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.     

Quantitative studies on prostaglandin synthesis in man. II. Determination of the major urinary metabolite of prostaglandins F1 alpha and F2 alpha

A method was developed for quantitative determination of 5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α in man. The method was based on the use of the O-methyloxime derivative of [5β-³H; 10,10,12,12-²H₄]5α,7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid as internal standard and determination of ratios between unlabeled and deuterium-labeled molecules by multiple-ion analysis. Excretion values found for healthy human subjects were: males, $\text{10.8–59.0 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 24.0 ± 17.2 (SD) μg) and females, $\text{7.6–13.6 μ}\text{g}\text{24 hr}$ (n = 10, mean value, 10.5 ± 2.1 (SD) μg).     

A modified method for the estimation of pregnanediol, pregnanetriol and seven common 17-ketosteroids in urine by gas-liquid chromatography

A method is described for simultaneous determination of seven common urinary 17-ketosteroids, pregnanediol (5β-pregnane- 3α, 20α-diol) and pregnanetriol (5β-pregnane-3α,17α,20α-triol) by gas-liquid chromatography. The essential steps include hydrolysis of conjugates by an extract from $\text{Helix}$$\text{pomatia}$, use of 5β-cholestan-3α-ol as an internal standard, silylation and gas chromatography using 3% XE-60 as a stationary phase. The chromatograms were clear and the peaks easily identifiable. Laborious purification procedures were not necessary. 5β-pregnane-3α,20α-diol and 5β-pregnane-3α, 17α,20α-triol were measured in 22 adult males and 17 adult females. The subjects were trained hospital personnel, healthy and without medications. The results, in general, were in agreement with those reported by others.     

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.     

Intramolecular catalysis. VII: The nature of side chain shielding of the 12alpha-hydroxyl group of steroids

A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one ($\text{lb}$) was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one ($\text{2}$) and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid ($\text{5d}$). 21-Benzyl-5β-pregnan-12α-ol ($\text{1g}$) was synthesized by hydrogenation of the 21-benzylidine derivative of ketone $\text{1b}$. 23-Pheny1-5β-norcholan-12α-ol ($\text{1k}$) was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone $\text{1b}$, dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol ($\text{lm}$). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol ($\text{14}$), was synthesized by a Grignard reaction on the 21-bromo intermediate $\text{11b}$. Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups.     

Rate and diel periodicity of pheromone emission from female gypsy moths, (Lymantria dispar) determined with a glass-adsorption collection system

The rate of pheromone emission from wild and laboratory-reared gypsy moth (Lymantria dispar) (Lepidoptera: Lymantriidae) virgin females was determined with an all-glass aeration apparatus. This device incorporated a bed of 1-mm glass beads to extract entrained pheromone from the air flowing over the protruded gland. The temporal pattern of emission was established by monitoring individual females after eclosion for 24 consecutive 2-hr intervals.At a constant 24°C, both wild and laboratory females exhibited a similar diel periodicity of pheromone emission. The mean release rate increased after onset of photophase, generally attained maximal levels between 1600 and 2200 hr and declined during scotophase. Pheromone was released continuously and the mean daily emission increased with age for both wild and laboratory moths. The mean emission rate over the 48-hr monitoring interval was $\text{15.4 ng}\text{2 hr}$ for wild females vs $\text{14.7 ng}\text{2 hr}$ for laboratory moths. The peak emission from 2-day-old laboratory moths was ca.$\text{28 ng}\text{2 hr}$ compared with the ca.$\text{25 ng}\text{2 hr}$ released by their wild counterparts.The calling periodicity of laboratory females was determined at a constant 24°C and under a natural temperature rhythm. At 24°C, the proportion of females calling exceeded 45% throughout the diel period, whereas under the temperature rhythm, calling was virtually eliminated by temperatures below 15°C, indicating that temperature acts as an exogenous cue to modify the expression of the calling rhythm and thus potentially the periodicity of pheromone emission.     

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.     

