Quantitative studies on prostaglandin synthesis in man. 3. Excretion of the major urinary metabolite of prostaglandins F 1 alpha and F 2 alpha during pregnancy

The mean urinary excretion of 5α, 7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α, in eight women in pregnancy weeks 37–40 was 32.5 ± 12.2 μg/24 hours, representing a 2–5 fold increase compared to non-pregnant women. Determination of the metabolite throughout pregnancy in three subjects showed that there was a gradual increase in the urinary excretion as pregnancy progressed with maximum excretion (4–5 times the individual pre-pregnant value) at the end of pregnancy. After pregnancy there was a rapid normalization back to the pre-pregnant excretion value.     

Radioimmunoassay for urinary metabolites of prostaglandin F2α

Antibodies against the main urinary metabolite of PGF_2α in the human, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the ω position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the ω carbon, and thus the assay could be used also for other metabolites of PGF_2α, e.g. the main urinary metabolite in the guinea pig, 5α,7α-dihydroxy-11-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either $\text{in vivo}$ by injection of |17,18-³H|-PGF_2α into humans after several days' treatment with indomethacin, or $\text{in vitro}$ by incubation of |17,18-³H|-15-keto-13,14-dihydro-PGF_2α with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively.The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF_2α; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment.The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.     

Determination of zinc by flameless atomic absorption spectrophotometry

The flameless atomic absorption method described here is a simple, rapid, accurate microtechnique for determining zinc in aqueous solutions, serum, or urine. It requires no sample pretreatment, only 1.0 μl of sample per determination, no correction for viscosity differences between sample and standard solutions, and is not subject to ionic or organic interference. The average recovery of added zinc in serum is 97.5% and in urine is 97.6%. The values obtained for serum (mean ± SD: 94.6 ± 11.0 μAg/100 ml; N = 25) and urine (range: $\text{sol}\text{600–1000 μg}\text{24}\text{hr}$; N = 4) are comparable to the values reported in the literature. The coefficient of variation was less than 5.0% in all cases. The qualitative concentration limit was $\text{0.009}\text{μg}\text{100}\text{ml}$. The techniques and instrumentation described are also applicable to a number of trace minerals of common interest.     

Radioimmunoassays of prostaglandin F2α and prostaglandin F2α-main urinary metabolite with prostaglandin-125I-tyrosine methylester amide

Radioimmunoassays for measuring prostaglandin F_2α (PGF_2α) and 5α, 7α-dihydroxy-11-keto tetranorprosta-1,16-dioic acid, PGF_2α-main urinary metabolite (PGF_2α-MUM), with ¹²⁵I-tyrosine methylester amide (TMA) of PGF_2α and PGF_2α-MUM were developed.Antibody to PGF_2α was produced in rabbits immunized with conjugates of PGF_2α coupled to bovine serum albumine. Antibody to PGF_2α-MUM was also produced in rabbits immunized with conjugates of PGF_2α-MUM coupled to bovine serum albumin.PGF_2α-¹²⁵I-TMA had an affinity to antiserum to PGF_2α. PGF_2α-MUM-¹²⁵I-TMA also responded to antiserum to PGF_2α-MUM.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2α in normal subjects

Radioimmunoassay of 5α,7α-dihydroxy-11-keto-tetranorprosta-1, 16-dioic acid, main urinary metabolite of prostaglandin F F_2α (PGE_2α-MUM), was performed in normal subjects. Twenty-four hours secretion of PGF_2α-MUM were 29.04 ± 9.73 μg in males and 18.22 ± 5.88 μg in females on an average. And an oral administration of aspirin resulted in the remarkable decrease of PGF_2α-MUM in both sexes.     

Inhibition of prostaglandin synthesis in man

7α-Hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins E₁ and E₂ in man, was determined in human urine by a method based on the use of the bis (O-²H₃-methyloxime) derivative of dimethyl 7α-hydroxy-5,11-diketotetranor-prostane-1,16-dioate as internal standard and determination of the ratio between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Male subjects excreted larger amounts of the metabolite (6.5–46.7 μg/24 hours, n=10) than did female subjects (2.5–5.3 μg/24 hours, n=10). The excretion rate was strongly suppressed following oral administration of therapeutic doses of indomethacin, aspirin and sodium salicylate.     

