Age-Related decrease in calcitonin gene-related peptide mRNA in the dorsal root ganglia of spontaneously hypertensive rats.

Age-related changes in levels of calcitonin gene-related peptide (CGRP) mRNA of the dorsal root ganglia was studied in 8-, 12- and 15-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). CGRP mRNA levels in SHR but not in WKY decreased with age. The contents of CGRP-like immunoreactivities in the atrium and mesenteric artery of 15-week-old SHR were greater than those in age-matched WKY. These results suggest that the outflow of CGRP-containing nerves from the spinal cord and CGRP release from CGRP nerve terminals decreases in SHR.

Decreased depressor response mediated by calcitonin gene-related peptide (CGRP)-containing vasodilator nerves to spinal cord stimulation and levels of CGRP mRNA of the dorsal root ganglia in spontaneously hypertensive rats.

The depressor response to electrical stimulation of the spinal cord and the level of calcitonin gene-related peptide (CGRP) mRNA in the dorsal root ganglion (DRG) in the spontaneously hypertensive rat (SHR) was compared with the normotensive Wistar Kyoto rat (WKY) and Wistar rat (WR). The animals were pithed by inserting a stainless-steel rod into the spinal cord. Pithed rats were treated with hexamethonium (2 mg/kg/min i.v.) to block autonomic outflow, and mean arterial blood pressure (MBP) was maintained at approximately 100 mmHg with continuous infusion of methoxamine (10 to 15 microg/kg/min i.v.). Electrical stimulation (2 and 4 Hz for 30 s) of the lower thoracic spinal cord (T9-12) via the pithing rod caused a frequency-dependent depressor response without a change in heart rate. The depressor response to spinal cord stimulation was significantly smaller in SHR than in WKY and WR. Long-term treatment of 8 week-old SHR with captopril (0.1% in drinking water) for 7 weeks restored the reduced depressor response to spinal cord stimulation. The level of CGRP mRNA in DRG of SHR was significantly lower than that in WKY. These results suggest that the function of CGRP-containing nerves from the spinal cord decreases in SHR and captopril treatment prevents its reduction.

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats.

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 10(10) counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7alpha-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-beta-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an important role in the formation of secondary bile acids and coprostanol in rats.

Analysis of bile acids in urine specimens from healthy humans: determination of several bile acids with beta-hydroxyl and carbonyl groups.

Urinary bile acids of 39 healthy male undergraduates were analyzed by capillary gas chromatography and capillary gas chromatography-mass spectrometry. 3 alpha-Hydroxy-12-oxo-5 beta-cholanoic acid, 3 alpha, 12 beta-dihydroxy-5 beta-cholanoic acid, 3 beta, 7 alpha-dihydroxy-5 beta-cholanoic acid, and 3 alpha, 7 alpha, 12 beta-trihydroxy-5 beta-cholanoic acid, in addition to known bile acids, were identified and then quantified. The major part of the urinary bile acids was occupied by secondary bile acids. Every 7 beta-hydroxylated bile acid species was found in more than 80% of the subjects. The bile acid detected in the largest amount was 3 alpha-hydroxy-12-oxo-5 beta-cholanoic acid. The metabolites of cholic acid were quantitatively more predominant than those of chenodeoxycholic acid. These results indicate that bile acids with beta-hydroxyl and carbonyl groups at the C-3,7 and/or 12 positions are usual bile acids usually found in the urine of healthy humans. It is concluded that the occurrence of these bile acids is an effect of the intestinal bacterial flora and living conditions.

Conjugation types of 7 beta-hydroxylated bile acids detected in healthy human urine.

Bile acids extracted from the urine of a healthy volunteer who excreted 7 beta-hydroxylated bile acids were fractionated to nonamidated, glycine-conjugated, taurine-conjugated, and sulfated bile acid fractions. The chemical conjugation types of the 7 beta-hydroxylated bile acids were then determined by treatment with several enzymes and by capillary column gas chromatography. Large amounts of 3 alpha,7 beta,12 alpha-trihydroxycholanoic acid were present as nonamidated and nonconjugated bile acids, while 3 beta,7 beta-dihydroxycholanoic acid formed nonamidated bile acid N-acetylglucosaminide. In addition, ursodeoxycholic acid formed both glycine-conjugated bile acid and glycine-conjugated bile acid N-acetylglucosaminide. Bile acid N-acetylglucosaminides were hydrolyzed by solvolysis.

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system.

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.

Determination of 17-hydroxyprogesterone in plasma by gas chromatography-mass spectrometry with high-resolution selected-ion monitoring.

A method for determining 17-hydroxyprogesterone in plasma by isotope dilution-mass spectrometry is described. For the internal standard 17-hydroxy [2H4]progesterone is used. Extraction of plasma is followed by conversion into the 3,20-dienol,17-tristrimethylsilyl ether derivative and analysis by capillary gas chromatography-mass spectrometry with selected-ion monitoring, at a resolution of 6000. The lower limit of quantitation was 1 pg, judged from a criterion of a signal-to-noise ratio of 10. The precision and accuracy of the method were satisfactory.

Bile acids of patients with renal failure (possibility of bile acid secretion in the distal tubule).

Bile acids of patients with renal failure and of healthy subjects were analyzed by capillary gas chromatography after group separation. The amount of bile acids in the total dialysate (150 L) of the patients was smaller than that in the 24-h urine of healthy subjects. Polar bile acid sulfates constituted 17.3% and 30.9% of the total bile acids in serum and urine of healthy subjects, respectively, 26.0% in predialysis serum of patients, and only 11.3% in dialysate of patients. The amount of bile acid sulfates in the hemodialysate converted during a 24-h period dialysis, was still smaller than that in 24-h urine of healthy subjects. We propose that the distal tubule secretes bile acid sulfates.

Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry.

A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with 2H2O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using [2H]diethylene glycol and [2H]hydrazine hydrate afforded [11,11,12,12,23,23(-2)H]lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-[11,11,12,12(-2)H]pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-[11,11,12,12(-2)H] progesterone (XIV), consisting of 0.3% 2H0-, 1.1% 2H1-, 8.6% 2H2-, 37.1% 2H3-, 52.1% 2H4-, and 0.8% 2H5-species.

Internal standards for quantitative gas chromatography of individual bile acids after group separation of bile acids in urine.

Sodium glyco-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oate, sodium tauro-7 beta,12 beta-dihydroxy-5 beta-cholan-24-oate and disodium glyco-7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oate 7 alpha-sulphate have been synthesized for the first time. These compounds, together with 7 beta,12 beta-dihydroxy-5 beta-cholan-24-oic acid, which were added to a sample prior to extraction, were found to be useful as internal standards for determination by gas chromatography of individual bile acids in each fraction after group separation of urinary bile acids.

Bile acids of patients with renal failure receiving chronic hemodialysis.

Bile acids in serum, urine and dialysate of 8 patients with renal failure in chronic hemodialysis were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following results were obtained: 1. Lithocholic acid, 3 beta-hydroxy-5-cholen-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were identified in hemodialysate as well as in serum and urine. 2. The serum bile acid concentration of the patients was 2.78 +/- 0.57 micrograms/mL before hemodialysis and 1.34 +/- 0.48 micrograms/mL after a 5-h period hemodialysis with cuprophane membrane. The proportions of secondary bile acids in predialysis and postdialysis serum of patients were significantly higher than those of healthy subjects. 3. Two out of 8 patients excreted urine. But the amounts of bile acids in urine of the patients were very small compared to those of healthy subjects. 4. The amount of bile acids removed from blood by hemodialysis was 0.70 +/- 0.25 mg. In dialysate, cholic acid constituted a larger proportion of the total bile acids, and lithocholic acid a smaller proportion, when compared to those in urine of patients and healthy subjects.

7 beta,12 beta-Dihydroxy-5 beta-cholan-24-oic acid as an internal standard for quantitative determination of bile acids by gas chromatography.

In order to find an artificial internal standard compound for quantitative determination of bile acids by gas chromatography, 7 alpha,12 alpha-, 7 alpha, 12 beta-, 7 beta, 12 alpha- and 7 beta,12 beta-dihydroxy-5 beta-cholan-24-oic acids were chemically synthesized with cholic acid (1) as the first starting material. The gas chromatographic retention time of 7 beta,12 beta-dihydroxy-5 beta-cholan-24-oic acid (beta beta-isomer) was more different from that of natural bile acids than the other isomers. Moreover, beta beta-isomer was extracted in the same fraction as the bile acids from urine, and no urinary substance had the same retention time as beta beta-isomer. No artifact was produced from beta beta-isomer during the analysis procedure. It was concluded that the beta beta-isomer is an internal standard compound with certain advantages for the quantitative determination of bile acids in urine by gas chromatography, irrespective of the recovery rate during the analysis procedure.

Intraocular fate of dexamethasone disodium phosphate topically applied to the eyes of rabbits.

After instillation of 3H-dexamethasone into the eyes of a rabbit, 3H-9alpha-fluoro-11beta-hydroxy-16alpha-methyl-1,4-androstadiene-3,17-dione was found in the aqueous humor. The same metabolite was also formed by incubating 3H-dexamethasone with the anterior ocular tissues of rabbit. Identification of 3H-9alpha-fluoro-11beta-hydroxy-16alpha-methyl-1,4-androstadiene-3,17-dione was performed by its mobility on a thin layer plate and by proving its radiochemical homogeneity after recrystallization with the unlabeled sample which had been synthesized from dexamethasone by oxidation with sodium bismuthate. When dexamethasone disodium phosphate was instilled into rabbit's eyes, it was hydrolyzed to free dexamethasone and then metabolized to 9alpha-fluro-11beta-hydroxy-16alpha-methyl-1,4-androstadiene-3,17-dione.

Characterization of the microsomal steroid-8-ene isomerase of cholesterol biosynthesis.

Rat liver microsomes contain an enzyme that catalyzes the isomerization of the nuclear double bond of steroids from the 8(9) position to the 7(8) position. The enzyme is most active with zymosterol, 5alpha-cholesta-8,24-dien-3beta-ol, which is a precursor of cholesterol. Properties of the microsomal isomerase have now been studied, and preliminary data are reported on both regulation of enzymic activity and first steps in the solubilization of the enzyme from membranes. After a brief lag period, the velocity of isomerase is relatively constant for about 5 min of incubation, and then isomerization subsides. The apparent Michaelis constant (52-70 micro M) is difficult to determine accurately, due to these complex kinetic changes. V(max) is 4.0-4.7 nmol/min per mg of microsomal protein. The apparent specific activity is more than ten times that of liver microsomal methyl sterol oxidase. The maximal specific activity of microsomal isomerase is approximately doubled when rats are fed an intestinal bile acid sequestrant, cholestyramine. Changes in specific activity of isomerase parallel changes in activities of other microsomal enzymes of cholesterol biosynthesis, such as 3-hydroxy-3-methylglutaryl-CoA reductase and 4-methyl sterol oxidase. Isomerase activity is destroyed by phospholipase A digestion, high concentration of bile salts, and solvent extraction, all of which are known either to remove phospholipid or to alter microsomal membrane integrity. On the other hand, isomerase remains active in the presence of a mild, nonionic detergent, Triton WR-1339; thus, solubilization with nonionic detergents is under study.