Semiautomated enzymic micro methods for blood glucose and lactic acid on a single filtrate

Methods have been modified and adapted to permit enzymic automated micro analysis of glucose on 0.021 ml of blood, and of lactic acid on 0.0115 ml of blood. By means of a single 1:20 Somogyi filtrate with a final volume of 1–1.4 ml prepared from 0.1–0.15 ml blood, both determinations can be accomplished individually on the autoanalyzer.The range of concentrations of each method has been extended so that glucose may be determined between 5 and $\text{200 mg}\text{100 ml}$ (or higher depending on dilution) and lactic acid between 5 and $\text{125 mg}\text{100 ml}$.The methods are sensitive, reproducible, and stable. The recoveries and the comparison studies with manual methods are well within the limits of clinical investigation procdures.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Bile acid induction specificity of 7α-dehydroxylase activity in an intestinal Eubacterium species

The addition of cholic acid to growing cultures of $\text{Eubacterium}$ species V.P.I. 12708 caused a 25 and 46-fold increase in 7α-dehydroxylation activity using cell extracts or whole cell suspensions, respectively. Bile acid conversion rates using either [¹⁴C]-cholic acid or [¹⁴C]-chenodeoxycholic acid as substrates increased at approximately the same rate when either cholic or chenodeoxycholic acid was added to growing cultures as inducer. The induction of 7α-dehydroxylase activity was highly specific requiring a free C-24-carboxyl group and an unhindered 7α-hydroxy group on the B ring of the steroid nucleus. Unexpectedly, cholic acid also rapidly induced NADH:flavin oxidoreductase activity in growing cultures of this bacterium.     

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.     

Retro-steroids. I. Metabolism of the progestogen 6-chloro-9 , 10 -pregna-1,4,6-triene-3,20-dione in man

The metabolism of the retro-steroid 6-chloro-9β,10α:-pregna-1,4,6-triene-3,20-dione (I) has been investigated following oral administration of C-7 tritium labeled drug to a normal woman. Of the total radioactive dose, 47% and 30% were excreted in the urine and feces respectively within 7 days. About 20% of the urinary radioactivity was unconjugated while 70% was released following hydrolysis with β-glucuronidase. Qualitative analysis of a large urine pool from patients receiving I has resulted in the isolation and identification of three metabolites; 6-chloro-20α-hydroxy-9β, 10α-pregna-1,4,6-trien-3-one (II), 6-chloro-20α hydroxy-9β,10α-pregna-4,6-dien-3-one (III) and 20α-hydroxy-9β, 10α-pregna-1,4,6,8(14)-tetraen-3-one (IV). No intact I could be found in the urine indicating that the steroid undergoes complete metabolism $\text{in vivo}$.     

Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with methyl methanesulphonate

Rats treated with methyl methanesulphonate (MMS) excreted significantly higher quantities of deoxycytidine, thymidine, uracil, 1-methylnicotinamide (1-meNmd) and 1-methyl-6-pyridone-3-carboxylamide (6-pyr-1-meNmd) in their urine 0–24 h after MMS injection (100 $\text{mg}\text{kg}$). Excretion of thymidine, which was not detectable in untreated rats, was dose-dependent. No increase in urinary 7-methylguanine was found, and creatinine excretion was decreased by MMS treatment. Experiments with methyl-¹⁴C-labelled MMS showed transfer of ¹⁴C-label to 7-methylguanine and 1-meNmd. X-Irradiation (500 rad) caused increased excretion of pyrimidines, like MMS, but did not increase excretion of the nicotinamide derivatives.     

Dinoflagellate sterols IV: Isolation and structure of 4α,23ξ,24ξ-trimethylcholestanol from the dinoflagellate Glenodiniumhallii

The dinoflagellate $\text{Glenodinium}$$\text{hallii}$ was investigated for its sterol composition. Five of the six sterols were isolated and identified as cholest-5-en-3β-ol, (24ξ)-24-methylcholest-5-en-3β-ol, stigmasta-5,22-dien-3β-ol, (22E,24R)-4α,23,24-trimethyl-5α-cholest-22-en-3β-ol, and 4α,23ξ,24ξ-trimethyl-5α-cholestan-3β-ol.     

A rapid,sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol

A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at $\text{m}\text{e}$ 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.     

