Unconjugated Fraction Research Articles

MEASUREMENT OF THYROXINE AND TRIIODOTHYRONINE IN HUMAN URINE

Urinary excretion of unconjugated Summary thyroxine (T 4) was 2·0 μg. per day (mean) in thirty-six normal subjects. The renal clearance of serum unbound T4 was 26 ml. per minute (mean) in ten subjects. Hydrolysis of conjugated T4 in urine, however, yielded a further 2·8 μg. per day (mean) by enzyme hydrolysis or 3·7 μg. per day by acid hydrolysis. Mean figures for percentages of unconjugated and conjugated thyroxine were 39% and 61% of total T4, respectively. The unconjugated fraction should reflect the prevailing serum level of unbound T4, and be a useful thyroid-function test. Urinary immunoassayable triiodothyronine (T3) was 0·8 μg. per day (mean) in thirty-eight normal subjects. This fraction was 52% (mean) of the total T3, which was no more than 1·4 μg. per day (mean), in nine subjects. It is probably close to the amount of unconjugated T3. Renal clearance of serum unbound T3 seemed to be greater than glomerular filtration-rate in some cases, raising the possibility of tubular excretion of T3.

Metabolism of ethynyloestradiol in man.

SUMMARY The metabolism of 17α-ethynyloestradiol was studied in one man and 16 women. After intravenous or oral administration, 22·6–46·9% of the dose was excreted in the urine over a 5-day period. Up to 36% of the urinary radioactivity may be present in the unconjugated form with a further 64% being extractable after enzymic hydrolysis. Sulphate conjugates formed only 11·4% of the urinary radioactivity. Semi-quantitation of these extracts showed ethynyloestradiol itself to be the major metabolite, accounting for 3·2–8·4% of the administered dose in the unconjugated fraction with a further 2·4–16·5% in the enzyme hydrolysed fraction. Up to 16·5% of the dose was therefore excreted unchanged, and metabolites less and more polar than ethynyloestradiol were also found. Excretion via the faeces accounted for up to 30% of the administered dose, with most of the extractable radioactivity being in the unconjugated form. Ethynyloestradiol accounted for up to 29·6% of faecal radioactivity. No significant amounts of radioactivity were detected in sweat collected from one subject 2 or 24 h after administration of the dose. Plasma levels of 3·32% and 1·72% of the administered dose per litre were found 1 and 24 h after administration. Metabolites in plasma were present mainly as sulphate conjugates.

Metabolism of 14C-estradiol-17 injected intravenously into the pregnant baboon (Papio cynocephalus).

After 14C—estradiol–17β was injected into a hand vein of a pregnant baboon in the 157th day of gestation, the following steroids were isolated from the maternal urine: estrone and estradiol from the unconjugated fraction, estrone, 2–methoxyestrone, estradiol, 15α–hydroxyestradiol, 16–ketoestradiol and estriol from the sulfate fraction, and estrone, 2–methoxyestrone, 16α–hydroxyestrone, estradiol, 16–ketoestradiol and estriol from the glucosiduronate fraction. Estrone was the most abundant estrogen in all three fractions. From the metabolites isolated it can be concluded that estradiol can be converted by the pregnant baboon to estrone and both estrone and estradiol–17β can undergo hydroxylations and conjugation. These data fit well with the observations that estradiol–17β is the most abundant estrogen found in the placenta of the baboon and estrone is found in the largest amount in pregnancy urine. (Endocrinology 91: 523, 1972)

Metabolism of 1,2-3H-cortisol perfused through human liver in vivo.

ABSTRACT The liver of a 38-year-old man was injected via the portal vein with 100 μc-1,2-3H-cortisol over a period of 1 minute. Blood samples were withdrawn via the liver vein with a heart catheter at 1-, 3-, 5-, 10- and 20-minute intervals. Metabolism of cortisol was rapid (at 5 min.). The glucuronides represented 20% of the recovered metabolites. Cortols, cortolones, tetrahydrocortisone and tetrahydrocortisol predominated in this fraction. Only the ring-A reduced metabolites were found in the conjugate fraction, indicating a limitation by the Δ4-3-keto-reducing system. There appeared to be an influence by the oxygen at C-11 on the configuration of the 20-hydroxyl. There were a considerable number of metabolites in the unconjugated fraction. Cortisol, cortisone and their 20-reduced analogues, as well as the 6-hydroxylated derivatives, were in this portion. Five 11-oxygenated-17-ketosteroids were also indicated. The sulfates appeared in such low concentration that their analysis was not attempted.

Secretion rate and metabolism of the sulfates of cortisol and corticosterone in newborn infants.

