Vitamin D and its metabolites in human breast milk.
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Animals↗
Biomedical subjects
H L Makin
Publications and source records attributed to H L Makin.
The quantitative estimation of bile acids and their conjugates in human biological fluids.
This review attempts to provide a concise and critical summary of modern methods for the analysis of bile acids and their conjugates in human biological fluids. Most emphasis is given to more up-to-date procedures that have been applied to the study of human disease and attention is drawn to previous reviews in areas that have not been covered here. An increasing awareness of the possibility that bile acids may be involved in the etiology of a number of disorders, or that such disorders may give rise to changes in bile acid concentration, has stimulated the study of bile acid methodology. Although many procedures have been described using, for example, high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), gas-liquid chromatography--mass spectrometry (GLC-MS), and radioimmunoassay (RIA), no simple but comprehensive procedure for the estimation of bile acids and their conjugates has yet been published. Further study in this area is still required in order to establish the role of bile acid estimations in the routine diagnosis and treatment of disease.
Bile Acids and Salts↗
A rapid and simple method for the measurement of plasma 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 using Sep-Pak C18 cartridges and a single high-performance liquid chromatographic step.
A simple one step high-performance liquid chromatographic (HPLC) procedure for the analysis of plasma concentrations of 25-hydroxyvitamin D3 (25-OHD3) and 25-hydroxyvitamin D2 (25-OHD2) is described. Plasma (2-4 ml) was extracted with methyl cyanide which was passed through a Sep-Pak C18 cartridge, washed with methanol:water (70:30, v/v) and the 25-OHD fraction eluted with methyl cyanide. After isomerisation to their isotachysterol derivatives, the secosteroids were estimated using a straight-phase HPLC system, monitoring the eluent at 301 nm. Radioactive 25-OHD3, added to plasma at the start of the procedure, was used to correct for losses. Recovery of added 25-OHD3 was quantitative and values obtained using this method were similar to those obtained on the same plasma samples using a mass fragmentographic technique. Normal ranges were similar to those described by other workers and within- (5.8% for 25-OHD3) and between-(8.0% for 25-OHD3) batch reproducibilities were satisfactory.
25-Hydroxyvitamin D 2↗
A semi-automated computerised gas-liquid chromatographic system for urinary steroid profile analysis.
A simple gas-liquid chromatographic method for the measurement of some of the major steroids in urine is described. The system uses conventional packed columns, automatic injection and is linked to an integrator. Calculations are all carried out by computer. The system is recommended as a simple screening procedure, producing a urinary steroid 'profile', which can process large numbers of urine samples cheaply and quickly, enabling patients worthy of more intensive investigation to be identified.
17-Hydroxycorticosteroids↗
High-performance liquid chromatography of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma. Use of isotachysterols and a comparison with gas chromatography--mass spectrometry.
A high-performance liquid chromatographic (HPLC) method for estimating plasma 25-hydroxyvitamin D2 (25-OHD2) and 25-hydroxyvitamin D3 (25-OHD3) is described. The method involves plasma extraction, Lipidex 5000 chromatography and HPLC on straight-phase Zorbax-SIL, collecting the 25-OHD2 + 25-OHD3 fractions. These secosteroids are isomerised to their isotachysterol derivatives and re-run in the same HPLC system, monitoring at 290 nm. 3H-Labelled 25-OHD3 is used as an internal standard. The method was evaluated in terms of reproducibility, and recovery of added secosteroids was quantitative. Values obtained using this method were in close agreement with those values obtained on the same plasma sample using gas chromatography--mass spectrometry.
25-Hydroxyvitamin D 2↗
A rapid and simple method for the esterification of fatty acids and steroid carboxylic acids prior to gas-liquid chromatography.
A simple procedure for the esterification of steroid carboxylic acid groups using an acetyl chloride/alcohol reagent has been carefully evaluated. The ease with which these esters can be formed using this reagent depends on the acid and the ester required. C24 bile acids form methyl esters using acetyl chloride/methanol (1:20, v/v) almost instantaneously at 50 degrees C, whereas the C20 aetienic acid studied requires 2 h at 50 degrees C. The formation of isopropyl esters of 5 beta-cholanic acid takes longer (4 h at 50 degrees C) than the formation of n-propyl esters (1 h at 50 degrees C). The identity of various esters fo cholanic acids formed using this reagent have been checked by gas chromatography-mass spectrometry and various other procedures used for the methylation of fatty acids have been examined to establish whether they can be used for the quantitative esterification of bile acids.