Synthesis and bioassay of 3-deoxy-1α-hydroxyvitamin D3, an active analog of 1α,25-dihydroxyvitamin D3

Two synthetic routes to 3-deoxy-1α-hydroxyvitamin D₃, an analog of 1α,25-dihydroxyvitamin D₃, are described. One involved the six-step conversion of 1α,2α-epoxy-6,6-ethylenedioxy-5α-cholestan-3- one to 1α-acetoxycholest-5-ene, whereas, in the second, the same intermediate was prepared from 1α-hydroxycholesterol. Conversion of the Δ⁵-sterol to the required 5,7-diene was accomplished most efficiently via 7-keto and 7-tosylhydrazone intermediates. Bioassay of 3-deoxy-1α-hydroxyvitamin D₃ in the rat establishes that the analog can fulfill all common vitamin D functions including stimulation of intestinal calcium transport, mobilization of calcium and phosphate from bone, stimulation of growth, and calcification of bone. Direct comparison indicates the compound to have $\text{1}\text{20}$ to $\text{1}\text{50}$ of the activity of 1α-hydroxyvitamin D₃, but it acts with a time course indistinguishable from the latter.     

Isolation of 4α-methyl-5α-Ergosta-8, 24 (28)-dien-3β-ol from ester fractions of nutritionally deprived neurospora crassa

A method of isolating pure fractions of 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol for sterol intermediate studies is described. Starvation cultures of $\text{Neurospora crassa}$ readily incorporate exogenous mevalonic acid into the sterol ester fraction. Isolation involves a simple solvent extraction and two chromatograms. Only the ester fraction yielded the required purity. Radioactive 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol is readily produced from DL-[2-¹⁴C] mevalonic acid.     

Effect of bombesin-stimulated gastrin on gastric acid secretion in man

The effect of bombesin on gastrin release and gastric acid secretion was investigated in 10 healthy volunteers. Bombesin (0.6 μg · Kg^?1 · hr^?1) produced a significantly higher $\text{(}\text{p}\text{<}\text{0.001)}$ increase in plasma gastrin levels (86.7 11.1 pmo/1 than after a protein meal (39.6 ± 5.6 pmol1/1). The gastric acid secretory response to bombesin (12.1 ± 2.9 mEq · hr^?1) was however significantly lower $\text{(}\text{p}\text{<}\text{0.005)}$ than the maximal response produced by pentagostrin (20.9 ± 3.5 mEq · hr^?1) at the dose of 6 μg · Kg^?1. Atropine did not modify gastrin release induced by bombesin but significantly reduced gastric acid secretion $\text{(}\text{p}\text{<}\text{0.01)}$. From the data presented it may be hypothesized that less biologically active forms of gastrin and/or other peptides inhibiting the gastrin effect upon gastric acid secretion may be released by bombesin.     

Minor and trace sterols in marine invertebrates. 34 Isolation and structure elucidation of 24-methyl-5α-cholesta-7,9(11), 24(28)-trien-3β-ol,the first naturally occurring Δ7, 9(11) unsaturated marine sterol

The lipid fraction of a Black Sea sponge, $\text{Haliclona flavescens}$, was investigated for its sterol composition. Twenty-three sterols were identified, including the hitherto unknown 24-methyl-5α-cholesta-7, 9(11), 24(28)-trien-3β-ol ($\text{3}$). Identification and structure elucidation were based upon 360 MHz ¹H-NMR and MS analyses.     

Identification of a new glutathione S-transferase from rat liver cytosol

A new glutathione $\text{S}$-transferase has been purified to homogeneity from 105,000 × g supernatant of Sprague-Dawley rat liver homogenates. The purified enzyme exhibited specific activities of approximately 1.8, and 0.12 μmoles. min^?1. mg^?1 toward 1-chloro 2,4-dinitrobenzene and cumene hydroperoxide respectively. The SDS gel electrophoresis data on subunit composition revealed that the new transferase is composed of two subunits with an identical M_r of 24,400 (Y_α Family). Our $\text{in}\text{vitro}$ translation experiments with rat liver poly(A) RNAs and substrate specificity data suggest that this subunit is different from the previously reported Ya, Yb and Yc subunits of rat liver glutathione $\text{S}$-transferases. Comparatively, the new isozyme showed significant activity toward 1,2 epoxy-3-(P-nitrophenoxy)-propane, ethacrynic acid and P-nitrophenyl acetate, 0.4, 0.34 and 0.18 μ moles. min^?1. mg^?1 respectively.