Isolation and identification of alpha-methyloctanylcarnitines from human urine

Medium-chain acylcarnitines were isolated from human urine using a combination of chloroform-methanol extraction, silicic acid column and molecular sieving chromatography and preparative HPLC. Three purified acylcarnitines were analyzed by fast atom bombardment mass spectrometry and were also saponified and the free fatty acids analyzed by gas chromatography and mass spectrometry. Combined electron impact mass spectrometry and fast atom bombardment mass spectrometry and periodate oxidation for location of double bonds, demonstrated the occurrence of delta 6-octenylcarnitine, 2-methyloctanylcarnitine and 2-methyl-delta 6-octenylcarnitine. These acylcarnitines were present in the thirteen urines obtained from normal humans, but were not detected in urines from three individuals who had been on total parenteral nutrition for more than a year. The occurrence of alpha-methyl medium-chain acylcarnitines in human urine indicates a role for carnitine in excretion (detoxification) of these acyl derivatives.     

Isolation and identification of L-beta-aspartyl-Llysine and L-gamma-glutamyl-L-ornithine from normal human urine.

L-beta-Aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine were isolated from pooled normal human urine and each peptide was shown to be identical with the authentic peptides. The concentrations of these dipeptides in the urine of individual subjects were determined directly by using a new buffer sequence on a standard ion exchange chromatographic amino acid analyzer with a sensitivity of 10(-10) mol. In urine from normal subjects ranging in age from 12 to 64 years, mean values of 1.47 mumol/g of creatinine of L-gamma-glutamyl-L-ornithine and 8,24 mumol/g of creatinine of L-beta-aspartyl-L-lysine were found. The urine of children under 10 years of age contained, relative to creatinine excretion, more L-beta-aspartyl-L-lysine and L-gamma-glutamyl-L-ornithine than that of older children and adults. All urines contained substantially larger concentrations of L-beta-aspartyl-L-lysine than of L-gamma-glutamyl-L-ornithine. Both peptides were found in urine collected after 21 hr of fasting in lower concentrations than found in urine from nonfasting subjects. The urinary concentrations of both peptides did not appear to be influenced by race or sex.     

Isolation and identification of an endogenous metabolite of 18-oxocortisol from human urine

The naturally occurring mineralocorticoid agonist, 18-oxocortisol, is secreted in increased amounts in two hypertensive syndromes. One is primary aldosteronism and the other a genetic disorder first described by Sutherland and co-workers in which aldosterone secretion is ACTH-dependent and the mode of inheritance is autosomal dominant. 18-Hydroxy and -oxocortisol are the components of the cortisol oxidation pathway which arise when cortisol becomes an alternate substrate for corticosterone methyl oxidase. This enzyme system normally resides in the glomerulosa zone of the mammalian adrenal cortex. In an effort to account for a larger fraction of 18-oxocortisol and provide a reliable index of its secretion and of the expression of the cortisol C-18 oxidation pathway, metabolites were sought in the urine of a patient with the ACTH-dependent autosomal dominant form of aldosteronism. Using a variant of the technique of reverse isotope dilution, a pool of [3H]-labeled urinary metabolites form a normal subject was mixed with the patient's urine and subjected to customary methods of hydrolysis for urinary steroids. The radiolabeled glucuronide fraction was the most abundant and was subjected to repeated HPLC fractionation to yield the predominant component. The evidence from gas chromatography-mass spectrometry indicated that this metabolite was a tetrahydro derivative. The structure of the isolated tetrahydro 18-oxocortisol was confirmed by a biosynthesis of a reference standard from 18-oxocortisol and a 5 beta-pregnane reductase preparation.     

Isolation and identification of hydroxyproline analogues of bradykinin in human urine

Hydroxyproline (Hyp) analogues of bradykinin and lysyl-bradykinin, in which the third residue of bradykinin, proline, is replaced by hydroxyproline, were isolated from human urine. Their amino acid sequences were confirmed by both amino acid and sequence analyses, and also by comparison of their chromatographic behavior with that of synthetic peptides. The possibility that Lys-Ala3-bradykinin, isolated by Mindroiu et al. [(1986) J. Biol. Chem. 261, 7407-7411] from human urine, was actually Lys-Hyp3-bradykinin is discussed.     

