The Use of Single Drop Microextraction and Field Amplified Sample Injection for CZE Determination of Homocysteine Thiolactone in Urine.

Two cheap, simple and reproducible methods for the electrophoretic determination of homocysteine thiolactone (HTL) in human urine have been developed and validated. The first method utilizes off-line single drop microextraction (SDME), whereas the second one uses off-line SDME in combination with field amplified sample injection (FASI). The off-line SDME protocol consists of the following steps: urine dilution with 0.2 mol/L, pH 8.2 phosphate buffer (1:2, v/v), chloroform addition, drop formation and extraction of HTL. The pre-concentration of HTL inside a separation capillary was performed by FASI. For sample separation, the 0.1 mol/L pH 4.75 phosphate buffer served as the background electrolyte, and HTL was detected at 240 nm. A standard fused-silica capillary (effective length 55.5 cm, 75 µm id) and a separation voltage of 21 kV (~99 µA) were used. Electrophoretic separation was completed within 7 min, whereas the LOD and LOQ for HTL were 0.04 and 0.1 µmol/L urine, respectively. The calibration curve in urine was linear in the range of 0.1-0.5 µmol/L, with R2 = 0.9991. The relative standard deviation of the points of the calibration curve varied from 2.4% to 14.9%. The intra- and inter-day precision and recovery were 6.4-10.2% (average 6.0% and 6.7%) and 94.9-102.7% (average 99.7% and 99.5%), respectively. The analytical procedure was successfully applied to the analysis of spiked urine samples obtained from apparently healthy volunteers.

Estimated prevalence of moderate to severely elevated total homocysteine levels in the United States: A missed opportunity for diagnosis of homocystinuria?

Classical homocystinuria (HCU) is a genetic disorder caused by mutations in the cystathionine beta synthase gene, which results in impaired metabolism of the sulfur-bearing amino acid homocysteine and its accumulation in blood and tissues. Classical HCU can be detected via newborn screening in the United States, but the test is widely acknowledged to miss many patients. While severely elevated homocysteine levels (>100 µmol /L) frequently lead to a classical HCU diagnosis, intermediate levels (>30 to 100 µmol /L), though linked to many of the known complications of HCU, are not always recognized as associated with HCU. We aimed to identify and describe potentially undiagnosed classical HCU patients using a nationally-representative database of administrative claims and laboratory results. We estimated the national prevalence of patients with homocysteine >30 µmol /L, and compared their demographic and clinical characteristics to those of patients with homocysteine levels ≤30 µmol/L. Among 57,580 patients with a homocysteine test result, 1.8% had a value >30 µmol /L. Patients with homocysteine >30 µmol /L were more frequently diagnosed with hypothyroidism (39.2% vs. 20.7%, p < .001) and renal disease (9.7% vs. 5.5%, p < .001), and were more likely to have a prescription for an anxiolytic/antidepressant (44.5% vs. 38.9%), opioid (58.4% vs. 53.1%), steroid (46.4% vs. 42.5%), or thyroid hormone (38.8% vs. 18.8%), compared to patients with homocysteine ≤30 µmol /L (all p < .05). Both groups were equally likely to have a diagnosis of homocystinuria or another disorder of sulfur-bearing amino acid metabolism (3.8% vs. 4.0%, p = .752). The age-adjusted national prevalence of homocysteine >30 µmol /L was estimated at 33,068 (95% CI: 1033 - 35,104). These findings suggest that thousands of people in the US may be living with intermediate to severely elevated homocysteine levels and may require further evaluation for the presence of classical HCU.

Determination of homocysteine thiolactone in human urine by capillary zone electrophoresis and single drop microextraction.

