Red wine prevents the postprandial increase in plasma cholesterol oxidation products: a pilot study.

Moderate wine consumption has been shown to lower cardiovascular risk. One of the mechanisms could involve the control of postprandial hyperlipaemia, a well-defined risk factor for atherosclerosis, reasonably by reducing the absorption of lipid oxidised species from the meal. The objective of the present study was to investigate whether wine consumption with the meal is able to reduce the postprandial increase in plasma lipid hydroperoxides and cholesterol oxidation products, in human subjects. In two different study sessions, twelve healthy volunteers consumed the same test meal rich in oxidised and oxidisable lipids (a double cheeseburger), with 300 ml of water (control) or with 300 ml of red wine (wine). The postprandial plasma concentration of cholesterol oxidation products was measured by GC-MS. The control meal induced a significant increase in the plasma concentration of lipid hydroperoxides and of two cholesterol oxidation products, 7-ß-hydroxycholesterol and 7-ketocholesterol. The postprandial increase in lipid hydroperoxides and cholesterol oxidation products was fully prevented by wine when consumed with the meal. In conclusion, the present study provides evidence that consumption of wine with the meal could prevent the postprandial increase in plasma cholesterol oxidation products.

Determination of aminoethylcysteine ketimine decarboxylated dimer in human plasma and cultured cells by high-performance liquid chromatography with electrochemical detection.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural compound with antioxidant properties of a new family of sulfur-containing amino acids. It has been detected in human urine and plasma, in mammalian cerebellum and, more recently, in dietary vegetables. In the present study, a simple, highly sensitive method using a high-performance liquid chromatography system with electrochemical detection (ECD) has been developed. The method showed excellent precision and accuracy. It has been found to be about 100-fold more sensitive than gas chromatographic method and 2000-fold more sensitive in respect to the liquid chromatography method with UV detection. The method showed the required features of specificity and sensitivity to detect aminoethylcysteine ketimine decarboxylated dimer in human plasma and in cultured cells after in vitro supplementation.

Effects of traditional and ultrasonic liposuction on adipose tissue: a biochemical approach.

Little is known about the interaction of ultrasonic liposculpture with fat tissue. The surgical technique is well established and its clinical effects are satisfactory. However, the in vivo effects on adipose tissue remain to be determined. Previous studies have shown that ultrasound waves break fat cells. The purpose of this study was to ascertain whether ultrasound waves can cause the release of fatty acids from the molecular structure of triglycerides. A double-blind study was designed with samples obtained from traditional and ultrasonic liposuction of an equivalent area in the same patient. Samples were checked for triglycerides and for free fatty acids. Triglyceride values were always higher in the sample that had undergone ultrasonic procedure. No significant differences were observed between the free fatty acid chromatograms of the two kinds of samples analyzed. Data showed that no changes occurred in the triglyceride molecule when using ultrasound waves in the experimental conditions.

Identification of aminoethylcysteine ketimine decarboxylated dimer in human plasma.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD) is a natural sulfur-containing tricyclic compound detected, until now, in human urine and bovine cerebellum. Recently, the antioxidant properties of this compound, and particularly its protective effect on the in vitro oxidation of low-density lipoproteins, have been demonstrated. In this paper, the identification of AECK-DD in human plasma by means of gas chromatography, high-performance liquid chromatography and gas chromatography-mass spectrometry, performed after a simple and fast purification procedure, is reported.

Antioxidant activity of aminoethylcysteine ketimine decarboxylated dimer on copper-induced LDL oxidation.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD), a member of a family of natural cyclic sulfur-containing aminoacids, recently detected in biological samples, protects low density lipoprotein (LDL) against copper-mediated oxidation, as assessed by monitoring the kinetics of conjugated diene formation, thiobarbituric acid-reactive substances production and LDL-tryptophan fluorescence quenching. Moreover, AECK-DD exerts a protective effect also against metal-independent, peroxyl radical-induced lipoprotein oxidation. It is of note that the concentrations exerting a protective effect against LDL oxidation are similar to those found in biological samples.

Detection of a decarboxylated dimer of aminoethylcysteine ketimine in bovine cerebellum.

Aminoethylcysteine ketimine is a sulfur-containing cyclic compound produced by the enzymatic alpha-deamination of the parent aminoethylcysteine that has been detected in bovine brain and cerebellum. Aminoethylcysteine ketimine is known to dimerize spontaneously and easily lose one carboxyl group. This decarboxylated compound, simply named the dimer, has been recently detected in normal human urine. In this article we provide evidence on the occurrence of the dimer in the bovine cerebellum.

Aminoethylcysteine ketimine decarboxylated dimer detected in normal human urine by gas-liquid chromatography, selected-ion monitoring and mass spectrometry.

