Extracts Of Human Urine Research Articles

An assay of the capacity of biological fluids to stimulate renal glucose-6-phosphate dehydrogenase activity in vitro as a marker of their ability to inhibit sodium potassium-dependent adenosine triphosphatase activity.

A highly sensitive cytochemical method for the assay of the ability of plasma and extracts of human urine to stimulate renal glucose-6-phosphate dehydrogenase (G6PD) activity in vitro is described. In the proximal convoluted tubules there was a linear increase of G6PD activity with the logarithm of concentration of a highly purified natriuretic extract from normal human urine (0·384–384 ng active material/l) which was used as a standard. The stimulation of G6PD obtained with dilutions of normal human plasma was parallel to that produced by the standard. The sensitivity of the assay permitted the measurement of as little as 0·384 ng active material/l of the natriuretic extract (0·001 units/ml) and dilutions of 1/10 000 could be detected using normal human plasma. The mean ± s.e.m. index of precision was 0·068± 0·003 (n = 9). It is known that inhibition of sodium potassium-dependent adenosine triphosphatase (Na+-K+-ATPase) is associated with a rise in G6PD activity. We have confirmed this observation by demonstrating that ouabain, a potent inhibitor of Na+ -K+-ATPase, stimulates renal G6PD activity in our assay and that natriuretic extract, human plasma and ouabain stimulated renal G6PD activity in vitro and simultaneously inhibited renal Na+-K+-ATPase activity in vitro. The plasma from 12 normal subjects (five of whom were previously shown to inhibit renal Na+-K+-ATPase activity in vitro in a manner related to sodium intake) stimulated renal G6PD activity in vitro, and this activity was also directly related to sodium intake. It is suggested that the change in the capacity of plasma to stimulate renal G6PD activity in vitro is a marker of the concentration of a circulating sodium transport inhibitor.

(R)-mevalonate excretion in human and rat urines.

(R)-Mevalonate was identified by radioenzymatic assay in human and rat urines. This was confirmed by spectrophotometric enzymatic assay of an ether extract of human urine. The average excretion rate of (R)-mevalonate in humans was 1.7 mumol/24 hr which corresponds to 29% of the glomerular filtration rate. In anesthetized rats the average rate of (R)-mevalonate excretion was 350 pmol/min, corresponding to 44% of the glomerular filtration rate. These rates were not affected by the sex of the subjects or animals. After bilateral nephrectomy, the concentration of (R)-mevalonate in rat serum increased within 2 hr to a new level that was 5 times that of sham-operated controls. Nephrectomized rats showed a decrease in their tolerance to an intravenous load of mevalonate. These data show that (R)-mevalonate is normally excreted in the urine of humans and rats. Urinary excretion of (R)-mevalonate should therefore be taken into account in in vivo studies on the metabolism of this compound.

The metabolic fate of Sormodren (bornaprine hydrochloride) in animals and humans.

1. Single oral doses of the anticholinergic drug [14C]Sormodren to rats (1 mg/kg), dogs (0.3 mg/kg) and humans (0.03 mg/kg) were well absorbed. Excreted in urine and faeces were means of 31 and 70%, 53 and 39%, and 78 and 4% in rats, dogs, and humans, respectively, during five days: excretion was prolonged and still incomplete at five days in humans. 2. Peak plasma levels of 14C (scaled for dose) were generally reached within 1-2 h after oral doses in rats, 49 (ng/ml)/(mg/kg), dogs 290 (ng/ml)/(mg/kg) and humans 410 (ng/ml)/(mg/kg), and declined with half-lives of approx. 5, 12 and 30 h, in these species respectively. Repeated oral doses of [14C]Sormodren to dogs resulted in some accumulation of 14C in the plasma. 3. Tissue concn. of 14C in dogs were generally higher than those in rats, particularly in the brain, lungs and eyes. The tissue distribution of 14C in rats and dogs was consistent with that of a compound readily eliminated by both renal and hepatic routes. 4. Basic metabolites in dog and human, urine and plasma were investigated using a combination of h.p.l.c. and g.l.c.-mass spectrometry. Unchanged Sormodren was not detected in the dog samples and was only a minor component in human urine and plasma. Some metabolites were present as conjugates. 5. A basic extract of enzyme-hydrolysed dog urine (5 mg/kg dose) contained 42% of the urine 14C. The major metabolites in this fraction were identified as three isomers of monohydroxy-N-desethyl-Sormodren and three isomers of monohydroxy-Sormodren, resulting from hydroxylation in the bicyclic ring. The positions of oxidation were not determined. A similar extract from dog urine (0.3 mg/kg dose) contained 26% of the urine 14C and the major metabolites were identified as isomers of monohydroxy-N-desethyl-Sormodren. 6. A basic extract of enzyme-hydrolysed human urine (0.03 mg/kg dose) contained 23% of the urine 14C. The unchanged drug was only a minor component and most of the radioactivity was associated with five isomers of monohydroxy-Sormodren, hydroxylation having occurred in the bicyclic ring. 7. Basic extracts of dog and human plasma only contained about 10% of the plasma 14C. Metabolites were chromatographically similar to the hydroxylated metabolites identified in the corresponding urine samples.

