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Determination of 17-hydroxycorticosteroids in human urine.

A study was undertaken to develop a method that could be used an an indication of the absorption of steroids. It was demonstrated that 17-hydroxycorticosteroids in urine could be quantitatively determined via absorptivity values obtained through the use of standard hydrocortisone-alcohol solutions or standard solutions of hydrocortisone in urine utilizing the blue tetrazolium assay method. This method is dependent upon the hydrolysis of conjugated urinary steroids by beef liver glucuronidase. The resultant concentration of 17-hydroxycorticosteroids is determined colorimetrically using blue tetrazolium.

17-Hydroxycorticosteroids↗

Circadian pattern of plasma 17-hydroxycorticosteroid: alteration by anticholinergic agents.

Atropine, administered to cats just prior to the time of the expected circadian rise in levels of 17-hydroxycorticosteroid in plasma, blocks this rise. Atropine does not alter this circadian pattern when administered at other times in the circadian cycle. Results similar to those obtained with atropine have been observed with short-acting barbiturates. Dibenzyline administered just prior to the time of the expected circadian rise is ineffective in blocking this rise. These findings support the hypothesis that the circadian pattern of plasma 17-hydroxycorticosteroid levels reflects activation, by the central nervous system, of the hypothalamicpituitary-adrenal axis during a "critical time period" in the circadian cycle.

17-Hydroxycorticosteroids↗

A comparative study of urinary 17-hydroxycorticosteroids, urinary free cortisol, and the integrated concentration of plasma cortisol.

The discriminatory power of three clinical tests for adrenal activity are compared by measuring the variability of the results obtained in 40 normal control subjects, 35 patients with mild essential hypertension, and 13 patients with Cushing syndrome. The variance of the 24-h integrated plasma concentration of cortisol was significantly (P < 0.0001) smaller than the variance of the 24-h urinary excretion of 17-hydroxycorticosteroids and free cortisol. While the 24-h urinary excretion of 17-hydroxycorticosteroids and free cortisol of the patients with Cushing syndrome overlapped with the corresponding values of the normal and hypertensive subjects, their integrated cortisol concentration did not exhibit any overlap.

17-Hydroxycorticosteroids↗

The relationship of 17-hydroxycorticosteroid to acute necrotizing ulcerative gingivitis.

Eleven patients presenting with acute necrotizing ulcerative gingivitis (ANUG) collected 24-hour urine samples during and after the course of their ANUG. The Porter-Silber technique was used to determine the 17-hydroxycorticosteroid concent of the urine, a physiological measure of stress. All of the patients had significantly higher levels of 17-hydroxycorticosteroid during the course of their ANUG than after it had resolved.

17-Hydroxycorticosteroids↗

Interference of bencyclan with 11-hydroxycorticosteroids determinations.

Falsely high fluorimetric readings were obtained in plasma and urine 11-hydroxycorticosteroids (mostly cortisol) assays during the administration of bencyclan to hypertensive patients. No valid conclusion can be drawn from 11-hydroxycorticosteroids assays in bencyclan treated patients.

11-Hydroxycorticosteroids↗

Improved hydrolysis of urinary 17-hydroxycorticosteroid glucuronides with beta-glucuronidase from Helix pomatia, on adding sodium sulfate.

Sodium sulfate increases the hydrolysis of urinary 17-hydroxycorticosteroid glucuronides with beta-glucuronidase preparations derived from Helix pomatia because it removes the inhibitory activity of urinary high-molecular-weight substances. For maximum hydrolysis of urinary 17-hydroxycorticosteroid glucuronides, the hydrolysis [5 ml of urine, 0.5 ml of 2 mol/liter acetate buffer (pH 5.0)] should be conducted in the presence of sodium sulfate (final concentration: 80 g/liter) with (a) 600 Fishman units of the enzyme per milliliter of urine (18 h at 52 degrees C) or (b) with 1500 units of the enzyme per milliliter of urine (3 h at 57 degrees C). Under conditions a, analytical recovery of steroid glucuronides added to 12 urine samples was 99 +/- 2.1% (96-102%). Values obtained for 20 urine samples with this method were 99 +/- 2.7% (93-104%) as great as those yielded by a method in which 600 units of the enzyme from bovine liver are used together with sodium sulfate (18 h at 48 degrees C).

17-Hydroxycorticosteroids↗

Natrium, potassium, cholesterol and total free 11-hydroxycorticosteroids variations in thymectomized and thymic extract treated rats.