Aminoethylcysteine ketimine is a biochemical product known to be converted spontaneously in the decarboxylated dimer. Since the ketimine has been detected in a mammalian brain, it was assumed that also the dimer could be present in the mammalian body and eventually excreted in the urine. Using human urine as the biological source, an extract was prepared which, submitted to gas-liquid chromatography, selected-ion monitoring and mass spectrometry, indicated the presence of the dimer.

Sulfur-containing cyclic ketimines and imino acids. A novel family of endogenous products in the search for a role.

Aminoethylcysteine, lanthionine, cystathionine and cystine are mono-deaminated either by L-amino-acid oxidase or by a transaminase exhibiting the properties described for glutamine transaminase. The deaminated products cyclize producing the respective ketimines. Authentic samples of each ketimine were prepared by reacting the appropriate aminothiol compound with bromopyruvate, except cystine ketimine which required the interaction of thiopyruvate with cystine sulfoxide. Reduction of the first three mentioned ketimines with NaBH4 yields the respective derivatives with the saturated rings of thiomorpholine and hexahydrothiazepine. The same reduction is carried out enzymically by a reductase extracted from mammalian tissues. Properties of the members of this family of compounds are described. Gas chromatography followed by mass spectrometry permits the identification of most of these products. HPLC is very useful for the determination of the ketimines by taking advantage of specific absorbance at 380 nm obtained by prior derivatization with phenylisothiocyanate. Adaptation of these and other analytical procedures to biological samples disclosed the presence of most of these compounds in bovine brain and in human urine. By using [35S]lanthionine ketimine as a representative member of the ketimine group, the specific, high-affinity, saturable and reversible binding to bovine brain membranes has been demonstrated. The binding is removed by aminoethylcysteine ketimine and by cystathionine ketimine indicating the occurrence in bovine brain of a common binding site for ketimines. The reduced ketimines are totally ineffective in competing with [35S]lanthionine ketimine. Alltogether these findings are highly indicative for the existence in mammals of a novel class of endogenous sulfur-containing cyclic products provided with a possible neurochemical function to be investigated further.

Dimerization and other changes of aminoethylcysteine ketimine.

High performance liquid chromatography and gas liquid chromatography have been used for the study of the stability of aminoethylcysteine-ketimine (AECK) in different experimental conditions. Concentration and acidic pH lead to the formation of the dimer of AECK which is very sensitive to temperature and is slowly converted to the corresponding decarboxylated dimer even at room temperature. In the presence of air at neutral and alkaline pH AECK is converted to an unknown compound having spectral characteristics similar to AECK and to other compounds not detectable by HPLC. Under nitrogen at neutral pH AECK is more stable and only undergoes the dimerization reaction to some extent.

Detection of cystathionine ketimine in bovine cerebellum.

A new sulfur-containing cyclic imino acid, cystathionine ketimine, has been detected in bovine cerebellum by gas chromatography, gas chromatography-mass spectrometry, and high pressure liquid chromatography procedures. Gas chromatography and gas-mass analyses are based on derivatization of endogenous cystathionine ketimine with diazomethane after a simple enrichment procedure. The high pressure liquid chromatography procedure takes advantage of the selective absorbance at 380 nm of the phenyl isothiocyanate-ketimine interaction product. The concentration of this new sulfur imino acid found in a pool of four bovine cerebella is approximately 0.5 nmol/g.

Detection of 2H-1,4-thiazine-5,6-dihydro-3-carboxylic acid (aminoethylcysteine ketimine) in the bovine brain.

2H-1,4-Thiazine-5,6-dihydro-3-carboxylic acid (trivial name: aminoethylcysteine ketimine) is a cyclic sulfur-containing imino acid detected in bovine brain extracts by means of three different procedures. Gas liquid chromatography of protein-free extracts of five bovine brains revealed the presence of this compound at concentrations ranging from 2 to 3 nmol/g wet weight of tissue. The enzymatic method based on the inhibition of D-amino acid oxidase activity by aminoethylcysteine ketimine together with an high-performance liquid chromatography procedure confirm the identification and quantitations obtained with gas liquid chromatography. The discovery of this compound structurally similar to pipecolic acid opens the question of its physiological role in the central nervous system.

Selective oxidation of methionine beta(55)D6 at the alpha 1 beta 1 interface in hemoglobin completely destabilizes the T-state.

When methionine beta(55)D6 in human hemoglobin is oxidized to its sulfoxide derivative, the modified protein appears to maintain most of the chemical and structural properties typical of the native protein. On the contrary, the functional behavior is drastically changed, being characterized (like that of the isolated chains) by high oxygen affinity (p50 = 0.47 torr in 0.1 M Tris (pH 7.3) + 0.1 M NaCl at 25 degrees C), absence of cooperativity (n = 1), and lack of Bohr effect. The complete destabilization of the T-state as a result of this modification is related to a perturbation of the alpha 1 beta 1 subunit interface, which in native hemoglobin remains static during the quaternary ligand-linked transition. Results also suggest that methionyl sulfoxide-containing hemoglobin, obtained under different conditions, assumes functionally different R-states, none of which is exactly comparable with that typical of the native protein.

