Olle Lantto

Gradients of monoamine metabolites and cortisol in cerebrospinal fluid of psychiatric patients and healthy controls

Amine metabolites and cortisol were measured in four consecutive 3 ml samples of lumbar cerebrospinal fluid (CSF). There were pronounced concentration gradients for both 5-hydroxyindoleacetic acid and homovanillic acid, and there was no difference in the gradients of 11 controls and 17 psychiatric patients (10 depressed). As there was a close correlation between the mean of the first two fractions and the mean of all four fractions, a concentration obtained in 6 ml can be used to calculate that in a 12 ml CSF sample. In psychiatric patients there was a slight, but statistically significant concentration gradient for 3-methoxy-4-hydroxyphenylglycol (unconjugated as well as conjugated). No gradient was found for cortisol in CSF.

Plasma cortisol determination by mass fragmentography

Abstract A reference method for the estimation of plasma cortisol with high accuracy and specificity is described. A fixed amount of [4- 14 C] cortisol (usually 0.2 μg) is added to a fixed amount of plasma (usually 2 ml) and the mixture is extracted with dichloromethane. The extract is treated with methoxylamine hydrochloride and trimethylsilylimidazol to yield the dimethoxime-tri-trimethylsilyl derivative of cortisol. The amount of unlabeled cortisol is determined from the ratio between the recordings at m / e 605 and m / e 607 obtained after analysis with a combined gas Chromatograph—mass spectrometer equipped with an MID unit (multiple ion detector). The two ions used correspond to the M-31 peak in the mass spectrum of unlabeled and (4- 14 C) labeled cortisol, respectively. The relative standard deviation of the method is about 3.5%. The method was compared with a fluorometric method and a competitive protein binding method used in routine analysis. The results indicate that the competitive protein binding technique is to be preferred in routine work.

Studies on the ethanol-induced decrease of fatty acid oxidation in rat and human liver slices.

Abstract In order to elucidate the mechanisms involved in the acute ethanol-induced liver triglyceride accumulation, the oxidation, esterification and β-keto acid formation have been studied in rat and human liver slices after incubation with albumin bound, long chain fatty acids (palmitic. oleic and linoleic acids). The addition of alcohol to rat and human liver slices depressed the formation of 14 CO 2 from palmitic acid-1- 14 C, oleic acid-1- 14 C and linoleic acid-1- 14 C. The esterification to triglycerides and phospholipids was increased and the formation of β-keto acids was decreased by alcohol. Addition of 4-methylpyrazole, an inhibitor of liver alcohol dehydrogenase, almost prevented the alcohol effect on the lipid metabolism of the liver slices. The oxidation of alcohol is thus obligatory for the decreased β-oxidation of fatty acids, the increased esterification and for the decreased formation of β-keto acids. The results suggest that ethanol oxidation inhibits β-oxidation of fatty acids and that this primary effect leads to accumulation of liver triglycerides by increased esterification of plasma free fatty acids.

Validation of routine methods for serum progesterone determination using mass fragmentography

A mass fragmentographic reference method for determination of serum progesterone is described. A fixed amount of [4-14C]progesterone (usually 7.5 ng) is added to a fixed amount of serum (usually 1 ml) and extracted with hexane. The extract is purified by means of thin-layer chromatography. The purified progesterone is converted into the dienol heptafluorobutyrate derivative by treatment with heptafluorobutyric acid anhydride. The amount of unlabeled progesterone is determined from the ratio between the recordings at m/e 510 and m/e 512 obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). The two ions used correspond to the molecular peak in the mass spectrum of the dienol heptafluorobutyrate derivative of unlabeled and labeled progesterone, respectively. The relative standard deviation of the method was 3.7% provided that serum progesterone concentration was in the range 10-70 nM. Two radioimmunoassay techniques with commercial kits were compared with the mass fragmentographic method. The correlation coefficient obtained was 0.93 and 0.76 respectively, and the regression coefficient 0.93 and 1.05, respectively.

Serum testosterone determination by mass fragmentography.

A mass fragmentographic reference method for the determination of plasma or serum testosterone is described. A fixed amount of [4--14C]testosterone (usually 10 ng) is added to a fixed amount of serum (usually 1 ml) and extracted with ether. The ether extract is purified by means of thin-layer chromatography. The purified testosterone is converted into the di-trimethylsilyl derivative by treatment with trimethylsilylimidazole. The amount of unlabeled testosterone is determined from the ratio between the recordings at m/e 432 and 434, obtained after analysis with a combined gas chromatograph-mass spectrometer equipped with a MID-unit (multiple ion detector). The two ions used correspond to the molecular peak in the mass spectrum of unlabeled and 4--14C-labeled testosterone, respectively. The relative standard deviation of the method was about 2.7 percent. The method was compared with a radioimmunoassay technique. There was a good correlation and the regression coefficient was about 0.83. A diurnal rhythm in testosterone secretion was confirmed both with the mass fragmentographic technique and with radioimmunoassay.