The authors investigated the effect of thymectomy and TP2 thymic polypeptide extract administration on total, esterified and free cholesterol in the plasma and the adrenals, as well as on plasma Na and K and plasma free total 11-hydroxycorticosteroids. The results showed that total, esterified and free cholesterol decrease in the adrenals by 23.20%, 28.57% and 17.70% maximum respectively, in the thymectomized rat and that under the influence of the TP2 extract the values increase by 14.81%, 15.38% and 12.67%, respectively. Plasma cholesterol did not change. Thymectomy caused an increase in the plasma 11-hydroxycorticosteroids value by 13.4% and administration of the TP2 extract lowered their value by 58.11%. The plasma Na and K values did not change.

11-Hydroxycorticosteroids↗

Cefoxitin interferes with the "Clini-Skreen" column method for urinary 17-hydroxycorticosteroids.

Cefoxitin interferes with determination of urinary 17-hydroxycorticosteroids. The apparent concentration of hormone is increased from three- to 10-fold in samples from patients receiving cefoxitin when the Amberlite XAD-2 "Clini-Skreen" column is used. To determine the mechanism of interference, we reacted aqueous solutions of cefoxitin, cortisol, cortisone, and 11-deoxycortisol with phenylhydrazine; recorded the adsorption spectra; and determined the molar absorptivities and the equilibrium and rate constants. Also, we recorded the absorption spectra of phenylhydrazine with eight other cepha antibiotics and benzylpenicillin. Cortisol, cortisone, 11-deoxycortisol, and cefoxitin react with phenylhydrazine and absorb light with superimposable spectra and absorption maxima of 410 nm. The other antibiotics react with phenylhydrazine but absorbance maxima of the products vary, none being at 410 nm. Cortisol, cortisone, and 11-deoxycortisol react with phenylhydrazine 35-fold faster, have equilibrium constants ninefold greater, and have molar absorptivities 1.6 times that of cefoxitin. Thus, cefoxitin interferes with determination of urinary 17-hydroxycorticosteroids by forming a chromophore with the same absorbance maximum and with a molar absorptivity similar to cortisol, but much more slowly.

17-Hydroxycorticosteroids↗

[Mediation of the lithium increase in rat liver tryptophan pyrrolase activity by 11-hydroxycorticosteroids].

After a single intraperitoneal administration lithium carbonate (50 and 100 mg per kg of body weight) caused an increase in the tryptophane pyrrolase activity in rat liver tissue by 50-80% within 5 hrs and did not affect the enzyme within 1, 3 and 20 hrs. Content of lithium was quite unaltered in rat blood serum after administration of lithium carbonate at a dose of 50 mg/kg; it slightly exceeded the therapeutic level at a dose of 100 mg/kg. Addition of the lithium preparation (7-10(-3)-7-10(-5) M) to liver homogenates did not change the tryptophane pyrrolase activity in vitro. The same doses of lithium (50 and 100 mg/kg) caused a 2.5-fold increase in content of 11-hydrocorticosteroids in rat blood plasma; the effect was prevented by previous administration of dexamethasone. In experiments with adrenalectomized rats lithium did not affect the enzymatic activity. Repeated administration of lithium (50 mg/kg) within 10 days did not affect the tryptophane pyrrolase activity and did not alter the content of 11-hydroxycorticosteroids in rat blood plasma. The data obtained suggest that the increase in the tryptophane pyrrolase activity, caused by lithium, is mediated through the elevation in content of 11-hydroxycorticosteroids in blood.

11-Hydroxycorticosteroids↗

Failure of industrial noise to change the patterns of vanilmandelic acid and 17-hydroxycorticosteroids in the urine of the female weavers with hearing loss.

The field experiments were undertaken on two consecutive working days on seven female weavers with noise-induced hearing loss (NIHL), and a control group of seven sewers with normal hearing. The first day, urine was collected during the shift (06:00-14:00 h) and at home (17:00-05:00 h) for measurements of 17-hydroxycorticosteroids (17-OH) levels. The following day, the procedure was repeated, but for vanilmandelic acid (VMA) analysis. No significant changes in the hormone levels were found, both concerning the different occupational noise exposure and the periods of a working day.

17-Hydroxycorticosteroids↗

A fluorometric determination of urinary 17-hydroxycorticosteroids using benzamidine.

A fluorometric determination of urinary 17-hydroxycorticosteroids using a reaction of benzamidine with compounds carrying the dihydroxyacetone side chain is described. The fluorescent compounds have excitation and emission maxima at 370 and 480 nm, respectively. The method includes enzymatic hydrolysis with beta-glucuronidase (EC 3.2.1.31, from Escherichia coli) and extraction with methylene chloride and generation of fluorescence in alkaline solution (pH 13.4). The specificity of the reaction was examined and the results were compared with those of an accepted method based on the Porter-Silber reaction (C. C. Porter and R. H. Silber, 1950, J. Biol. Chem. 185, 201-207). The coefficient of correlation was 0.945 with regression line of y = 0.91x + 0.7 mg/day (y, present method; x, Porter-Silber reaction method). Sensitivity of the reaction was 0.5 microgram/ml of standard or sample, mean recovery of cortisol added to five urine samples (5-micrograms addition) was 95%, and the coefficient of variation of the method (five repeated assays of sample with a value of 5.2 mg/liter) was 6.2%.