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH 4 + and/or H + excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH 4Cl-induced acidosis were prepared. These extracts were tested for their effect on NH 4 + excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH 4 + excretion in the toad urinary bladder. We found no effect on H + excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH 4 +. The factor could act as a permease in the bladder cell or as a stimulator of an NH 4 + transport system.

Isolation of 15α-hydroxypregnenolone and 15α-hydroxy-dehydroisoandrosterone from human pregnancy urine

15α-Hydroxydehydroisoandrosterone and 15α-hydroxypregnenolone were isolated from hydrolyzed extracts of human late pregnancy urine and identified by means of the isotope dilution technique. In two separate determinations the excretion rate of 15α-hydroxydehydroisoandrosterone was found to be 1.7 and 3.2 μg per day while that of 15α-hydroxypregnenolone was 1.7 and 2.9 μg per day. It is postulated that 15α-hydroxydehydroisoandrosterone might serve as a precursor of 15α-hydroxylated estrogens already isolated from late pregnancy urine. Similarly, 15α-hydroxypregnenolone might be an endogenous precursor of 15α-hydroxyprogesterone.

The metabolism of the neuroleptic agent 1 (4'-fluorophenyl)-4-(cyclohexyl-1'-piperzinyl-4'-carboxylated)-butan-1-one hydrochloride in rats and man.

1. An oral dose the neuroleptic agent 1-(4'-fluorophenyl)-4-(cyclohexyl-1'-(14C)piperazinyl-4'-carboxylate)butan-1-one was mainly eliminated in the urine within 12 h by rats and man. During 5 days, 63-6% and 83-3% was eliminated in the urine of rats and man respectively. 2. Plasma concentrations in man related a maximum during 30 min to 1 h, representing 1-43 microgram equiv./ml. The proportion of unchanged drug in plasma decreased from 48% at 15 min to less than 10% after 1 h. 3. Seven major radioactive components were detected in the chloroform extract of basified rat urine and five major components in similar extracts of human urine. The major rat metabolites were isolated and identified by mass spectrometry as components resulting from mono- and dihydroxylation in the cyclohexane ring, reduction of the keto group to a secondary alcohol and hydrolysis and decarboxylation of the cyclohexylcarbamoyl group. The major metabolite in the rat urine extract was the dihydroxylated secondary alcohol derivative while the major human metabolite was the monohydroxylated secondary alcohol derivative. The metabolites were also partly eliminated as conjugates.

The isolation of the urogastrones - inhibitors of gastric acid secretion - from human urine.

It has been known for over thirty years that extracts of human urine could cause inhibition of gastric acid secretion. The active principle was called urogastrone and this has now been isolated using a series of twelve simple stages of partition, gel and ion-exchange chromatography. In fact two products were obtained, each in a yeild of less than 1 mg per 1 000 l urine, which represented an overall recovery of 3 - 5%. These materials were biologically indistinguishable, causing inhibition of gastric acid secretion in a variety of circumstances at doses of less than 1 mug/kg. The purified urogastrones were found to be acidic polypeptides composed of 53 and 52 amino acid residues with three internal disulphide bonds, and they differed by only one arginine residue. Neither threonine nor phenylalanine residues were present in the urogastrones.

Identification of methaqualone metabolites from urine extract by gas chromatography-mass spectrometry

A rapid and sensitive method for identifying methaqualone metabolites bv means of gas chromatography-mass Spectrometry is presented. The mass spectra of TMS derivatives of ten synthetic metabolites of methaqualone have been studied and compared with the components of three silylated human urine extract. Four to five metabolites were identified in each extract.

