Nuclear magnetic resonance to study the interactions acting in the enantiomeric separation of homocysteine by capillary electrophoresis with a dual system of γ-cyclodextrin and the chiral ionic liquid EtCholNTf2.

The enantiomeric separation of 9-fluorenylmethoxycarbonyl chloride (FMOC)-homocysteine (Hcy) by CE was investigated using γ-CD and the chiral ionic liquid (R)-(1-hydroxybutan-2-yl)(trimethyl)azanium-bis(trifluoromethanesulfon)imidate (also called (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate) (EtCholNTf2 ) as chiral selectors. Using 2 mM γ-CD and 5 mM EtCholNTf2 in 50 mM borate buffer (pH 9), FMOC-Hcy enantiomers were separated with a resolution value of 3.8. A reversal in the enantiomer migration order in comparison with the single use of γ-CD in the separation buffer was obtained. Then, NMR experiments were carried out to elucidate the interactions taking place in the enantiomeric separation of FMOC-Hcy. NMR analyses highlighted the formation of an inclusion complex since the hydrophobic group of FMOC-Hcy was inserted into the γ-CD cavity. Moreover, interactions between EtCholNTf2 and γ-CD were also observed, suggesting that the chiral ionic liquid would also enter the cavity of the γ-CD.

Application of GC-MS technique for the determination of homocysteine thiolactone in human urine.

It is well established that homocysteine thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC-MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min-1 and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01-0.20 nmol mL-1 urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL-1, respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from

Effect of the combined use of γ-cyclodextrin and a chiral ionic liquid on the enantiomeric separation of homocysteine by capillary electrophoresis.

The enantioseparation of the non-protein amino acid homocysteine by CE was investigated in this article using eleven neutral CDs and five chiral ionic liquids as chiral selectors. Using a previous derivatization step with FMOC and the subsequent separation under neutral conditions, homocysteine enantiomers were only separated when γ-CD or (R)-N,N,N-trimethyl-2-aminobutanol-bis(trifluoromethane-sulfon)imidate (EtCholNTf2) were employed as sole chiral selectors in the separation buffer. On the one hand, γ-CD gave rise to the enantiomeric separation in 10min with a resolution value of 1.9, whereas EtCholNTf2 let to obtain a resolution value of 1.4 in more than 50min. Then, the evaluation of the combined use of both selectors was also carried out, resulting in a considerable increase in the Rs. The best enantioseparation for homocysteine was obtained when 10mM EtCholNTf2 was added to 50mM phosphate buffer (pH 7.0) containing 2mM γ-CD. In an attempt to discriminate specific chiral cation effect from the salt effect, the influence of adding LiNTf2 to the separation medium was also evaluated, resulting in lower resolution values for homocysteine when compared to those achieved with the addition of EtCholNTf2, indicating a synergistic effect between EtCholNTf2 and γ-CD. Interestingly, the enantiomer migration order changed depending on the use of a single chiral selector or dual systems. When EtCholNTf2 or γ-CD were employed as sole chiral selectors, D-enantiomer was the first-migrating enantiomer. However, an inversion in the migration order was observed when both selectors were combined in a dual system being the L-enantiomer the first-migrating one.

A Novel Dicyanoisophorone-Based Ratiometric Fluorescent Probe for Selective Detection of Cysteine and Its Bioimaging Application in Living Cells.

A selective and ratiometric turn-on fluorescent probe was designed and synthesized by using a novel dicyanoisophorone-based derivative and acrylate moiety. The probe displayed high stability and good selectivity to cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). It also exhibited rapid response to Cys within 180 s. Most importantly, the fluorescence intensity ratio at 590 and 525 nm (I590/I525) was linearly dependent on the Cys concentration in the range from 0 to 40 µM and the detection limit calculated to be 0.48 µM. This probe was also applied for bioimaging of intracellular Cys in living HeLa cells with low cytotoxicity.

Capillary electrophoresis tandem mass spectrometry determination of glutamic acid and homocysteine's metabolites: Potential biomarkers of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects both lower and upper motor neurons, leading to muscle atrophy, paralysis, and death caused by respiratory failure or infectious complications. Altered levels of homocysteine, cysteine, methionine, and glutamic acid have been observed in plasma of ALS patients. In this context, a method for determination of these potential biomarkers in plasma by capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is proposed herein. Sample preparation was carefully investigated, since sulfur-containing amino acids may interact with plasma proteins. Owing to the non-thiol sulfur atom in methionine, it was necessary to split sample preparation into two methods: i) determination of homocysteine and cysteine as S-acetyl amino acids; ii) determination of glutamic acid and methionine. All amino acids were separated within 25min by CE-MS/MS using 5molL-1 acetic acid as background electrolyte and 5mmolL-1 acetic acid in 50% methanol/H2O (v/v) as sheath liquid. The proposed CE-MS/MS method was validated, presenting RSD values below 6% and 11% for intra- and inter-day precision, respectively, for the middle concentration level within the linear range. The limits of detection ranged from 35 (homocysteine) to 268nmolL-1 (glutamic acid). The validated method was applied to the analysis of plasma samples from a group of healthy individuals and patients with ALS, showing the potential of glutamic acid and homocysteine metabolites as biomarkers of ALS.