3H-cortisol and 14C-cortisol sulfate, and 3H-corticosterone and 14C-corticosterone sulfate, were injected intravenously to human newborns aged 2 days. From 49 to 79% of 3H and from 62 to 100% of 14C were accounted for in their urine. Of the total amount of tritium present in the urine only small proportions were extractable. The amounts extracted in both the sulfate (3–18%) and glucuronide (1.3–13%) fractions were substantially greater than in the unconjugated fraction. Almost negligible amounts of 14C appeared in either the unconjugated or glucuronide fractions. The sulfate fraction consisted of 2 main components: the sulfates of cortisol and corticosterone, which were devoid of tritium; and a highly polar fraction which contained both 14C and most of the tritium extractable in this fraction. From the specific activities of tetrahydrocortisone and tetrahydro-11-dehydrocorticosterone purified from the glucuronide fraction, and from those of the sulfates of cortisol and corticosterone, the secretion rate of cortisol was calculated as ranging from 12.6 to 16.4 mg/m2/day; that of corticosterone from 4.6 to 18.3; those of their respective ester sulfates: 0.12–0.35 and 1.8–5.4. The findings indicate that cortisol and corticosterone are not sulfurylated prior to being metabolized; that a relatively substantial amount of their metabolites may be excreted as sulfate esters; and that corticosterone and its sulfate may represent very significant secretory products of the adrenal in the human newborn.

Determination of conjugated and unconjugated bilirubin by methods based on direct spectrophotometry and chloroform-extraction. A reappraisal.

The direct spectrophotometry method (15) for determination of conjugated and unconjugated bilirubin is seriously sensitive to hemoglobin admixture. In samples without hemolysis both the direct spectrophotometry method and the chloroform-extraction method (16) include a bilirubin conjugate (most probably bilirubin monoglucuronide) in the unconjugated fraction. The effect of pH on the partition of this bilirubin conjugate between a water and a chloroform phase is studied.

The metabolism of betamethasone.

SUMMARY The metabolism of [3H]betamethasone has been studied in normal subjects and in patients receiving high therapeutic doses of the steroid. About 70% of the dose was excreted in the urine in 48 hr.; 15–30% of the dose was found in the unconjugated fraction. Six metabolites as well as unchanged betamethasone were isolated and identified by paper chromatography using reference compounds prepared from [3H]betamethasone. The transformations were: oxidation of the 11β-hydroxyl group, hydroxylation at the 6β-position, reduction of the carbonyl group at C-20 and removal of the side chain to give 17-oxosteroids.

Oestrogens in the plasma of the sheep foetus.

Oestradiol-17α is the only oestrogen which has been identified in sheep foetal tissues (Velle, 1963). Oestrogens have been detected in blood (Skrzeczkowski, Dobrowolski & Domanski, 1967) and urine (Fevre & Rombauts, 1966) of the pregnant ewe. The oestrogen values reported have been very low relative to those in man. In contrast, this paper reports very considerable oestrogen concentrations in the plasma of the sheep foetus. Blood was drawn by heart puncture from foetuses within about 15 min. of slaughtering ewes at the city abbatoirs. The blood was collected into ice-cold heparinized bottles, centrifuged, and the plasma stored at −15° until analysed. Gestational ages were estimated from crown-rump lengths. To determine the oestrogen conjugates present, plasma was extracted with ether ('unconjugated fraction'), then incubated with molluscan β-glucuronidase (Koch-Light) in the presence of sodium dihydrogen phosphate to inhibit sulphatases (Dodgson & Spencer, 1953), and re-extracted with ether to give the 'glucosiduronate

Excretion of Unconjugated Bilirubin in the Bile of Gunn Rats

Conjugation is a generally accepted prerequisite for biliary excretion of bilirubin, but small amounts of unconjugated bilirubin regularly are identifiable in bile. This unconjugated pigment fraction usually is present in the bile together with much larger amounts of conjugated bilirubin suggesting a relationship in excretion between these two pigment types. Data are presented which suggest that the biliary excretion of unconjugated bilirubin is enhanced approximately 30-fold by the concomitant forced excretion of conjugated pigment in Gunn rats. Complex formation between the two pigment types (“bilirubin monoglucuronide”) is postulated to explain this phenomenon.

6β-hydroxycortisol metabolism in man

6β-hydroxycortisol (6β-0H-F) metabolism was studied by i.v. administration of 1,- 3H-6β-0H-F to a normal male. Of the excreted radioactivity, 80% was found in the unconjugated fraction and there was no evidence for conjugation with either glucuronic acid or sulfuric acid. The principal urinary steroid was 6β-OH-F and constituted about half the radioactivity present. Reduction of the 20-ketone to yield the 20β-dihydro derivative of 6β-OH-F and oxidation of the 11β-hydroxyl group to give 6β-hydroxycortisone (6β-0H-E) proved to be minor metabolic transformations. The presence of several other, as yet unidentified, minor urinary metabolites of 6β-OH-F has also been noted.