11-dehydrocorticosterone (compd. A) formation by the Microtus adrenal

No 11β-hydroxysteroids were detected after 30 minutes incubations of progesterone-4-¹⁴C and pregnenolone-7α-³H with adrenals of $\text{Microtus pennsylvanicus}$. 11-Dehydrocorticosterone (Compd. A) was isolated as the major product and its identity confirmed by crystallization to constant specific activity. A tetrahydro derivative, 3α, 21-dihydroxy-5β-pregnane-11, 20-dione and an 18-hydroxy derivative, 18, 21-dihydroxy-4-pregnene-3,11, 20-trione were tentatively identified based-on Chromatographic behavior. The same products were observed with male adrenal and NADPH and with female adrenal using a NADPH generating system. Since the plasma manifested the typical fluorescence characteristics of corticosterone, the in vitro production of 11-keto steroids is considered to be the result of unusually high activity of the 11β-hydroxysteroid dehydrogenase in the $\text{Microtus}$ adrenal.     

Assay of liver microsomal cholesterol 7 alpha-hydroxylase using deuterated carrier and gas chromatography-mass spectrometry

The activity of cholesterol 7α-hydroxylase in rat liver microsomes was assayed by measuring the mass of 5-cholestene-3β, 7α-diol formed from endogenous cholesterol under standardized incubation conditions. After termination of incubations, a known amount of 5-[24,25,7β-²H₃]cholestene-3β,7α-diol was added. A chloroform extract of the incubation mixture was subjected to thin layer chromatography and the fraction containing 5-cholestene-3β,7α-diol was converted into trimethylsilyl ether. The trimethylsilyl ether was subjected to combined gas chromatography-mass spectrometry and the amount of unlabeled 5-cholestene-3β,7α-diol in the mixture was calculated from the ratio between the relative intensitics of the peaks at $\text{m}\text{e}$ 456 (M-90) and $\text{m}\text{e}$ 459 [M-(90 + 3)]. The precision of the method was ±2.2% (SD). The results with this method of assay of cholesterol 7α-hydroxylase were compared with those obtained with a method based on conversion of a trace amount of added [4-¹⁴C]cholesterol into 5-cholestene-3β,7α-diol.     

The content of water and potassium in fat cells

The distribution spaces at equilibrium for ³H₂O, [¹⁴C]urea and $\text{3-O-[}{}^{14}\text{C]-}\text{methylglucose}$ were measured in white fat cells using centrifugation through silicone oil at 2500 × g; no significant differences were observed. l-[¹⁴C] Glucose added immediately before the centrifugation was used as a marker for the extracellular water space. The calculated intracellular water content of the cells after the centrifugation through oil (e.g. ³H₂O space minus l-[¹⁴C] glucose space) is an unbiased measure of the water content of the cells in suspension as judged by the following criteria: (1) The intracellular distribution space for $\text{3-O-[}{}^{14}\text{C}\text{]}$methylglucose at equilibrium (methylglucose space minus l-glucose space) was not different from that calculated from a methylglucose wash-out curve. (2) The intracellular content of l-[¹⁴C]glucose (half time of efflux about 60 min) in cells preloaded during incubation of the tissue with collagenase was not different in cells recovered by (a) centrifugation through oil at 2500 × g, (b) centrifugation through oil at 600 × g, (c) centrifugation at 600 × g in the absence of oil and (d) filtration on Millipore filters.The intracellular content of water determined on cells from single rats weighing 120–150 g was 2.75 ± 0.55 μl/100 μl fat cells (± S.D., n = 30). The intracellular content of potassium, determined on cells from the same rats, was 252 ± 62 nmols/100 μl fat cells (± S.D., n = 30). The concentration of potassium in the intracellular water was calculated as 104 ± 15 mM (± S.D., n = 30).     