A fluorescent probe for the efficient discrimination of Cys, Hcy and GSH based on different cascade reactions.

A fluorescent probe (1) for distinguishing amongst biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), is developed based on different cascade reactions. The key design feature of fluorescent probe 1 is the integration of two potential reaction groups for the thiol and amino groups of biothiols in one molecule. By reacting with the halogen atom and α, ß-unsaturated malonitrile in probe 1, Cys, Hcy and GSH can generate a total of three main products with distinct photophysical properties. Probe 1 shows a strong fluorescence turn-on response to Cys with blue-green emission by using an excitation wavelength of 390nm. At an excitation wavelength of 500nm, probe 1 responds to GSH over Cys and Hcy and emits strong orange fluorescence. The discrimination of biothiols can be demonstrated by cell imaging experiments, indicating that probe 1 can be a useful tool for the selective imaging of Cys and GSH in living cells.

Toxinas unidas a proteínas: valor añadido en su eliminación con altos volúmenes convectivos / Protein-bound toxins: added value in their removal with high convective volumes

La enfermedad renal crónica tiene mayor riesgo de eventos cardiovasculares. En los últimos años, han ido adquiriendo mayor importancia las toxinas unidas a proteínas, que han sido asociadas a mayor morbimortalidad y que se caracterizan por la dificultad para su depuración en diálisis. El objetivo de este estudio es valorar la influencia de altos volúmenes convectivos en HDF-OL posdilucional sobre la eliminación de medianas moléculas, pequeñas moléculas y moléculas unidas a proteínas. Material y métodos: Se realizaron 40 sesiones de HDF-OL posdilucional en 13 pacientes y se midió el porcentaje de reducción de toxinas de distinto peso molecular y entre ellas, moléculas unidas a proteínas como el p-cresyl sulfato, indoxyl sulfato y homocisteína. Resultados: El volumen convectivo total fue de 28,3(5,1) litros con un rango entre 16,3 y 38,0 litros. La reducción media de moléculas unidas a proteínas fue de 44,4 (15,7) % para el p-cresyl sulfato, de 48,7(14,1) % para el indoxyl sulfato y de 58,6(8,8) % para la homocisteína. Además, se encontró una relación directa y estadísticamente significativa entre el porcentaje de reducción de las tres moléculas con el volumen de sustitución y con el Kt/V. Conclusión: Altos volúmenes convectivos totales en HDF-OL en posdilución se asocian a una mayor eliminación de toxinas urémicas unidas a proteínas (AU)

Chronic kidney disease is associated with an increased risk of cardiovascular events. In recent years, protein-bound toxins have become more important due to their association with increased morbidity and mortality, characterised by inadequate clearance during dialysis. The purpose of this study is to assess the influence of high convective volumes on postdilution online haemodiafiltration (OL-HDF) on the removal of medium-sized molecules, small molecules and protein-bound molecules. Material and methods: In forty postdilutional OL-HDF sessions, the reduction rates of toxins of different molecular weights were measured in 13 patients, including protein-bound molecules such as p-cresyl sulphate, indoxyl sulphate and homocysteine. Results: Total convective volume was 28.3 (5.1) litres (range 16.3-38.0 litres). Mean reduction rate of protein-bound molecules was 44.4% (15.7%), 48.7% (14.1%) and 58.6% (8.8%) for p-cresyl sulphate, indoxyl sulphate and homocysteine, respectively. Moreover, a statistically significant direct association was found between the reduction rates of all three molecules, the replacement volume and the Kt/V. Conclusion: High convective volumes during postdilution OL-HDF are associated with increased removal of protein-bound uraemic toxins (AU)

An aldehyde group-based P-acid probe for selective fluorescence turn-on sensing of cysteine and homocysteine.