Identification of 1,4-thiomorpholine-3-carboxylic acid (TMA) in normal human urine.

The sulfur-containing cyclic imino acid 1,4-thiomorpholine-3-carboxylic acid (TMA) has been identified in normal human urine. After the enrichment procedure with ion-exchange chromatography, the urine extracts were reacted with diazomethane. Gas-liquid chromatography revealed the presence of two peaks with the same retention times exhibited by authentic TMA after the same derivatization. The two compounds have been identified by mass-spectrometry as the monomethylated and dimethylated derivatives of TMA. This result represents the first indication of the occurrence of TMA in a mammalian sample.

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid are present in normal human urine.

Hexahydro-1,4-thiazepine-3,5-dicarboxylic acid and thiomorpholine-3,5-dicarboxylic acid, simply referred to as cyclothionine and TMDA, respectively, are two cyclic sulfur-containing imino acids detected in bovine brain. Human urine has been investigated to establish the occurrence of these imino acids as common constituents under normal conditions. The morning urine of healthy subjects has been analyzed for enrichment of these compounds by using an ion-exchange procedure. Gas/liquid chromatography of the final extracts revealed the presence of peaks coeluting with authentic cyclothionine and TMDA. The latter compound eluted very close to an unknown sulfur-containing compound. A resolved peak of TMDA has been obtained by high-performance liquid chromatography of the final extracts derivatized with phenylisothiocyanate. Selected ion monitoring with multiple-ion detection applied to the compounds separated by gas chromatography revealed the presence of the respective molecular ions and of the decarboxylated fragments, thus confirming the identification of cyclothionine and TMDA in human urine.

Gas-chromatographic mass-spectrometric detection of 1,4-hexahydrothiazepine-3,5-dicarboxylic acid (cyclothionine) in bovine brain.

Cystathionine has been reported to undergo enzymatic changes leading to the formation of seven membered cyclic products (Ricci, G., Santoro, L., Achilli, M., Matarese, R. M., Nardini, M., and Cavallini, D. (1983) J. Biol. Chem. 258, 10511-10517; Cavallini, D., Costa, M., Pensa, B., and Coccia, R. (1985) Biochem. Int. 10, 641-646). Gas-chromatographic and mass-spectrometric evidence reported in this paper indicates that the cyclic derivative of cystathionine, 1,4-hexahydrothiazepine-3,5-dicarboxylic acid, here simply named cyclothionine, is a normal component of bovine brain. This finding together with the detection of the same compound in the urine of cystathioninuric patients (Kodama, H., Sasaki, K., Mikasa, H., Cavallini, D., and Ricci, G. (1984) J. Chromatogr. 311, 183-188) supports the conclusion that cystathionine, apart from its role in trans-sulfuration, is converted also into cyclic compounds whose biochemical significance is as yet unknown.

1,4-Thiomorpholine-3,5-dicarboxylic acid, a novel cyclic imino acid detected in bovine brain.

Gas-liquid chromatography of enriched bovine brain extract revealed the occurrence of several sulfur-containing compounds. By co-chromatography with authentic product and by mass-spectrometric analysis, one of these compounds has been identified as 1,4-thiomorpholine-3,5-dicarboxylic acid (TMDA). The possible derivation of TMDA from lanthionine is discussed. This represents the second S-containing cyclic amino acid so far discovered in a mammalian brain whose physiological significance has not yet been explored.

Similarity of the oxidation products of L-cystathionine by L-amino acid oxidase to those excreted by cystathioninuric patients.

L-Cystathionine is oxidized by snake venom L-amino acid oxidase at a rate about half that with L-leucine at pH 8.5. The appearance of an absorbance at 296 nm and quantitation of the products of oxidation in the presence of catalase indicate formation in the solutions of a seven-membered ketimine ring produced by cyclization of the monoamino monoketo derivative of cystathionine. A limited double deamination has also been observed. In the absence of catalase, S-(carboxymethyl)homocysteine and S-(beta-carboxyethyl)cysteine have been identified together with ninhydrin-unreactive compounds yielding the above mentioned carboxy compounds upon hydrolysis with HCl. Authentic samples of the monoamino monoketo analogs of cystathionine have been prepared and compared with the enzymatic products. Cyclization of the synthetic products into the ketimine ring is pH-dependent as established by UV spectrum and other assays. Compounds derived from either the oxidation or the reduction of the ketimine have been prepared. It was found that many products of enzymatic and chemical changes of cystathionine and its ketimine described in the present paper are identical with those identified in the urine of cystathioninuric patients. This result indicates the occurrence in humans of secondary metabolic routes of cystathionine centered on the production of cystathionine ketimine, in equilibrium with the open form, which in cystathioninurics is revealed by the lack of cystathionase.