17-Hydroxycorticosteroids↗

17-Hydroxycorticosteroids in urine.

A new method has been devised to extract 17-hydroxycorticosteroids (17OH-CS) from a hydrolyzed urine using an EXTUBE fiber column. This extraction was combined with a modified Porter-Silber color reaction to quantitate total urinary 17OH-CS. The new method takes only half the time required in older methods. Recovery of tetrahydrocortisone in the new method was improved to 85--95% compared with 70% in the previous method. Between-run precision (C.V.%) was concentration dependent in both methods and was improved to 5--7% compared with 10--18% in the previous method. Evidence was obtained to demonstrate that the new methodology would provide the same information and interpretation as the older, more cumbersome method.

17-Hydroxycorticosteroids↗

Hydrolysis of conjugated steroids by beta-glucuronidase from Ampullaria and application to the determination of urinary 17-hydroxycorticosteroids.

The usefulness of Ampullaria beta-glucuronidase as a reagent for the hydrolysis of steroid glucuronides was examined. The purified enzyme showed an optimum pH of 4, and was stable between pH 4.5 and 7.5. The enzyme from Ampullaria and Helix pomatia hydrolyzed various artificial substrates and conjugated steroids. Affinity of the Ampullaria enzyme for 17-hydroxycorticosteroid glucuronide was 7-fold the affinity of the Helix pomatia enzyme. Hydrolysis of urinary steroid conjugates was completed by an incubation with 320 Fishman units of Ampullaria beta-glucuronidase at 60 degrees C for 1 h. A close correlation was obtained between the values of steroid conjugates hydrolyzed by the Ampullaria enzyme and those by the bovine liver enzyme used conventionally. Ampullaria beta-glucuronidase is suitable for hydrolysis of urinary steroid conjugates because of its higher affinity for steroid substrate and the thermal stability.

17-Hydroxycorticosteroids↗

The excretion of 17-ketosteroids and 17-hydroxycorticosteroids in night urine of elite rowers during altitude training.

During an altitude training camp (23 days, 1850 m above sea level) we collected night urine from 36 German elite rowers in order to measure the excretion of urea, creatinine, 17-ketosteroids (17-KS), and 17-hydroxycorticosteroids (17-OHCS). 17-KS and 17-OHCS represent the major metabolites from endogenous anabolic and catabolic steroid hormone systems. There was no significant change in the excretion of 17-OHCS during the training camp (mean value: females 0.0025 +/- 0.0011, males 0.0033 +/- 0.0012 mg/kgbw.h). Significant increases in the excretion of 17-KS (mean value: females 0.007 +/- 0.003, males 0.0092 +/- 0.0039 mg/kgbw.h) and in the ratio 17-KS/17-OHCS (mean value: females 3.13 +/- 1.65, males 3.11 +/- 1.71) were found during the first week and towards the end of the training camp. To investigate training effects on the excretion of these hormone metabolites, we used a recently described classification for rowing training (categories I to IV of rowing-specific training according to lactate levels and non-rowing specific categories) and a corresponding rowing data base. More than 80% of training was performed at a lactate level lower than 2 mmol/l. Using multiple regression analysis, the general finding was that in males the ratio of 17-KS/17-OHCS increased with rowing specific and non specific training regimes where a lactate level below 2 mmol/l was observed. Training at higher lactate levels caused a decrease in the ratio that may be interpreted as a shift in the production from endogenous androgenic steroid hormones to cortisol. No significant effects of single training variables were found in female rowers, which indicates major training influences on testosterone metabolism. The influence of further factors such as a relationship between urine urea and 17-KS in males are described and need further explanation.

17-Hydroxycorticosteroids↗

The effect of sodium bisulfite on the removal of drugs and their metabolites interfering with the Porter-Silber reaction in the determination of urinary 17-hydroxycorticosteroids.