Inhibitory substances of isolated smooth muscles in human urine

Weak acid and neutral fractions separated from the extracts of male and female human urine showed an inhibitory action against contraction of smooth muscle preparations. Their inhibitory action was tested on contraction of isolated guinea-pig ileum induced by histamine and isolated rat uterus by oxytocin. It was suggested that one of the principles of inhibitory activity in the urine might be the metabolites of steroid hormones.

Differentiation of Urinary N-Acetyl-p-Aminophenol from Endogenous Phenolic Compounds

Abstract An unknown spot, observed on two-dimensional paper chromatograms of extracts of human urine, was identified as N-acetyl-p-aminophenol (APAP), a drug metabolite. This finding suggests that APAP is clearly separable from other endogenous phenolic compounds in urine by a two-dimensional paper chromatographic method. This simple technique is feasible for the specific routine analysis of APAP in urine.

Immunoreactive luteinizing hormone in acetone precipitates and kaolin extracts of human urine

Urinary luteinizing hormone (LH) was precipitated with acetone and measured in a radioimmunoassay specific for LH. Recovery experiments were done adding the Second International Reference Preparation of Human Menopausal Gonadotropin (2nd IRP-HMG) to the urine of a hypophysectomized patient. Parallelism of the dilution curves of 2nd IRP-HMG and several dilutions of acetone precipitates of urine confirmed the validity of the assay. Direct acetone precipitation and acetone precipitation of kaolin extracts were compared for LH and follicle-stimulating hormone (FSH) recoveries. The methods gave comparable results for immunochemical FSH, but LH values for acetone precipitates were 3.7 to 4.3 times higher than those in the kaolin extracts and the discrepancies were comparable for all age groups. Measurement of serum LH and urinary LH by direct acetone precipitation during four normal menstrual cycles and in 2 patients with gonadal agenesis before and after estrogen administration confirmed that the urinary excretion reflected the serum LH concentration.

Characteristics of in vitro colony formation by cells from haemopoietic tissues

The characteristics of stimulation of colony formationin vitro from cells of mouse haemopoietic tissues has been briefly reviewed. Mouse kidney or embryo feeder cells, media conditioned by the cells from these tissues, normal or leukemic mouse sera, sera from leukemic or infectious mononucleosis patients, human urine and mouse embryo extracts are all sources of colony stimulating activity and their properties have been described.

The ovarian ascorbic acid depletion test for luteinizing hormone. Lack of specificity.

ABSTRACT Urinary extracts from two women hypophysectomized for metastasizing mammary adenocarcinoma and from 11 children of either sex between 5 and 9 years were found to produce marked ovarian ascorbic acid depletion in the Parlow test for luteinizing hormone. As these urines cannot contain significant amounts of luteinizing hormone, it is concluded that the depletion must be due to unspecific substances, probably proteins in the urines. From the literature other indications of non-specificity of the Parlow test are mentioned, and the main conclusion of the present investigation is that for the assay of luteinizing hormone in extracts of human urine the test is unreliable.

Inhibition of toad bladder sodium transport and Na+ plus K+ stimulated adenosinetriphosphatase by extracts of human urine: studies of patients with virilizing adrenal hyperplasia.

ABSTRACT In order to detect the presence of a possible sodium transport inhibitor, urine extracts were tested directly in 2 in vitro systems: (A) sodium transport across the urinary bladder of the toad and (B) Na++K+ stimulated adenosine triphosphatase (Na-K-ATPase). The sodium transport across the toad bladder, which remained 60 min after urine extract was added, was compared to the sodium transport before the addition and expressed as a ratio, R60. The R60 in children with virilizing adrenal hyperplasia (VAH) (R60=.33±.05) and in normal adults (R60=.39±.08) was significantly lower than normal prepubertal children (R60=.79±.03), indicating more inhibitory activity in the former 2 groups. The effect of urine extracts on Na-K-ATPase was similar to the effect on the bladder (children with VAH-46.9±7.0% of control ethanol additions, normal adults 46.8±7.7% of control, and normal children 75.6±2.6% of control). Among the children with VAH, there was no difference in the inhibitory activity of urine extracts f...

Phosphaturic activity of urine extracts not associated with parathormone.

