Desmosine (D) and isodesmosine (I), the intramolecular crosslinking amino acids that occur in chains of elastin, have now been found in free form in human urine. Until now, these amino acids (Mr ‫؍‬ 526) were found to occur in urine only as higher molecular weight (Mr ‫؍‬ 1,000 -1,500) peptides. Thus, the previously used analytical methods required, as the first step, acid hydrolysis of the urine at elevated temperature to liberate D and I from their peptides. The analytical method described here uses HPLC followed by electrospray ionization MS for the detection and quantitation of free D and I in unhydrolyzed urine. Identities of both D and I were established by their retention times on LC and by their mass ion at 526 atomic mass units, characteristic of each compound. The sensitivity of the method is 0.10 ng. The average values of free D and I in the urine of seven healthy subjects were 1.42 ؎ 1.16 and 1.39 ؎ 1.04 g͞g of creatinine, respectively. After acid hydrolysis of the urine, the amounts of D and I were 8.67 ؎ 3.75 and 6.28 ؎ 2.87 g͞g of creatinine, respectively. The method was also successfully used to measure peptide-bound D and I levels in the sputum of patients with chronic obstructive pulmonary disease. T wo pyridinium amino acids, desmosine (D) and isodesmosine (I), are positional isomers that serve as crosslinking molecules binding the polymeric chains of amino acids into the 3D network of elastin (1-3). The degradation of elastin-containing tissues that occurs in several widely prevalent diseases, such as pulmonary emphysema, chronic obstructive pulmonary disease (COPD), cystic fibrosis, atherosclerosis, aortic aneurysm, etc., has been associated with the excretion in the urine of peptic derivatives of these two pyridinium compounds (4-13). Previously identified (14) urinary metabolites are a group of high molecular weight polypeptic derivatives of D and I ranging in M r from 1,000 to 1,500. In this paper, we report that D and I are also excreted in urine in free form. Moreover, after hydrolysis, D and I have been found in sputum obtained from patients with COPD.Previously used techniques for the analysis of urinary D and I have used RIA (15, 16), HPLC (17-19), and capillary zone electrophoresis (20,21). In each case, acid hydrolysis (treatment of the urine with HCl at elevated temperature for 24 h) was used as the first step to liberate D and I from their peptidic conjugates. The analytical method described here is sufficiently sensitive and specific so that it is possible to analyze for free D and I even though they are less abundant in urine than their peptidic forms. The method involves HPLC followed by electrospray ionization MS (ESIMS). Besides, avoiding the drastic HCl hydrolysis (which severely complicates the subsequent purification process), this method makes possible the identification of D and I with high specificity and sensitivity, such that the analysis of only 1-3 ml of urine is possible. Moreover, other fluids of concern (sputum, lung lavage, etc.) where only small samples are av...

Five steroids-38-hydroxypregn-S-en-20-one (pregnenolone; P), 3fi-hydroxy-5a-pregnan-20-one (3.3-AP), 3a-hydroxy-5a-pregnan-20-one (3a-AP), 3.-hydroxyandrost-5-en-17-one (dehydroepiandrosterone; D), and 3fi-hydroxy-5a-androstan-17-one (EpiA)-were extracted from the brains of adult male rats, rabbits, and dogs. The steroids exist in this organ as uncoajugated compounds and as sulfates, lipoidal esters, and sulfolipids. The techniques for separating these four classes ofsteroids from each other and for separating the five steroids from each other are described. In all cases, the steroids were identified by their retention time (Re) on HPLC, theirRt by gas chromatography, and by selected ion monitoring of their mass spectra. The latter were also used for quantification. In their reaction toward organic bases, the sulfolipid conjugates resemble previously described sulfolipids of cholesterol and sitosterol. These conijugates are relatively abundant in brain, particularly those of P and D, and this suggests that, in the search for the physiological significance of these brain constituents, these conjugates warrant attention.also seem to indicate that the above-mentioned steroids may be present in brain in amounts somewhat larger than those previously reported. Furthermore, until now only the sulfate and lipoidal esters of both P and D have been detected in the brain. Our findings reveal that another steroidal conjugate(s), still to be characterized, is also present. In particular, evidence is given here that indicates that steroidal conjugates comparable in nature to those that we previously described (11) for cholesterol and sitosterol are also present in substantial amounts in brain. We have designated this conjugated form a sulfolipid, with the possible structure StO-SO2-OL (where St is a steroid and L is a lipid moiety), because this nonpolar conjugate, when treated with an organic base such as triethylamine, can cleave to give either a product that behaves like the sulfuric acid ester of a steroid or an unconjugated steroid, like P. Although their exact structures have yet to be determined, the abundance of these conjugates in brain suggests that their relationship to brain function warrants corsideration.The most recent facet of steroid hormone biochemistry to evoke intense interest, astonishing activity, and discoveries that presage previously unforeseen scientific advances is that which deals with the relationship between steroids and neuronal-especially brain-function. One ofthe factors that has been responsible for stimulating the interest in this chapter of biochemistry has been the detection of steroids in brain (neurosteroids) by Baulieu and his colleagues (1). Another impetus for the development was the finding of Majewska et al. (2) that certain steroid derivatives are potent modulators of the Cl-channel associated with the type A receptor for y-aminobutyric acid. More recent findings have shown that other ion channels, particularly those associated with N-methyl-D-aspartate receptors, may also ...

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