Carboxylic Acids↗
The estimation of vitamin D and its metabolites in human plasma.
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24,25-Dihydroxyvitamin D 3↗
The in vivo metabolism of Althesin (alphaxalone + alphadolone acetate) in man.
Gas chromatograph-mass spectrometry has been used to study the metabolism of Althesin (alphaxalone and alphadolone acetate) in man. Two metabolites, 20 alpha-reduced alphaxalone and alphadolone, as well as the two parent steroids, have been detected in the plasma during and after an infusion of Althesin. The main urinary metabolites were alphaxalone and 20 alpha-reduced alphaxalone, with smaller amounts of alphadolone, all of which were excreted mainly as the glucuronide conjugates. No alphadolone acetate was detected in the urine. In 3 patients in whom bile was collected over the 1st 24 h post-operation from an indwelling T-tube, it has not been possible using the methods described to detect in bile the presence of the parent steroids, or any of their probable metabolites.
Adult↗
High-performance liquid chromatography of vitamin D: enhanced ultraviolet absorbance by prior conversion to isotachysterol derivatives.
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Chromatography, High Pressure Liquid↗
The estimation of vitamin D and some metabolites in human plasma by mass fragmentography.
A mass fragmentographic assay for vitamin D3, 25-hydroxyvitamin D3, 25-hydroxyvitamin D2 and 24,25-dihydroxyvitamin D3 is described. After extraction of plasma with methanol and dichloromethane, Lipidex 5000 chromatography was used to separate the plasma extract into three fractions (a vitamin D fraction, a 25-hydroxyvitamin D fraction and a 24,25-dihydroxyvitamin D fraction). Cholesterol was removed from the vitamin D fractio by thin-layer chromatography. :After addition of vitamin D2 to each fraction, isotachysterol trimethylsilyl ethers were formed. Recoveries prior to gas chromatography-mass spectrometry were assessed by the use of tritiated standards added to the plasma before extraction. Using this procedure, normal ranges were established (vitamin D3: 3--17.7, 25-hydroxyvitamin D3: 5.9--35.2, 25-hydroxyvitamin D2: 0.6---1.0 and 24,25-dihydroxyvitamin D3: 0.6--2.9 micrograms/l).
24,25-Dihydroxyvitamin D 3↗
A new procedure for the formation of isotachysterol derivatives of subnanomole quantities of ergocalciferol, cholecalciferol and its metabolites prior to gas liquid chromatography.
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Cholecalciferol↗
Use of hydroxyapatite in radioimmunoassay.
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Animals↗
Plasma concentrations of alphaxalone by gas chromatography: comparison with other gas chromatographic methods and gas chromatography-mass spectrometry.
Existing methods for the measurement of plasma or tissue alphaxalone concentrations by gas chromatography are not wholly satisfactory because of the problems of interfering peaks and detector contamination by the silylation reagent. This paper describes an alternative gas chromatographic method using the nitrogen selective alkali flame ionization detector. Plasma samples from patients given Althesin (alphaxalone-alphadolone acetate) were extracted with light petroleum. O-Methyl oximes were formed using methoxyamine hydrochloride, and the O-methyloxime-3-acetate prepared by heating the extract with acetic anhydride-pyridine. Gas liquid chromatography was carried out on a column of 3% OV 17 ON Celite 545 (mesh 80-100), initial temperature 235 degrees C and increasing by a degree C min-1. Comparison has been made of the O-methyl oxime acetates with trimethylsilyl ethers using both gas chromatography and gas chromatography-mass spectrometry. The results of the two methods were not significantly different. The coefficient of variation for the O-methyloxime-3-acetates at a plasma concentration of 1.09 microliter-1 was 6.9%. This method has an improved sensitivity and selectivity over the existing method, and allows determination of the plasma alphaxalone concentrations found in patients receiving Althesin-supplemented anaesthesia.
