PubMed Health⌕ Search

Biomedical subjects

R Knuppen

Publications and source records attributed to R Knuppen.

At least 91 records · Page 5Linked to original sources
PubMed1979

Radioimmunoassay of 2-hydroxyesterone and 2-methoxyestrone in human urine.

An assay for the quantitative determination of 2-hydroxyestrone and 2-methoxyestrone in human urine is described. The analytical procedure involves several purification steps: XAD-2 column chromatography of urine (1 ml), hot acid hydrolysis under reducing conditions, extraction with benzene/ethyl acetate, separation of monophenolic steroids from catecholestrogens by the formation of borate complexes, and partition between different solvents. Quantitation is achieved by radioimmunoassay using highly specific antibodies. For correction of procedural losses [6, 7-3H2] 2-hydroxyestrone and [6, 7-3H2] 2-methoxyestrone are used as internal standards. The method is highly specific as checked by comparison to a double-isotope-derivative method and a newly developed gas chromatography-mass spectrometry procedure. Using this method the urinary excretion of 2-hydroxyestrone and 2-methoxyestrone was studied in children, men, cycling, pregnant and postmenopausal women. Special interest was focussed on the molar ratios of 2-hydroxyestrone to 2-methoxyestrone and the so called "total estrogens" which vary markedly within the different groups investigated. The excretion of 2-hydroxyestrone is especially favoured in women during the menstrual cycle and pregnancy.

Adult↗
PubMed1979

Radioimmunoassay for 2-methoxyoestrone in human plasma.

A bovine serum albumin conjugate of 2-methoxyoestrone was used for the preparation of highly specific antibodies in rabbits. Cross-reactivity for catecholoestrogens and monophenolic steroids was below 0.3%. Only 2-methoxyoestradiol cross-reacted with 44%. An assay procedure for the determination of unconjugated and conjugated 2-methoxyoestrone in human plasma is described. The following mean plasma concentrations (pg/ml) were found (unconjugated/conjugated): children 61/1130, young men 74/1320, elderly men 109/1260, cycling women 131/1040, post-menopausal women 102/1420, and pregnant women 3980/5850.

Adolescent↗
PubMed1978

Radioimmunoassay of 2-hydroxyestrone.

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectometry. Using this antigen highely specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50-95 pg/ml), pregnant women (105-220 pg/ml), men (45-65 pg/ml) and children(20-40 pg/ml).

Adult↗
PubMed1978

Formation of 2- and 4-hydroxyestrogens by brain, pituitary, and liver of the human fetus.

When [6,7-3H]estradiol was incubated with tissue homogenates of the brain, the pituitary, and the liver of two human female fetuses, a number of radioactive metabolites more "polar" than the incubated substrate were detected. Among these, the identification of two types of catecholestrogens, i.e. the 2- and 4-hydroxyestrogens, was of major interest. Compared on the basis of wet weight of tissues (250 mg), the conversion of estradiol to 2-hydroxyestrogens (2-hydroxyestradiol and 2-hydroxyestrone) was 0.8% in the frontal cortex, 1.0% in the hypothalamus, 2.1% in the pituitary, and 7.8% in the liver. For the first time, the formation of 4-hydroxyestrogens was demonstrated. The percentages of incubated estradiol hydroxylated at C-atom 4 (4-hydroxyestradiol and 4-hydroxyestrone) were 0.5 in the cortex, 0.4% in the hypothalamus, .1% in the pituitary, and 0.5% in the liver. The results show that fetal brain and pituitary tissue can hydroxylate estradiol in positions 2 and 4 to a similar extent, whereas in the liver, about 15 times more 2-hydroxy than 4-hydroxy compounds are formed. Moreover, the 2-hydroxylating capacity of the liver is definitely greater than that of the brain, whereas the 4-hydroxylating capacity is about the same as that of the brain.

Brain↗
PubMed1978

Comparative studies on the metabolism of oestradiol in the brain, the pituitary and the liver of the rat.

Radioactive oestradiol was incubated with homogenates of hypothalamus, pituitary and liver and slices of cortex, hippocampus, hypothalamus, pituitary and liver of ovariectomized rats. 2- and 4-substituted oestrogens were the main metabolites in the brain and were found in the same proportions. In the liver only the 2-substituted oestrogens were the main metabolites, while the 4-substituted ones were of minor quantitative importance. On the basis of wet weight the 2-hydroxylating capacity of the liver was approximately 50 fold greater than that of the brain and the 4-hydroxylating capacity about the same as that of the brain. Only slight differences in the 2- and 4-hydroxylating capacity of various brain areas were observed, although apparently the hypothalamus and the pituitary were more active in the formation of catecholestrogens than the cortex and the hippocampus.

Animals↗
PubMed1977

Influence of rifampicin treatment on the metabolic fate of [4-14C] mestranol in women.

[4-14C] Mestranol was administered to 2 rifampicin treated and to 3 untreated hysterectomised women with normal liver functions. The urinary excretions of mestranol, ethynylestradiol, 2-hydroxy-ethynylestradiol and of the total radioactivity were measured within the following 5 days. After this period the total urinary excretion of radioactivity amounted to approx. 43% (38%-50%) of the administered dose and no difference was found for the rifampicin treated and untreated women. Moreover, in the urines of both of these groups the same amounts of radioactive mestranol (1.1%-6.3% of the urinary radioactivity) and 2-hydroxy-ethynylestradiol (0.9%-3.9% of the urinary radioactivity) were measured. In contrast, the urinary excretion of ethynyl-estradiol was definitely lower in the rifampicin treated women (3.5% and 4.9% of the urinary radioactivity) as compared to the control group (15%-23%).