In the determination of urinary 17-hydroxycorticosteroids, the urine is saturated with NaHSO3 after hydrolysis with beta-glucuronidase and then extracted with methylene chloride. Sodium bisfulfite removes almost all non-steroidal impurities in the urines from patients medicated with acetylspiramycin, leucomycin, erythromycin, triacetylolenadomycin, rifampicin and tranquilizers such as chlorpromazine, which interfere with the absorption at 410 nm. in the subsquent Porter-Silber reaction. In order to increase the specificity of a routine method, a procedure conducted by Allen has been often employed: The sum of 370- and 450-nm. absorbances is subtracted from twice absorbance at 410 nm. However, the procedure could not be used in the medicated urines mentioned above, because the spectral absorption curve of these drugs and their metabolites in the Porter-Silber reaction was not a straight but a strongly convex or concave line in the 370-450-nm. range. Using the present method, interference with the Porter-Silber reaction was not found in the urines from patients medicated with chloramphenicol, minocycline, chlordiazepoxide, meprobamate, methyprylon, nitrazepam, synthetic penicillins such as hetacillin, oxacillin and cloxacillin, or cephalosporins such as cephalexin and cephalothin. However, to obtain correct values in urines from patients medicated with spironolactone, it was necessary to subject the urines to treatment with methylene chloride before enzyme hydrolysis.

17-Hydroxycorticosteroids↗

The effect of real and simulated time-zone shifts upon the circadian rhythms of body temperature, plasma 11-hydroxycorticosteroids, and renal excretion in human subjects.

1. Observations were made upon five subjects who flew through 4(1/2)-6 time zones, four of them returning later to their starting point, and upon twenty-three subjects experiencing simulated 6 or 8 hr time zones shifts in either direction in an isolation unit.2. Measurements were made of plasma concentration of 11-hydroxycorticosteroids, of body temperature, and of urinary excretion of sodium, potassium and chloride. Their rhythm was defined, where possible, by fitting a sine curve of period 24 hr to each separate 24-hr stretch of data and computing the acrophase, or maximum predicted by the sine curve.3. The adaptation of the plasma steroid rhythm was assessed by the presence of a sharp fall in concentration after the sample collected around 08.00 hr. The time course of adaptation varied widely between individuals; it was usually largely complete by the fourth day after westward, and rather later after eastward, flights. After time shift the pattern often corresponded neither to an adapted nor to an unadapted one, and in a subject followed for many months after a real flight a normal amplitude only appeared 2-3 months after flight.4. Temperature rhythm adapted by a movement of the acrophase, without change in amplitude, although on some days no rhythm could be observed. This movement was always substantial even on the first day, and was usually nearly complete by the fifth.5. High nocturnal excretion of electrolyte was often seen in the early days after time shift, more notably after simulated westward flights. Adaptation of urinary electrolyte rhythms usually proceeded as with temperature, but the movement of the acrophase was slower, more variable between individuals, more erratic, and sometimes reversed after partial adaptation. On a few days there were two maxima corresponding to those expected on real and on experimental time.6. Sodium excretion was much less regular than that of potassium, but adapted more rapidly to time shift, so that the two often became completely dissociated. Chloride behaved much as sodium.7. The time course of adaptation of the plasma steroid and urinary potassium rhythms were sufficiently similar to suggest a causal connexion. The time course of adaptation of the temperature rhythm did not coincide with that of any other component considered here.

Adrenal Cortex Hormones↗

Overnight urinary 11-hydroxycorticosteroid estimations in diagnosis of Cushing's syndrome.

Overnight specimens of urine were collected from control, obese, and hirsute women and from 11 women with Cushing's syndrome. Urinary 11-hydroxycorticosteroid levels in the group with Cushing's syndrome were significantly higher than in the other three groups. This simple test has proved invaluable when screening for Cushing's syndrome in a busy outpatient clinic.

11-Hydroxycorticosteroids↗

Angiotensin II infusion increases vasopressin, ACTH, and 11-hydroxycorticosteroid secretion.

The effects of intravenous infusion of Asp1. Ile5-angiotensin II on blood pressure, plasma vasopressin, ACTH and 11-hydroxycorticosteroid levels and on plasma renin activity were studied in five trained, conscious dogs. The dogs were prepared with bilateral carotid loops. Infusion of angiotensin II at rates of 5, 10, and 20 ng/kg.min raised its plasma concentration from 23 +/- 7 to 48 +/- 8, 125 +/- 8, and 187 +/- 21 pg/ml, respectively. The lowest rate of infusion was mildly pressor, the two higher rates more so. All rates of infusion promptly increased vasopressin levels and depressed renin levels. The two higher rates also stimulated ACTH, although with a latency of 30-45 min. Since the rates of infusion of angiotensin II employed produced plasma levels within the physiological range, it is suggested that peripherally generated angiotensin II may play an important role in the regulation of vasopressin, and ACTH secretion.

11-Hydroxycorticosteroids↗