Chromatography, Gas↗
Defective cortisol binding globulin affinity in association with adrenal hyperfunction: a case report.
A patient with an apparent abnormality of cortisol binding globulin (CBG) is reported. Investigations showed that while the plasma CBG binding capacity (equivalent to the plasma CBG concentration) was normal, the binding affinity was reduced by a factor of four. The significance of this observation is discussed with regard to its possible role in the development of the adrenocortical hyperfunction also seen in this patient.
Adrenocortical Hyperfunction↗
A rapid and specific method for the estimation of glucose using and oxygen electrode and simple differentiating circuit.
A simple differentiating circuit is described which, together with an oxygen electrode assembly, provides a rapid (20 sec) and cheap system for the estimation of glucose in body fluids using a sample volume of 20 microliter. Response was linear over the range 0-22 mmol/l. The complete system has been evaluated using plasma and whole blood. "Within-day" precision on control plasmas over the concentration range 2.73-8.33 mmol/l lies between 2.0 and 3.3% (C.V., n = 23); "day-to-day" precision was 4.6% (C.V., n = 23). On whole blood, to which nitrate had been added, precision was 4.5% (C.V., n = 15); precision using standard glucose solutions was never greater than 3.3% (C.V., n = 15) over the concentration range 1.1-22.2 mmol/l. Recoveries of added glucose to plasma and whole blood were greater than 90%. Good correlation between glucose values obtained by the oxygen electrode system described here and an automated system were obtained for plasma (r = 0.9902, n = 35) and whole blood (r = 0.9610, n = 35), although whole blood gave values on average 27% lower than plasma. The use of the system for the measurement of plasma cholesterol is also demonstrated.
Blood Glucose↗
Effect of anticonvulsants on plasma testosterone and sex hormone binding globulin levels.
Plasma sex hormone binding globulin (SHBG) and testosterone levels were measured in 29 patients with epilepsy (16 men and 13 women), most of them on chronic therapy with anticonvulsant drugs. Sex hormone binding globulin concentrations were increased in both sexes and testosterone levels in male patients. It is postulated that anticonvulsants may induce hepatic synthesis of SHBG.
Anticonvulsants↗
Male pseudohermaphroditism due to partial end-organ insensitivity to androgens.
A 46XY individual with incomplete male pseudohermaphroditism was studied. The phenotype was distinctive, with a masculine habitus despite a small phallus, gynaecomastia and diminished virilization. Plasma levels of testosterone, oestrogens, gonadotrophins and sex-hormone binding globulin were elevated. It is proposed that this syndrome is a variant of Type I incomplete male pseudohermaphroditism.
Adult↗
Hydroxyapatite - a reagent for the separation of free and antibody-bound steroid during the radioimmunoassay.
The use of hydroxyapatite to absorb antibody-bound steroid and thus separate free and antibody-bound steroid during radioimmunoassay has been examined using three steroid antisera (to testosterone, to 17-hydroxyprogesterone and to estradiol-17beta). For all three antisera studied the separation was shown to be independent of length of time in contact with hydroxyapatite (up to 1h); temperature variations from 4 degrees -37 degrees and pH over the range 4.9-8.0. The presence of protein affected the absorption of antibody-bound steroid but this effect could be overcome by the addition of increasing amounts of hydroxyapatite. Further increase in the amount of hydroxyapatite added had no effect on the separation of free and bound steroid. Sodium phosphate buffers of molarity greater than 0.01M eluted antibody-bount steroid from hydroxyapatite, but Tris-HC1 buffers up to molarities of 0.1 M had no effect. Hydroxyapatite when used as a dry powder had the same effects as suspensions. No effect on the cross-reactivities of the antisera used could be demonstrated when hydroxyapatite was used and plasma testosterone assays on 22 plasma samples using hydroxyapatite gave essentially the same results as assays on the plasma using a coated-tube assay. Hydroxyapatite can also be successfully pumped along small bore plastic tubing without settling and can thus be used in automated immunoassay systems.
Estradiol↗