A simple, fast, sensitive and reproducible capillary zone electrophoresis (CZE) method with single drop microextraction (SDME) for determination of homocysteine thiolactone (HTL) in human urine has been developed and validated. The method is characterized by good precision, high accuracy, short analysis time and low consumption of reagents. The procedure consists only of few steps: urine sample centrifugation, dilution with phosphate buffer and methanol, chloroform addition onto the top of donor phase, on-line SDME in CE system, sample separation by CZE and ultraviolet detection of HTL at 240 nm. The background electrolyte was 0.1 M pH 4.75 phosphate buffer. Effective separation was achieved within 6.04 min under the separation voltage of 24 kV (~110 µA). The LOQ and LOD for HTL were 50 and 25 nM urine, respectively. The calibration curve in urine showed linearity in the range of 50-200 nM, with R2 0.9995. The intra- and inter-day precision and recovery were 4.0-14.5% (average 8.7% and 9.3%) and 92.7-115.5% (average 103.6% and 104.8%), respectively. The procedure was successfully applied to analysis of urine samples.

A vinyl sulfone clicked carbon dot-engineered microfluidic paper-based analytical device for fluorometric determination of biothiols.

A microfluidic paper-based analytical device integrating carbon dot (CDs) is fabricated and used for a fluorometric off-on assay of biothiols. Vinyl sulfone (VS) click immobilization of carbon dots (CDs) on paper was accomplished by a one-pot simplified protocol that uses divinyl sulfone (DVS) as a homobifunctional reagent. This reagent mediated both the click oxa-Michael addition to the hydroxyl groups of cellulose and ulterior covalent grafting of the resulting VS paper to NH2-functionalized CDs by means of click aza-Michael addition. The resulting cellulose nanocomposite was used to engineer an inexpensive and robust microfluidic paper-based analytical device (µPAD) that is used for a reaction-based off-on fluorometric assay of biothiols (GSH, Cys, and Hcy). The intrinsic blue fluorescence of CDs (with excitation/emission maxima at 365/450 nm) is turned off via the heavy atom effect of an introduced iodo group. Fluorescence is turned on again due to the displacement of iodine by reaction with a biothiol. The increase in fluorescence is related to the concentration over a wide range (1 to 200 µM for GSH and 5-200 µM for Cys and Hcy, respectively), and the assay exhibits a low detection limit (0.3 µM for GSH and Cys and 0.4 µM for Hcy). The method allows for rapid screening and can also be used in combination with a digital camera readout. Graphical abstract Schematic representation of a µPAD based on click immobilized carbon dots and used for a reaction-based fluorometric off-on assay of biothiols. The intrinsic blue fluorescence of carbon dots is turned off via the heavy atom effect of an introduced iodo group and turned on by the displacement of this atom by reaction with a biothiol.

Higher Levels of Low Molecular Weight Sulfur Compounds and Homocysteine Thiolactone in the Urine of Autistic Children.

In this study, the levels of concentration of homocysteine thiolactone (HTL), cysteine (Cys), and cysteinylglycine (CysGly) in the urine of autistic and non-autistic children were investigated and compared. HTL has never been analyzed in autistic children. The levels of low molecular weight sulfur compounds in the urine of both groups were determined by validated methods based on high-performance liquid chromatography with spectrofluorometric and diode-array detectors. The statistical data show a significant difference between the examined groups. Children with autism were characterized by a significantly higher level of HTL (p = 5.86 × 10-8), Cys (p = 1.49 × 10-10) and CysGly (p = 1.06 × 10-8) in urine compared with the control group. A difference in the p-value of <0.05 is statistically significant. Higher levels of HTL, Cys, and CysGly in the urine of 41 children with autism, aged 3 to 17, were observed. The obtained results may indicate disturbances in the metabolism of methionine, Cys, and glutathione in some autistic patients. These preliminary results suggest that further research with more rigorous designs and a large number of subjects is needed.

Urinary excretion of homocysteine thiolactone and the risk of acute myocardial infarction in coronary artery disease patients: the WENBIT trial.