The effect of indomethacin on the biosynthesis and metabolism of prostaglandin F2α in man

Healthy volunteers received 60 μg of [8,10,10-²H₃] PGF_2α by intravenous infusion both before and during a course of treatment with indomethacin (200 mg/day). Excretion of deuterated 5α, 7α-dihydroxy-11-ketotetranor-prostane-1, 16-dioic acid in urine was quantified by GC-MS using a reverse stable isotope dilution procedure. Indomethacin was found to have no detectable effect on the metabolism of the labelled PGF_2α whereas output of the endogenous metabolite was markedly reduced by the effect of the drug on prostaglandin biosynthesis.     

Parathyroid hormone is not involved in 1,25-dihydroxyvitamin D regulation of 25-hydroxyvitamin D metabolism in vivo

1,25-Dihydroxyvitamin D₃ administration to vitamin D-deficient rats suppresses accumulation of 1,25-dihydroxy-[3α-³H]vitamin D₃ and stimulates accumulation of 24,25-dihydroxy-[3α-³3H]vitamin D₃ from 25-hydroxy-[3α-³H]vitamin D₃ equally well in the presence and absence of parathyroid glands. These results demonstrate that this regulatory action is not mediated by the parathyroid glands and support conclusions from $\text{in}\text{vitro}$ studies that this represents a direct action of 1,25-dihydroxyvitamin D₃.     

Differential block by 1-hyoscyamine of the salivary and vascular responses of the dog mandibular gland to prostaglandin F2alpha.

Prostaglandin F_2α (PGF_2α) (3–300 pmol) administered to the dog mandibular gland $\text{via}$ the glandular artery produced salivation and an increase in blood flow rate in a dose-related manner. The salivary responses to PGF_2α and to electrical stimulation of the chorda-lingual nerve were abolished by intra-arterial infusion of $\text{1}$-hyoscymine (30 nmol/min), whereas the vascular responses to both were not affected. The salivary and vasodilator responses to PGF_2α were not affected by intra-arterial infusion of hexamethonium (0.6–2 μmol/min) which abolished those to stimulation of the chordalingual nerve. These results support the prevous conclusion that PGF_2α produces the two responses by exciting the parasympathetic ganglion or postganglionic neurons in the dog mandibular gland.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Gas chromatographic assay of urinary pregnanediol

A simple micro method for the estimation of urinary pregnanediol (5β-pregnane-3α,20α-diol) is described. Chloroform extracts of acid-hydrolyzed urine are assayed gas chromatographically at 235° using Gas Chrom Z coated with 0.5 gm % NPGA. Thirty urine specimens may be prepared for gas chromatography in 1.5 hr. The rate of hydrolysis of pregnanediol glucuronide is proportional to the concentration of acid, and the rate of decomposition of pregnanediol to the square of acid concentration. Normal pregnant women excreted a mean of $\text{7 mg}\text{24 hr}$ pregnanediol in the first trimester, $\text{20 mg}\text{24 hr}$ in the second, and $\text{36 mg}\text{24 hr}$ in the third. During the luteal phase of the menstrual cycle the excretion of pregnanediol by normal women increased from $\text{<}\text{0.3 mg}\text{24 hr}$ to $\text{5–7}\text{mg}\text{24}\text{hr}$.     

Metabolism of two PGF2α analogues in primates: 15(S)-15-methyl-Δ4-cis-PGF1α and 16,16-dimethyl-Δ4-cis-PGF1α

The metabolism of the prostaglandin F_2α analogues, 15-methyl-Δ⁴-$\text{cis}$-PGF_1α and 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α, has been investigated in the cynomolgus monkey and the human female. The two analogues, tritium labelled in the 9β-position, were administered by intramuscular injections into the monkeys and by subcutaneous injections into the human. Excretion of tritium labelled products were followed in urine (in both species) and feces (in monkeys only) and several metabolites were identified by GC/MS. The analogues were found to be resistant to the 15-hydroxy dehydrogenase and furthermore the degradation by β-oxidation was delayed. About 13% of the given dose of 15-methyl-Δ⁴-$\text{cis}$-PGF_1α was excreted unchanged into urine and feces from the monkey. The corresponding figure for 16,16-dimethyl-Δ⁴-$\text{cis}$-PGF_1α was about 20%. In addition, a large part of the metabolites had the carbon skeleton intact and were only metabolized by ω-oxidation. The relative resistance to degradation of these two analogues is likely to be the basis for their prolonged pharmacological activity.     