In vivo 6β-hydroxylation of 20β-dihydrocortisol

Following the intravenous administration of l,2- 3H-20β-dihydrocortisol (20β-dihydro-F) to a patient with Cushing's Syndrome, 6β,11β,17α,20β,21-penta-hydroxypregn-4-ene-3-one (20β-dihydro-6β-OH-F) was isolated from the unconjugated fraction of the urine. The metabolite was identified by comparison of its mass spectrum and Chromatographie properties with those of authentic 20β-dihydro-6β-OH-F. Specific activity determinations using 1- 14C-acetic anhydride have demonstrated that 20β-dihydro-6β-OH-F is not solely derived from utilization of circulating 20β-dihydro-F.

The in vivo transformation of cortisol to 1β-hydroxytetrahydrocortisone

Following the oral administration of a tracer dose of 1,2–3H-cortisol to a patient with Cushin's syndrome, radioactive 1β-hydroxytetrabydrocortisone (1β-OH-THE) was isolated from the unconjugated fraction of the urine. The metabolite was identified by the comparison of its chromatographic properties, infrared and mass spectra with those of authentic 1β-OH-THE. The 24 hour urinary excretion of 1β-OH-THE was estimated to be 300 – 400 μg and this accounted for 2.5% of the total unconjugated radioactivity.

Early jaundice after open-heart surgery.

Two hundred and thirty-two patients subjected to open-heart surgery have been analysed regarding early post-operative jaundice. The overall incidence of jaundice was 13%, being 55% where the mitral valve and 5% where the aortic valve was predominantly involved. Only one out of 59 patients in whom a congenital lesion was repaired became jaundiced. Hyperbilirubinaemia without jaundice was common. The biochemical pattern of the jaundice was singularly constant, the most striking feature being the rise in serum bilirubin level. The highest value occurred between the second and tenth post-operative days. At total levels less than about 4 mg./100 ml. the unconjugated fraction usually equalled or exceeded the conjugated. The alkaline phosphatase was normal in the first post-operative week but was sometimes slightly raised in the second, when the bilirubin levels were usually falling. The S.G.P.T. was normal or very slightly raised. Bilirubinuria was present. Centrilobular congestion with no evidence of extrahepatic biliary obstruction was found at post-mortem examination, and liver biopsy in three very severely jaundiced patients revealed only intrahepatic cholestasis. Pre-operative evidence of liver dysfunction was found in 13 patients who subsequently became jaundiced. Twelve had lesions predominantly affecting the mitral valve. Haemolysis, heart failure, and infection probably all played a part, and the effect of operation <i>per se</i> on liver function has been discussed. Drugs and viral hepatitis do not seem implicated in our patients. Maintenance of lower venous pressures in the post-operative period and the use of less blood reduced both the incidence and severity of jaundice.

“Applejack” concentration of urinary chlorpromazine metabolites: A widely applicable and mild procedure

Urine specimens from patients on chlorpromazine therapy, frozen in batches from 750 to 4000 ml, were slowly thawed at refrigerator temperatures of 5–8° C. Aliquots of increasing volumes were collected sequentially for determinations of comjugated and unconjugated drug fractions. From 9 to 15 fold concentration of the drug content was obtained in the initial 1 2 % of collected volumes, and from 50 to 60% of the drug metabolites were recovered in 10 to 12% of the original volumes. The procedure therefore lends itself to a variety of purposes concerned with concentration and isolation of chlorpromazine drug metabolites.

The metabolism of [4-14-C]cortisol in patients with sarcoidosis.

SUMMARY The major metabolic pathways of cortisol in a patient with sarcoidosis have been studied after intravenous injection of a tracer dose of [4-14C]cortisol. Results from some preliminary experiments in four other patients are also presented. The effects of large doses of cortisol on its metabolism were also studied. In all the patients studied the adrenal secretion rate of cortisol was in the low normal range. The rate of disappearance of radioactivity from the plasma fractions containing the unconjugated steroids was normal before cortisol therapy but slower than normal during therapy. During therapy there was a significant decrease in the total urinary radioactivity. In the one patient in whom detailed studies were made, an unusually large proportion of the urinary metabolites was in the unconjugated fraction in which the amount of 6β-hydroxycortisol was greater than normal. Apart from an increased ratio of 11β-hydroxy- to their corresponding 11-oxo-metabolites there was no essential difference in the pattern of excretion of the metabolites in this patient during therapy. In this patient only 65% of the injected radioactivity was excreted in urine after 48 hr., whereas the other patients excreted a normal percentage of the injected radioactivity during this period.