Isolation and identification of urinary beta-aspartyl dipeptides and their concentrations in human urine.

beta-Aspartyl-methionine, -aspartic acid and -glutamic acid and gamma-glutamyl-threonine and -glycine were isolated and identified in human urine by ion-exchange chromatography, high-voltage paper electrophoresis, acid hydrolysis and determination of N-terminal amino acids of the isolated compounds, and comparison of their behaviors in paper electrophoresis and chromatography with those of the authentic compounds. The concentrations of acidic beta-aspartyl dipeptides in human urine were determined using an amino acid analyzer. Their concentrations were as follows: beta-aspartyl-glycine, male, 44.4 +/- 8.5, female, 61.4 +/- 18.9, child, 83.7 +/- 27.1; -alanine, male, 11.0 +/- 4.9, female, 20.7 +/- 12.0, child, 25.3 +/- 9.1; -glutamic acid, male, 10.0 +/- 3.7, female, 23.0 +/- 8.5, child, 20.4 +/- 7.5; -serine, male, 9.9 +/- 2.8, female, 13.6 +/- 3.8, child, 14.9 +/- 4.7; -aspartic acid, male, 4.3 +/- 1.0, female, 9.1 +/- 2.2, child, 18.4 +/- 6.5; -threonine, male 3.9 +/- 0.9, female, 5.8 +/- 1.1, child, 13.2 +/- 4.9 mumol/g creatinine (mean +/- S.D.). The order of the sum of their concentrations tended to be child greater than female greater than male. Patients receiving intravenous hyperalimentation also excreted acidic beta-aspartyl dipeptides into urine in amounts similar to those in females and in a pattern similar to that observed in healthy persons. This finding indicates that urinary beta-aspartyl dipeptides were probably of endogenous origin because oral nutrition was stringently excluded in these patients.     

Isolation and identification of androstanediol glucuronide from human plasma

[3H]Dihydrotestosterone (50 microCi) was infused into normal men and women for 8 h. It was previously shown that this was sufficient time for this material to reach a steady state. Venous plasma was obtained at 6 and 8 h, pooled, and the unconjugated steroids removed by ether extraction. The remaining plasma was adjusted to pH 4.9 and the steroid conjugate was extracted first with ethyl acetate and then with an ether-ethanol mixture. The extracts were combined and taken to dryness. Steroid sulfates were solvolyzed using dioxane, and the mixture partitioned between ether and 1% NaOH. The aqueous phase was acidified and added to an XAD-2 column, washed with water, and the glucuronide fraction eluted with methanol. The solvent was concentrated and the methanol extract was passed through a C18 Sep-Pak, filtered through an Acrodisc CR and then subjected to gradient high performance liquid chromatography [HPLC] (Nova-Pak C18, KH2PO4, pH 3, and methanol). The fractions containing steroid glucuronides were collected and esterified with diazomethane and then acetylated with acetic anhydride in pyridine. The glucuronide triacetyl methyl ester (GAME) derivatives were then run in a second HPLC system (3 Lichrosorb 5 mu columns, 4 mm x 25 cm) using a gradient of ethanol-heptane and heptane. We clearly established that this system separates 3 alpha-diol GAME conjugated at the 17 and 3 positions (44 vs 50 min) with authentic samples previously synthesized in our laboratory. We concluded that the pooled plasma contained only the 17-GAME conjugate. No significant activity of the 3-glucuronide was detected. The natural compound in circulation, therefore, is 5 alpha-androstane-3 alpha, 17 beta-diol 17-glucuronide.     

4-hydroxyestrone,isolation and identification in human urine

Portions of pregnancy and midcycle urines were submitted to hot acid hydrolysis, extracted with benzene/ethyl acetate and the extracts washed with ascorbic acid buffer. From the remaining organic phase the catecholestrogens were removed with borate buffer and further purified on Sephadex LH-20 columns. After derivatisation 4-hydroxyestrone was separated from the isomeric 2-hydroxy compound by gas chromatography. The mass spectra of the 4-hydroxyestrone peak were identical with that of authentic 4-hydroxyestrone. After treatment of the extracts with sodium borohydride 4-hydroxyestradiol-17β was identified by GC-MS. By the addition of trace amounts of tritiated 4-hydroxyestrone a recovery of 40% was calculated. On the basis of this recovery and the peak heights of the gas chromatograms an excretion of 4 μg (midcycle) and 40 μg (pregnancy) of 4-hydroxyestrone/24 h was estimated.