OBJECTIVES: No individual homocysteine (Hcy) metabolite has been studied as a risk marker for coronary artery disease (CAD). Our objective was to examine Hcy-thiolactone, a chemically reactive metabolite generated by methionyl-tRNA synthetase and cleared by the kidney, as a risk predictor of incident acute myocardial infarction (AMI) in the Western Norway B-Vitamin Intervention Trial. DESIGN: Single centre, prospective double-blind clinical intervention study, randomized in a 2 × 2 factorial design. SUBJECTS AND METHODS: Patients with suspected CAD (n = 2049, 69.8% men; 61.2-year-old) were randomized to groups receiving daily (i) folic acid (0.8 mg)/vitamin B12 (0.4 mg)/vitamin B6 (40 mg); (ii) folic acid/vitamin B12 ; (iii) vitamin B6 or (iv) placebo. Urinary Hcy-thiolactone was quantified at baseline, 12 and 38 months. RESULTS: Baseline urinary Hcy-thiolactone/creatinine was significantly associated with plasma tHcy, ApoA1, glomerular filtration rate, potassium and pyridoxal 5'-phosphate (positively) and with age, hypertension, smoking, urinary creatinine, plasma bilirubin and kynurenine (negatively). During median 4.7-years, 183 patients (8.9%) suffered an AMI. In Cox regression analysis, Hcy-thiolactone/creatinine was associated with AMI risk (hazard ratio = 1.58, 95% confidence interval = 1.10-2.26, P = 0.012 for trend; adjusted for age, gender, tHcy). This association was confined to patients with pyridoxic acid below median (adjusted HR = 2.72, 95% CI = 1.47-5.03, P = 0.0001; Pinteraction = 0.020). B-vitamin/folate treatments did not affect Hcy-thiolactone/creatinine and its AMI risk association. CONCLUSIONS: Hcy-thiolactone/creatinine ratio is a novel AMI risk predictor in patients with suspected CAD, independent of traditional risk factors and tHcy, but modified by vitamin B6 catabolism. These findings lend a support to the hypothesis that Hcy-thiolactone is mechanistically involved in cardiovascular disease.

Non-targeted mercapturic acid screening in urine using LC-MS/MS with matrix effect compensation by postcolumn infusion of internal standard (PCI-IS).

While the targeted analysis of mercapturic acid (MA) metabolites in human urine is used to assess exposure to selected chemicals, this compound class has only rarely been addressed in non-target screening utilizing diagnostic neutral loss liquid chromatography tandem mass spectrometry (LC-MS/MS). Additionally, this type of analysis is severely affected by matrix effects (MEs) causing poor comparability of samples and distortion of signal intensities. However, MEs have been neglected in urinary MA non-target screening so far. Therefore, we developed a non-target screening method relying on neutral loss scanning for MAs using post column infusion of an isotope-labelled standard. For signal correction, we synthesized a structural analogue to MAs, N-acetyl-S-methyl-homocysteine-D3, lacking the characteristic neutral loss of the MAs. For method development, 16 structurally different model MA compounds and 20 spiked urine samples were used. Twelve out of the 16 model compounds could be analysed by the developed method. We found severe matrix effects (largely signal suppression) for the spiked model compounds, with only 34% of all peaks' intensities changing by less than a factor of two. This could be compensated by the post column internal standard infusion with now 68% of all peaks' intensities changing by less than a factor of two. For three compounds, an over-compensation was observed resulting in an increase of signal of up to a factor of 16. In the 20 urine samples, altogether 558 native MAs (between 74 and 175 per sample) could be detected after ME compensation. These results indicate that a large number of so far uncharacterized MAs are present in urine, which yield a potential for biomarker discovery and pattern characterisation. Graphical Abstract.

Molecular Vise Approach to Create Metal-Binding Sites in MOFs and Detection of Biomarkers.