CORTISOL METABOLISM IN THE NEWBORN

ABSTRACT Following intravenous administration of cortisol-4-14C to seven full-term and five premature newborn infants, the urinary excretion of free and conjugated substances was measured in different fractions. Three normal adult subjects served as controls. The study comprised the following fractions: a) Free »lowpolar« fraction, extractable with methylene dichloride. b) Free »high-polar« fraction, extractable with ethyl acetate. c) »Low-polar« glucuronoside fraction, extractable with methylene dichloride following β-glucuronidase hydrolysis. d) »High-polar« glucuronoside fraction, extractable with ethyl acetate following β-glucuronidase hydrolysis. e) Solvolysis fraction, extractable with ethyl acetate after previous removal of the free and glucuronoside conjugated fractions and subsequent solvolysis. The excretion of total radioactivity in the group of normal newborns was found to be delayed as compared with the adult subjects. The unconjugated fractions were found to be somewhat higher in the newborn than in the adult subjects. On the other hand, the »low-polar« glucuronosides were essentially lower. These deviations from the findings in normal adults were still more pronounced in the group of premature newborns. An inverse relationship was demonstrated between the percentage excreted as glucoronosides and the maximum serum bilirubin level in the neonatal period.

Rotor's Syndrome and Chronic Idiopathic Jaundice

During the past decade increasing attention has been paid to a group of congenital or familial diseases of bilirubin metabolism, prompted largely by the hope that these diseases might provide some clues to the mysteries of bilirubin uptake, conjugation, and excretion by the liver cell. Among these diseases are two conditions characterized by jaundice and an increase in serum levels of both the conjugated and unconjugated fractions of bilirubin. These are Rotor's syndrome 1,2 and chronic idiopathic jaundice. 3-5 Some workers have suggested that these two conditions are close relatives, or even variants of a single disease. Others have scrapped the original criteria set down for these entities and have instituted radical changes in established terminology. Because such premature breaches in nomenclature may lead to obfuscation, I believe it desirable to reexamine the current status of these conditions. In our present state of knowledge, the major differences between these two

Adrenal cortical function in chronic pulmonary tuberculosis

1. The excretion of unconjugated and conjugated 17-OHCS and formaldehydogenic corticosteroids was studied in chronic pulmonary tuberculosis. 2. A significant correlation was noted between the excretion of total corticosteroids and severity of pulmonary tuberculosis. 3. The low value of the excretion of total corticosteroids seen frequently in patients with moderately and far advanced pulmonary tuberculosis was largely due to a reduction in the conjugated fraction, while the unconjugated fraction remained at normal level. 4. Some cases with far advanced pulmonary tuberculosis showed a decrease in the value of the unconjugated fraction as well as the conjugated fraction. This indicates that patients with moderately and far advanced pulmonary tuberculosis have a form of adrenal insufficiency. 5. Following ACTH administration an increase in the excretion of unconjugated and conjugated corticosteroids was seen in these cases, suggesting secondary hypofunction of the adrenal cortex.

Metabolism of 4-C14-testosterone in human subjects. III. Fate of androsterone and etiocholanolone.

C14-androsterone and C14-etiocholanolone were administered intravenously to 7 human female subjects; 3 received both steroids, and 4 (with bile fistulas) received only one. The radioactivity was excreted in the urine almost quantitatively in a few hours. The unconjugated steroids were cleared from the plasma rapidly, with a half-life of twenty minutes. The level of the glueuronidate fraction of the plasma was approximately ten times higher than that of the unconjugated fraction. The glucuronidates after administration of etiocholanolone were cleared faster than after administration of androsterone. Androsterone and etiocholanolone are not excreted in the bile in appreciable quantities; in 3 of the 4 bile-fistula subjects negligible amounts of radioactivity were recovered. Moreover, after administration of 4-C14-testosterone, no androsterone and only a small amount of etiocholanolone was recovered from the bile. This last observation is tempered by the fact that most of the radioactivity in bile is not ext...

Metabolic fate of 11-hydroxyandrostenedione-C14 in human subjects.

Summary11-Hydroxyandrostenedione-C14 was administered I.V. to 8 human subjects: 2 normal males, 2 subjects with bile-fistulas, 3 females without and one female with cancer. The radioactivity was excreted in urine almost quantitatively within a very short period of time (8-12 hours). Only negligible amounts of radioactivity were found in the bile of the 2 subjects with bile-fistulas or in stools of all subjects. The unconjugated steroids were cleared from the plasma at 2 rates, a fast rate with a half-life of 30 minutes and a subsequent slower rate with a half-life of 80 minutes. The glucuronidate fraction of the plasma contained much higher levels of radioactivity than the unconjugated fraction within 15 minutes following the injection of the steroid and even more strikingly four hours following the administration of the radioactive steroid. The significance of these findings is discussed.