A highly sensitive and selective turn on fluorescent probe P-acid-aldehyde (P-CHO) is developed for the determination of cysteine (Cys) and homocysteine (Hcy). The probe is designed and synthesized by incorporating the specific functional group aldehyde group for thiols into a stable π-conjugated material 4,4'-(2,5-dimethoxy-1,4-phenylene) bis(ethyne-2,1-diyl) dibenzoic acid (P-acid). The probe fluorescence is quenched through donor photoinduced electron transfer (d-PET) between the fluorophore (P-acid) and the recognition group (aldehyde group). In the presence of thiols, Cys and Hcy can selectively react with aldehyde group of the probe because the inhibition of d-PET between fluorophore and recognition group. Therefore, a turn-on fluorescent sensor was established for the fluorescence recovery. Under the optimized conditions, the fluorescence response of probe is directly proportional to the concentration of Cys in the range of 4-95 NM L(-1), with a detection limit 3.0 nM. In addition, the sensing system exhibits good selectively toward Cys and Hcy in the presence of other amino acids. It has been successfully applied for bioimaging of Cys and Hcy in living cells with low cell toxicity.

Label-free colorimetric detection of biothiols utilizing SAM and unmodified Au nanoparticles.

Herein, a sensitive and selective sensor for biothiols based on colorimetric assay is reported. S-adenosyl-L-methionine (SAM) could induce the selective aggregation of unmodified gold nanoparticles (AuNPs) by electrostatic interaction. In the presence of biothiols, such as glutathione (GSH), homocysteine (Hcy), and cysteine (Cys), AuNPs prefer to react with thiols of biothiols rather than SAM due to the formation of Au-S bond. Thus, the AuNPs turn from the aggregation to the dispersion state, and the corresponding color variation in the process of anti-aggregation of AuNPs can be used for the quantitative screening of biothiols through UV-vis spectroscopy or by the naked eye. Under optimized conditions, a good linear relationship in the range of 0.4-1.2 µM is obtained for Cys, 0.2-0.9 µM for GSH, and 0.6-3.0 µM for Hcys. The detection limits of this assay for GSH, Cys and Hcys are 35.8 nM, 21.7 nM, and 62.4 nM, respectively. This colorimetric assay exhibits rapid operation (within 5 min), high selectivity and sensitivity towards biothiols with tunable dynamic ranges.

Highly sensitive and selective colorimetric detection of glutathione based on Ag [I] ion-3,3',5,5'-tetramethylbenzidine (TMB).

Glutathione (GSH) plays an important role in the biological system and serves many cellular functions. Since all of the biothiols possess similar functional groups, it is still challenging to selectively detect GSH over cysteine (Cys) and homocysteine (Hcy). In this work, a novel and simple colorimetric method for discriminative detection of glutathione (GSH) over Cys and Hcy is developed. The proposed method is based on the fact that Ag [I] ion could oxidize 3,3',5,5',-tetramethylbenzidine (TMB) to the oxidized TMB to induce a blue color and an absorption peak centered at 652 nm. However, the introduction of GSH could cause the reduction of oxidized TMB and it could also combine with Ag(+), both of which result in a blue color fading and a decrease of the absorbance at 652 nm. Based on this finding, we propose a method to qualitatively and quantitatively detect GSH by naked eyes and UV-vis spectroscopy, respectively. The proposed method shows a low detection limit of 0.1 µM by naked eyes and 0.05 µM with the help of UV-vis spectroscopy. In addition, this method has great potential in discriminatively detecting GSH over other amino acid and biothiols. More importantly, this method is simple and fast without the preparation of nanomaterials and has also been successfully applied to the detection of GSH in biological fluids.

A new BODIPY-based long-wavelength fluorescent probe for chromatographic analysis of low-molecular-weight thiols.

A new long-wavelength fluorescent probe, 1,7-dimethyl-3,5-distyryl-8-phenyl-(4'-iodoacetamido)difluoroboradiaza-s-indacene (DMDSPAB-I), was designed and synthesized for thiol labeling in high-performance liquid chromatography (HPLC). The excitation and emission wavelengths of DMDSPAB-I are 620 and 630 nm, respectively, with a high fluorescence quantum yield of 0.557, which is advantageous in preventing interference of intrinsic fluorescence from complex biological matrices and enabling high sensitivity HPLC. Based on DMDSPAB-I, a reversed-phase HPLC method was developed for measuring low-molecular-weight thiols including glutathione, cysteine, homocysteine, N-acetylcysteine, cysteinylglycine, and penicillamine. After the specific reaction of DMDSPAB-I with thiols, baseline separation of all six stable derivatives was achieved through isocratic elution on a C18 column within 25 min, with the limits of detection (signal-to-noise ratio = 3) from 0.24 nmol L(-1) for glutathione to 0.72 nmol L(-1) for penicillamine. The proposed method was validated in part by measuring thiols in blood samples from mice, with recoveries of 95.3-104.3%.

Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood.

Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5-15, 200-300, and 1-5 µM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.

Sensitive and selective detection of biothiols based on target-induced agglomeration of silver nanoclusters.

In this work, we present a label-free biosensor for the sensing of biothiols such as cysteine, homocysteine, and glutathione. This biosensor is based on the fluorescent probe, polyethyleneimine-capped silver nanoclusters. The selective binding of silver nanoclusters to biothiols promotes the silver nanoclusters agglomeration to yield larger non-fluorescent silver nanoparticles. And the fluorescence intensity of silver nanoclusters was quenched efficiently with increasing concentration of biothiols. The other amino acids introduced to the silver nanoclusters probe solution did not quench the fluorescence of the probe as they do not have thiol group which has strong affinity to the silver nanoclusters. Thus, this biosensor allowed selectively investigation of biothiols. The as-proposed biosensor was sensitive for the detection of biothiols. The linear ranges for cysteine, homocysteine, and glutathione were 0.1-10 µΜ (R²=0.9930), 0.1-10 µΜ (R²=0.9924), and 0.5-6 µΜ (R²=0.9900), respectively. The detection limits for cysteine, homocysteine, glutathione were 42, 47, and 380 nΜ, respectively. The silver nanoclusters-based fluorescent biosensor provides a simple, cost-effective, and sensitive platform for the detection of biothiols.

Synthesis of (S)-naproxen-benzotriazole and its application as chiral derivatizing reagent for microwave-assisted synthesis and indirect high performance liquid chromatographic separation of diastereomers of penicillamine, cysteine and homocysteine.

(S)-Naproxen-benzotriazole was synthesized by the reaction of (S)-naproxen with 1H-benzotriazole using coupling reagent dicyclohexyl carbodiimide and 4-dimethylamino pyridine (DCC/DMAP). It was used as chiral derivatizing reagent for microwave irradiated synthesis of diastereomers of penicillamine, cysteine and homocysteine. The diastereomers were separated by reversed phase high performance liquid chromatography using gradient elution of triethylammonium phosphate (pH 3.5)-acetonitrile (30-65% within 30 min). The method was validated for accuracy, precision, and limit of detection.

Simultaneous, noninvasive, and transdermal extraction of urea and homocysteine by reverse iontophoresis.

BACKGROUND: Cardiovascular and kidney diseases are a global public health problem and impose a huge economic burden on health care services. Homocysteine, an amino acid, is associated with coronary heart disease, while urea is a harmful metabolic substance which can be used to reflect kidney function. Monitoring of these two substances is therefore very important. This in vitro study aimed to determine whether homocysteine is extractable transdermally and noninvasively, and whether homocysteine and urea can be extracted simultaneously by reverse iontophoresis. METHODS: A diffusion cell incorporated with porcine skin was used for all experiments with the application of a direct current (dc) and four different symmetrical biphasic direct currents (SBdc) for 12 minutes via Ag/AgCl electrodes. The dc and the SBdc had a current density of 0.3 mA/cm(2). RESULTS: The SBdc has four different phase durations of 15 sec, 30 sec, 60 sec, and 180 sec. It was found that homocysteine can be transdermally extracted by reverse iontophoresis. Simultaneous extraction of homocysteine and urea by reverse iontophoresis is also possible. CONCLUSION: These results suggest that extraction of homocysteine and urea by SBdc are phase duration-dependent, and the optimum mode for simultaneous homocysteine and urea extraction is the SBdc with the phase duration of 60 sec.

Fluorescent sensing of homocysteine in urine: using fluorosurfactant-capped gold nanoparticles and o-Phthaldialdehyde.

This study reports the development of a simple, sensitive, and selective-detection system for homocysteine (HCys) based on the combination of fluorosurfactant-capped gold nanoparticles (FSN-AuNPs) and o-Phthaldialdehyde (OPA). The proposed assay utilizes FSN-AuNPs as extractors for HCys and cysteine (Cys), which can then be collected by centrifugation. As long as the HCys and Cys are isolated from the initial sample, they can be liberated from the NP surface by 2-mercaptoethanol (2-ME). The derivatization of released HCys with OPA/2-ME has a strong fluorescence maximum at 485 nm, whereas the derivatization of released Cys with the same reagent shows an extremely weak fluorescence maximum at 457 nm. As a result, the selectivity of this system is more than 100-fold for HCys over any aminothiols when excited at 370 nm. The extraction and derivation efficiencies are monitored as functions of the concentration of FSN-AuNPs and OPA, respectively. The proposed system has a detection limit of 180 nM at a signal-to-noise ratio of 3 for HCys. This study validates the applicability of this system by analyzing the amount of HCys in urine samples.