We report a new approach to create metal-binding site in a series of metal-organic frameworks (MOFs), where tetratopic carboxylate linker, 4',4'',4''',4''''-methanetetrayltetrabiphenyl-4-carboxylic acid, is partially replaced by a tritopic carboxylate linker, tris(4-carboxybiphenyl)amine, in combination with monotopic linkers, formic acid, trifluoroacetic acid, benzoic acid, isonicotinic acid, 4-chlorobenzoic acid, and 4-nitrobenzoic acid, respectively. The distance between these paired-up linkers can be precisely controlled, ranging from 5.4 to 10.8 Å, where a variety of metals, Mg2+ , Al3+ , Cr3+ , Mn2+ , Fe3+ , Co2+ , Ni2+ , Cu2+ , Zn2+ , Ag+ , Cd2+ and Pb2+ , can be placed in. The distribution of these metal-binding sites across a single crystal is visualized by 3D tomography of laser scanning confocal microscopy with a resolution of 10 nm. The binding affinity between the metal and its binding-site in MOF can be varied in a large range (observed binding constants, Kobs from 1.56×102 to 1.70×104  L mol-1 ), in aqueous solution. The fluorescence of these crystals can be used to detect biomarkers, such as cysteine, homocysteine and glutathione, with ultrahigh sensitivity and without the interference of urine, through the dissociation of metal ions from their binding sites.

Capillary electrophoresis coupled with chloroform-acetonitrile extraction for rapid and highly selective determination of cysteine and homocysteine levels in human blood plasma and urine.

A rapid and selective method has been developed for highly sensitive determination of total cysteine and homocysteine levels in human blood plasma and urine by capillary electrophoresis (CE) coupled with liquid-liquid extraction. Analytes were first derivatized with 1,1'-thiocarbonyldiimidazole and then samples were purified by chloroform-ACN extraction. Electrophoretic separation was performed using 0.1 M phosphate with 30 mM triethanolamine, pH 2, containing 25 µM CTAB, 2.5 µM SDS, and 2.5% polyethylene glycol 600. Samples were injected into the capillary (with total length 32 cm and 50 µm id) at 2250 mbar*s and subsequent injection was performed for 30 s with 0.5 M KОН. The total analysis time was less than 9 min, accuracy was 98%, and precision was <2.6%. The LOD was 0.2 µM for homocysteine and 0.5 µM for cysteine. The use of liquid-liquid extraction allowed the precision and sensitivity of the CE method to be significantly increased. The validated method was applied to determine total cysteine and homocysteine content in human blood plasma and urine samples obtained from healthy volunteers and patients with kidney disorders.

Quantification of urinary S- and N-homocysteinylated protein and homocysteine-thiolactone in mice.

Homocysteine (Hcy) and its metabolites Hcy-thiolactone, N-Hcy-protein, and S-Hcy-protein are implicated in vascular and neurological diseases. However, quantification of these metabolites remains challenging. Here I describe streamlined assays for these metabolites based on their conversion to Hcy-thiolactone. Free Hcy-thiolactone is extracted from the urine with chloroform/methanol. Total Hcy is converted to Hcy-thiolactone in the presence of 1 N HCl. Major urinary protein (MUP)-bound S-linked Hcy is liberated from the protein by reduction with dithiothreitol and converted to Hcy-thiolactone. Acid hydrolysis of MUP with 6 N HCl liberates N-linked Hcy as Hcy-thiolactone, which is then extracted with chloroform/methanol. Ferritin is used as an N-Hcy-protein standard and an authentic Hcy-thiolactone is used to monitor the efficiency of extraction. Hcy-thiolactone (free, derived from total Hcy, or from MUP-bound N-linked or S-linked Hcy) is separated by a cation exchange high-performance liquid chromatography, post-column derivatized with o-phthaldialdehyde, and quantified by fluorescence. Using these assays with as little as 2-20 µL of urine I show that MUP carry N-linked and S-linked Hcy and that N-Hcy-MUP and S-Hcy-MUP and Hcy-thiolactone are severely elevated in cystathionine ß-synthase-deficient mice. These assays will facilitate examination of the role of protein-related Hcy metabolites in health and disease.

