratios.

Determination of proinsulin intermediates by means of an immunoradiometric method using polyclonal antibodies.

In our endeavour to develop a method for proinsulin determination, an immunoradiometric method was developed. Thereby guinea-pig antibodies to bovine insulin which were purified with an immunoadsorbent, were, in surplus, coupled to polyethylene tubes. These tubes were used to extract proinsulin and insulin from the sample and standard that or was to be determined. The proinsulin adsorbed onto the wall of the tube was distinguished from insulin by incubating, in a second step, a rabbit antibody to human C-peptide with the tubes. In order to render the proinsulin anti-C-peptide complex measurable, a donkey antibody to rabbit IgG was used, which had been purified via an immunoadsorbent and which was labelled with iodine-125. Since proinsulin extracted from human pancreata was available next to biosynthetic human proinsulin, it was striking to note that these substances were very differently recorded by the determination method applied. Thus biosynthetic human proinsulin dissolved in gelatin buffer could not be measured at all. After mild tryptic cleavage of the biosynthetic human proinsulin, a clear increase of the immunoreactivity was seen in this method. Therefore the claim could be made that partially cleaved proinsulin molecules with a retained C-peptide structure had come into existence. This could be verified by application of the proinsulin cleavage products 65/A1 and 32/33, which exhibited a behaviour very similar to that of pancreatic proinsulin in this method. In this way we could demonstrate that the originally planned immunoradiometric determination method for proinsulin, recorded partially hydrolized intermediates of the proinsulin, which represent a large part of the proinsulin immunoreactivity in the serum.

Effects of prosomatostatin on growth hormone and prolactin response to arginine in man. Comparison with somatostatin.

The effects of the hypothalamic 28 aminoacid peptide prosomatostatin (Pro-SS) on arginine-induced growth hormone (GH) and prolactin (PRL) release and blood glucose levels in man are compared with those obtained after an equimolar dose of somatostatin (SS). In comparison with SS, Pro-SS caused greater and more prolonged inhibition of GH release, a more marked reduction of the PRL response to arginine, and greater enhancement of the hyperglycaemic action of arginine. The greater potency and prolonged action of Pro-SS make it an interesting tool to study hormonal control mechanisms in a variety of physiological and pathological conditions.