SummaryButanol extracts of normal human and rat urine contained two types of substances, distinct from each other and unrelated to PTH, which interfered with the bio-assay of PTH in urine. One caused hypercalcemia, the other hyperphosphaturia in PTX rats. The latter material was dialyzable and equilibrated between 20% acetic and n-butanol with a distribution coefficient (K) about 10 times lower than that of PTH. The “hypercalcemic” material was nondialyzable and its K about 5 times lower than that of PTH. Thus, both materials could be separated by CCD from PTH added to the urine before extraction. From 100 ml of normal (human or rat) urine, no PTH activity could be detected in fractions expected to contain PTH in its known form.

Immunologic studies with urinary follicle stimulating hormone.

ABSTRACT Antiserum to human follicle stimulating hormone (FSH) partially purified from postmenopausal urine was produced in rabbits. The antiserum, after absorption with human chorionic gonadotropin (HCG) and pediatric urine extract (Peduex), was shown by immunodiffusion to react with the partially purified fraction of FSH and Pergonal-26 with a “line of identity,” but did not give a line against HCG or Peduex. The biologic activity of FSH in the ovarian augmentation assay was inhibited by the antiserum but that of HCG was not affected. As little as 0.035 IU of urinary FSH, in the form of Pergonal or Ortho-HMG, was capable of inhibiting the agglutination reaction between the FSH antiserum and latex particles coated with urinary FSH. HCG had only a slight inhibitory effect. The immunoactivity of these and other urinary FSH preparations was in good agreement with their biologic FSH activities. These results confirm the antigenicity of urinary FSH, indicate that an antiserum specific for FSH and free of anti...

ALDOSTERONE STIMULATION IN VITRO

ABSTRACT An extract of human urine, which was previously shown to stimulate aldosterone production by rat adrenal sections, was further purified. Evidence was obtained that its aldosterone-stimulating effect was due to the presence of ammonium ions. Addition of ammonium chloride and of urine extract to the incubation medium caused identical increases in aldosterone production in vitro. In addition to ammonium ions, rubidium and caesium ions also stimulated aldosterone production up to 250% that of control values without a significant effect on corticosterone production. Similar dose-response curves were obtained when increasing concentrations of potassium, ammonium, rubidium and caesium ions were tested. Aldosterone production was maximal at concentrations of 7 mval/1 and was significantly lower at higher concentrations. When ammonium chloride and ACTH were simultaneously added to the incubation medium, the production of aldosterone and of corticosterone was lower than with ACTH alone. On the other hand, the stimulating activity on aldosterone and corticosterone production by »TPN« (NADP) and glucose-6-phosphate was enhanced by the simultaneous addition of ammonium chloride.

Measurement of urinary testosterone by gas-liquid chromatography

A method has been developed which employs gas-liquid chromatography for the assay of testosterone from extracts of human urine prepared according to an earlier method described by Camacho & Migeon in 1963. The trimethyl silyl ethers of testosterone and an internal standard, cistestosterone, were separated on the nitrile silicone rubber stationary phase XE-60. This method has given results which correspond satisfactorily with those obtained with UV spectrophotometry or measurement of sulfuric acid chromogens after paper chromatography.

RESPONSE OF THE POLYCYTHEMIC MOUSE AND POLYCYTHEMIC GUINEA PIG TO HUMAN ERYTHROPOIETIN.

Fe59 RBC incorporation was increased in the hypertransfused guinea pig following administration of an extract of urine from a patient with chloramphenicol-induced hypoplastic anemia. This extract also had erythropoietic activity in the polycythemic mouse. Maximum Fe59 incorporation into erythrocytes of guinea pigs occurred when the Fe59 C13 was given 48 hours after administration of the erythropoietically active human urine extract.

THE USE OF THE OVARIAN ASCORBIC ACID DEPLETION TEST FOR MEASURING LUTEINIZING HORMONE IN HUMAN URINE.

SUMMARY The possibility of applying the ovarian ascorbic acid depletion assay or its variants to the measurement of luteinizing hormone in human urine extracts has been investigated. A 3 hr. i.v. technique proved the most sensitive. The method had limitations as a clinical assay, namely: a relatively low degree of precision, toxicity of some urinary extracts and the fact that about 20 % of assays were invalid owing to a lack of parallelism. Two of ten patients with amenorrhoea and raised gonadotrophin levels showed a relative lack of FSH as compared with the International Reference Preparation for human menopausal gonadotrophin.