Determination of vanilmandelic acid in pig urine and chicken feces by gas-liquid chromatography

A gas chromatography method for the determination of free and bound vanilmandelic acid (VMA) in pig urine and chicken feces has been developed. The method consisted of extraction of the free or bound acids by ethyl acetate under acidic conditions. The ethyl acetate extracts were dried under nitrogen, followed by complete silylation of the phenolic and carboxylic acid groups with BSA (N,O)-bis (trimethylsilyl) acetamid. The solution was distilled at 180°C in a sealed glass tube after which the sample was injected on a stainless steel column (6 ft × .125 in. o.d.) containing 4% SE-30 on $\text{80}\text{100}$ mesh chromosorb GHP. The recovery of the urinary VMA was 82%, and the fecal VMA was 84% through the outlined procedures. Pigs ranging in age from 8 to 12 weeks were found to excrete $\text{2–8 mg urinary VMA}\text{24 hr}$ with no significant difference between the free and bound. Commercial laying hens excreted bound VMA in a range of $\text{1–5 mg}\text{24 hr}$ with a $\text{F}\text{B}$ ratio of 1:3.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2α

Radioimmunoassay of 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, main urinary metabolite of prostaglandin F₂α (PGF₂α), was performed using an antiserum produced in the rabbit.The antibody in 100 μ1 of 1,600-fold diluted antiserum binds with 60 picograms of metabolite.The main urinary metabolite level fell when flufenamic acid, a prostaglandin synthetase inhibitor, was given to rats. In contrast, it was significantly elevated when PGF₂α was administered.     

Incorporation of [1-14C]-acetate into testosterone,androstenediol, dehydroepiandrosterone and progestogens by ram testis following human chorionic gonadotropin administration

Testicular steroidogenesis in rams was examined by constant infusion (3 hr) of [1-¹⁴C]-acetate into the testicular artery of four conscious standing animals.The following steroids (in order of decreasing levels of [¹⁴C] labeling) were secreted by the testis and found in testicular tissue: testosterone, dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one, androstenediol, 5-androsten-3β,17β-diol and 17-hydroxy-4-pregnene-3,20-dione. In addition, [¹⁴C] labeling of 17,20α-dihydroxy-4-pregnen-3-one occurred in testicular tissue but not in blood. This $\text{in vivo}$ system with the conscious standing ram demonstrated an operative Δ⁵ steroidal pathway to testosterone. The physiological significance of 17,20α-dihydroxy-4-pregnen-3-one is not yet explained in this species.     

The occurrence and subcellular localization of 3 -hydroxysteroid dehydrogenase in adult rat brain

The subcellular localization of 3α-hydroxysteroid dehydrogenase, the dependency of the rate of reaction on time, concentration of protein, cofactor requirements and the substrate stereospecificity were investigated in the adult rat brain. The $\text{in vitro}$ conversion of 3-keto-5β-cholanoic-24-¹⁴C acid to lithocholic acid was shown to occur in the cytosol without added cofactors. Incubation of ¹⁴C labeled 3α,7α-dihydroxy-5β-cholanoic and 3α,12α-dihydroxy-5β-cholanoic acids with adult rat brain cell-free preparations resulted in the production of less polar metabolites identified as 3-keto-7α-hydroxy-5β-cholanoic and 3-keto-12α-hydroxy-5β-cholanoic acids by TLC, GLC combined with a radioactive monitoring detection system and by cocrystallization to constant specific activity.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.