Adult↗
PubMed1977

[Thin layer chromatography screening test for semiquantitative detection of 4-hydroxy-3-methoxy mandelic acid (HMMA) in urine without extraction (author's transl)].

A thin layer chromatography (TLC) screening test is described, which allows the semiquantitative determination of HMMA in urine samples. From the collected 24 hour urine a measured sample is applied without extraction to a special TLC plate, which is developed in one dimension. In comparison to a series of graded standard solutions the daily HMMA output can be estimated semiquantitatively. In 102 patients over 18 years of age, suffering from high blood pressure, the daily HMMA output was estimated by both the Pisano method (Pisano, J.J., Crout, J.R. & Abraham, D. (1962), Clin. Chim. Acta 7,285--291) and the TLC method. Coincident results with both methods were obtained in 99 of these cases. The residual 3 cases show pathological results with the Pisano method and results in the normal range or the warning range, respectively, with the TLC method. These cases can probably be classified as false positive results of the Pisano method. The TLC method is recommended as a screening test for estimating the daily HMMA excretion.

Chromatography, Thin Layer↗
PubMed1976

Convenient large scale preparation of catechol estrogens.

2-Hydroxyestrone, 2-hydroxyestradiol-17beta, 2-hydroxy-17alpha-ethynylestradiol, 2-hydroxyestriol, 4-hydroxyestrone, 4-hydroxyestradiol-17beta, 4-hydroxy-17alpha-ethynylestradiol and 4-hydroxyestriol are prepared on a preparative scale from the corresponding aminophenols using a new inverse oxidation procedure. By the synthesis described both the 2- and 4-hydroxylated estrogens are available in high yields.

Catechols↗
PubMed1976

Pryogalloloestrogens -- a new group of oestrogen metabolites.

After incubation of radioactive catecholoestrogen monomethyl ethers with rat liver slices the following well known metabolic pathways were observed: 1) demethylation, 2) 16alpha-hydroxylation, 3) oxidoreduction at C-atom 17, and 4) conjugation with glutathione, sulphuric acid and glucuronic acid. In addition, for the first time a further aromatic ortho-hydroxylation, leading to pyrogalloloestrogen derivatives, was detected. Thus, the incubation of 2-methoxyoestrone yielded 2,4-dihydroxyoestrone 2-methyl ether as the main metabolite of the lipophile fraction. Under the same conditions, 4-methoxyoestrone was converted to 2,4-dihydroxyoestrone 4-methyl ether and 2,4-dihydroxyoestradiol-17beta 4-methyl ether; these compounds were the quantitatively most important metabolites not only in the lipophile but also in the sulphate and glucuronide fractions. The identity of these new metabolic products was established by chromatography, microchemical reactions and recrystallisation to constant specific radioactivity.

Animals↗
PubMed1976

Pyrogalloloestrogens: enzymic methylation of pyrogalloloestrogens and interaction of pyrogalloloestrogens with the enzymic methylation of catecholamines in rat liver in vitro.

During incubation of 2,4-dihydroxyoestrone with the 105000 X g supernatant of rat liver in the presence of S-adenosyl-[Me-14C]methionine, the formation of radioactive mono- as well as dimethyl ether derivatives was demonstrated. The products were identified as: 2,4-dihydroxyoestrone 2-methyl ether, 2,4-dihydroxyoestrone 3-methyl ether, 2,4-dihydroxyoestrone 4-methyl ether, 2,4-dihydroxyoestrone 2,3-dimethyl ether, 2,4-dihydroxyoestrone 2,4-dimethyl ether and 2,4-dihydroxyoestrone 3,4-dimethyl ether. The monomethyl ethers were the main products; within this group the 3-methyl ether of 2,4-dihydroxyoestrone was the main metabolite. Among the dimethyl ether derivatives, the 2,4-dihydroxyoestrone 2,3-dimethyl ether represented the quantitatively most important product. When 2,4-dihydroxyoestrone 2-methyl ether was incubated under the same conditions, 2,4-dihydroxyoestrone 2,3- as well as 2,4-dimethyl ether was formed. The 2,3-dimethyl ether was again the main metabolite. The incubation of 2,4-dihydroxyoestrone 4-methyl ether yielded the 2,4- and 3,4-dimethyl ethers, the first being the main product. In contrast, the 3-methyl ether of 2,4-dihydroxyoestrone was not further methylated by the catechol methyltransferase preparation. In further experiments, the effect of the pyrogalloloestrogen and its monomethyl ether derivatives on the enzymatic methylation of catecholamines was investigated. It was demonstrated that the methylation of adrenalin and dopamine was competitively inhibited by 2,4-dihydroxyoestrone and the 2,4-dihydroxyoestrone monomethyl ethers. Only a weak inhibitory effect was observed with the 3- and 4-monomenthyl ethers (Ki values 200 and 160muM). The unsubstituted pyrogalloloestrogen produced a marked inhibition (Ki value 50muM), but the strongest inhibition was found with the 2-monomethyl ether of 2,4-dihydroxyoestrone (Ki value 14muM). The extent of inhibition caused by the addition of the 2-monomethyl ether of 2,4-dihydroxyoestrone was thereby in the same range as the inhibition caused by pyrogallol and the catecholoestrogens.

Animals↗
PubMed1975

The excretion of 2-hydroxyestrone during the menstrual cycle.

A double isotope derivative determination technique was developed to investigate the excretion of 2-hydroxyestrone inhuman menstrual cycle. The method is highly specific, accurate and precise within the range of 0.2-20 nmol 2-hydroxyestrone/20ml of urine. The amounts of 2-hydroxyestrone excreted during menstrual cycle varied between 10 (proliferation phase) and 60 mug/24 h urine (ovulatory peak), which is comparable to that of estriol, supposed hitherto to be the main excretion product of estrogen metabolism.

Estrone↗