Simultaneous determination of prednisolone and cortisol in serum by HPLC and by isotope dilution-mass spectrometry

A reference method for the simultaneous assay of cortisol and prednisolone by isotope dilution-mass spectrometry has been developed. A fixed amount of 2H4-labelled cortisol is added to a fixed amount of serum. The mixture is then extracted, converted into methoxime-trimethylsilyl derivative, and subjected to analysis by combined gas chromatography-mass spectrometry. The ions at m/e 603, m/e 605 and m/e 609 are followed through the gas chromatography. These ions correspond to the M-31 ions in the mass spectrum of methoxime-trimethylsilyl ether derivative of prednisolone, unlabelled cortisol and 2H4-cortisol, respectively. With the use of a standard curve, the concentration of prednisolone can be calculated from the ratio between the peak at m/e 603 and the peak at m/e 609 and the concentration of cortisol can be calculated from the ratio between the peak at m/e 605 and the peak at m/e 609. The coefficient of variation was about 2% with respect to determination of prednisolone and about 3% with respect to determination of cortisol. This method was compared with a routine method based on high-performance liquid chromatography (HPLC) The coefficient of variation was 2-3% with respect to determination of cortisol and 1-3% with respect to determination of prednisolone. There was a good correlation between the isotope dilution method (x) and the HPLC method (y). In the assay of serum prednisolone the regression analysis gave the following equation: y = 1.02x -69 nmol/l, r = 0.99. In the assay of serum cortisol, the regression equation was: y = 1.07x-14 nmol/l, r = 0.95. It is concluded that the HPLC-method, used for routine determination of cortisol and prednisolone, has an accuracy sufficient for its intended use.

Detection and quantitation of methandienone (dianabol®) in urine by isotope dilution—mass fragmentography

Abstract A highly accurate method has been developed for detection and quantitation of methandienone (Dianabol) in urine. A suitable internal standard containing a CD3-group in the 17α-position was synthesized. A fixed amount of this internal standard was added to a fixed amount of urine and the mixture treated with Helix pomatia. After extraction, treatment with acetic acid anhydride, and purification by preparative thin-layer chromatography, the purified extract was converted into the methoxime-trimethylsilyl derivative and subjected to combined gas chromatography—mass spectrometry. Unlabeled methandienone could be quantitated from the ratio between the tracings of the molecular ions at m/e 401 and m/e 404. Under the conditions employed, methandienone could be identified and determined in a concentration exceeding 3 ng/ml of urine. The compound could be detected and quantitated for several days after ingestion of a single dose of 10 mg of methandienone. In preliminary experiments the method has been used on urine samples obtained from athletes in connection with a competition. Some of the results are presented. In combination with a positive radioimmunoassay test, the present method should give sufficient evidence that an athlete is guilty of ingesting this steroid.

Detection and quantitation of stanozolol (Stromba®) in urine by isotope dilution-mass fragmentography

Abstract A highly specific and sensitive method for the analysis of stanozolol (Stromba®) in urine, based on isotope dilution-mass spectrometry, has been developed. A fixed amount of deuterium labelled stanozolol was added to a fixed quantity of urine and the mixture was treated with Helix pomatia digestive juice. The hydrolysate was extracted with diethyl ether under alkaline conditions and the extract subjected to preparative thin-layer chromatography. The purified extract was treated with propylene oxide in order to alkylate the secondary amine in the heterocyclic ring. The material was then converted into the TMS ether derivative and subjected to gas chromatography-mass spectrometry. Unlabelled stanozolol was quantitated by selected monitoring of the ions at m/e 414 and at m/e 417. Under the conditions employed, stanozolol could be detected and quantified above a level of about 2 ng/ml of urine. After a single dose of 7.5–10 mg to human volunteers, stanozolol could be detected in urine for the next 2–3 days. The method had about the same sensitivity as the radioimmunoassay method commonly used and should be of value for the detection of the inappropriate use of the drug by athletes.

Determination of 4-methylpyrazol in serum with mass fragmentography☆

Abstract A mass fragmentographic method for determination of 4-methylpyrazol in serum is described. 4-Methylpyrazol with three atoms of deuterium in the methyl group is added to a fixed amount of serum as internal standard. After extraction of the serum with ether, the amount of unlabeled 4-methylpyrazol is determined from the ratio between the recordings at m e 82 and m e 85 obtained after analysis with a combined gas chromatograph—mass spectrometer equipped with a MID-unit (multiple ion detector). With this method 4-methylpyrazol could be determined in concentrations down to about 0.1 μg/ml corresponding to about 1 μM. The relative standard deviation of the method in the range 4–21 μM was less than 2%. The method could be used for determination of disappearance rate of 4-methylpyrazol in experimental animals as well as humans.

Determination of urinary estriol by radioimmunoassay. Modification of a commercial kit for serum analyses.

A simple and rapid method for urinary estriol determination is described. Modification of a commercially available kit for serum estriol gives a method for urinary estriol determination with a capacity of approximately 75 determinations a day. A high degree of accurary was revealed by indirect comparison with a mass fragmentographic method.