New method for the determination of protein N-linked homocysteine.

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy-thiolactone with epsilon-amino group of a protein lysine residue. This reaction leads to impairment and alteration of protein's function and has been implicated in atherothrombotic disease. However, the data regarding N-linked Hcy content in proteins are limited, mostly due to a lack of facile assays. Here I describe a new sensitive assay for the determination of protein N-linked Hcy and demonstrate its utility for individual proteins and biological fluids. N-linked Hcy is liberated from proteins by acid hydrolysis and converted to Hcy-thiolactone, which is then purified and quantified by high-performance liquid chromatography on a cation exchange column. The quantification is by fluorescence after postcolumn derivatization with o-phthaldialdehyde. Using this assay, the levels of N-linked Hcy in individual pure proteins were found to vary from as high as 0.470-0.515 mol/mol protein for human and equine ferritins to as low as 0.00006 mol/mol protein for chicken lysozyme. Hemoglobins from a variety of species contained more N-linked Hcy than did corresponding albumins (0.0127-0.0828 vs. 0.0027-0.0086 mol/mol). Normal human plasma and milk were found to contain submicromolar concentrations of protein N-linked Hcy, whereas cow milk and whey contained micromolar concentrations of protein N-linked Hcy.

Utilization of nanobiotechnology in haemodialysis: mock-dialysis experiments on homocysteine.

BACKGROUND: The utilization of modern achievements from nanobiotechnology has resulted in novel modalities for renal replacement therapy. For conventional intermittent haemodialysis (HD), sophisticated membranes are currently being manufactured that guarantee selective removal of target toxins. These membranes have a narrow pore-size distribution that is focused around a mean value at the nanometre level. For continuous HD, novel artificial renal devices are currently being designed and evaluated in in vitro experiments that will be both implantable and have continuous function. METHODS: We present mock-dialysis experiments using magnetically assisted HD (MAHD) that we very recently introduced for the selective removal of target toxins. MAHD is based on the preparation of conjugates (Cs) made up of biocompatible ferromagnetic nanoparticles (FNs) and a specifically designed targeted binding substance that must have a high affinity for a specific target toxin substance. The FN-targeted binding substance Cs should be administered to the patient prior to MAHD to allow for binding with the target toxin substance in the bloodstream. The complex FN-targeted binding substance-target toxin substance will then be removed by a 'magnetic dialyzer' that is installed in the dialysis machine in series to the conventional dialyzer. In the present work, we compared the in vitro efficiency of MAHD to conventional HD for the removal of homocysteine (Hcy) during mock-dialysis experiments. RESULTS: These mock-dialysis experiments performed on Hcy revealed that both the removal rate and the overall removal efficiency of MAHD were significantly greater than conventional HD. CONCLUSIONS: MAHD appears to be a promising method that can be employed for the selective and more efficient extraction of toxins that are not adequately removed by conventional HD.

Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method.

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-leucinamide (L-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a "meso" configuration such as diaminopimelic acid (A2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the DL-FDLA method, and the resulting elution behavior was summarized as follows: (1) the LL- and meso-isomers were eluted prior to the DD-isomer except for one case; (2) the LL- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the L- and D-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.

The determination of homocysteine-thiolactone in human plasma.

The thioester homocysteine-thiolactone, a reactive metabolite of homocysteine, has been implicated in human cardiovascular disease. However, data on the levels of homocysteine-thiolactone in humans are limited, mostly due to a lack of facile and reliable assays. Here we describe a sensitive assay for the determination of plasma homocysteine-thiolactone and demonstrate its utility with a cohort of 60 healthy human subjects. Plasma homocysteine-thiolactone is first separated from macromolecules by ultrafiltration and then selectively extracted with chloroform/methanol. Further purification of plasma homocysteine-thiolactone is achieved by high-performance liquid chromatography on a cation exchange microbore column. The detection and quantification is by monitoring fluorescence after postcolumn derivatization with o-phthaldialdehyde. The limit of detection is 0.36 nM. Using this assay, homocysteine-thiolactone concentrations in plasma from normal healthy human subjects (n=60) were found to vary from zero to 34.8 nM, with an average of 2.82+/-6.13 nM. In 29 of the 60 human plasma samples analyzed, homocysteine-thiolactone levels were below the detection limit. Homocysteine-thiolactone represented from 0 to 0.28%, on average 0.023+/-0.05%, of plasma total homocysteine.