A Sensitive Ratiometric Long-Wavelength Fluorescent Probe for Selective Determination of Cysteine/Homocysteine.

The development of sensitive fluorescence probes to detect biothiols such as cysteine and homocysteine has attracted great attention in recent times. Herein, we described the design and synthesis of coumarin based long-wavelength fluorescence probe, Bromo-2-benzothiazolyl-3-cyano-7-hydroxy coumarin (BBCH, 2) for selective detections of cysteine and homocysteine. The probe is rationally designed in such a way that both sulfhydryl and adjacent amino groups of thiols are involved in sensing process. Only cysteine/homocysteine able to react with BBCH to release fluorescence reporter (BCH, 1); while, glutathione and other amino acids unable to react with BBCH due to the absence of adjacent amino groups. In presence of cysteine, the color of BBCH is turns from colorless to red and thus BBCH is a naked eye fluorescence indicator for cysteine. Besides, BBCH can discriminate cysteine and homocysteine based on color changes and different reaction rates. The described sensing platform showed good sensing performances to detect cysteine and homocysteine with detection limits of 0.87 and 0.19 µM, respectively. Practical applicability was verified in biological and pharmaceutical samples.

Vitamin B-12 Status Differs among Pregnant, Lactating, and Control Women with Equivalent Nutrient Intakes.

BACKGROUND: Limited data are available from controlled studies on biomarkers of maternal vitamin B-12 status. OBJECTIVE: We sought to quantify the effects of pregnancy and lactation on the vitamin B-12 status response to a known and highly controlled vitamin B-12 intake. METHODS: As part of a 10-12 wk feeding trial, pregnant (26-29 wk gestation; n = 26), lactating (5 wk postpartum; n = 28), and control (nonpregnant, nonlactating; n = 21) women consumed vitamin B-12 amounts of ∼8.6 µg/d [mixed diet (∼6 µg/d) plus a prenatal multivitamin supplement (2.6 µg/d)]. Serum vitamin B-12, holotranscobalamin (bioactive form of vitamin B-12), methylmalonic acid (MMA), and homocysteine were measured at baseline and study-end. RESULTS: All participants achieved adequate vitamin B-12 status in response to the study dose. Compared with control women, pregnant women had lower serum vitamin B-12 (-21%; P = 0.02) at study-end, whereas lactating women had higher (P = 0.04) serum vitamin B-12 throughout the study (+26% at study-end). Consumption of the study vitamin B-12 dose increased serum holotranscobalamin in all reproductive groups (+16-42%; P ≤ 0.009). At study-end, pregnant (vs. control) women had a higher holotranscobalamin-to-vitamin B-12 ratio (P = 0.04) with ∼30% (vs. 20%) of total vitamin B-12 in the bioactive form. Serum MMA increased during pregnancy (+50%; P < 0.001) but did not differ by reproductive state at study-end. Serum homocysteine increased in pregnant women (+15%; P = 0.009) but decreased in control and lactating women (-16-17%; P < 0.001). Despite these changes, pregnant women had ∼20% lower serum homocysteine than the other 2 groups at study-end (P ≤ 0.02). CONCLUSION: Pregnancy and lactation alter vitamin B-12 status in a manner consistent with enhanced vitamin B-12 supply to the child. Consumption of the study vitamin B-12 dose (∼3 times the RDA) increased the bioactive form of vitamin B-12, suggesting that women in these reproductive states may benefit from vitamin B-12 intakes exceeding current recommendations. This trial was registered at clinicaltrials.gov as NCT01127022.

Adult-onset eculizumab-resistant hemolytic uremic syndrome associated with cobalamin C deficiency.

A 20-year-old man was hospitalized for malignant hypertension, mechanical hemolysis, and kidney failure. Kidney biopsy confirmed glomerular and arteriolar thrombotic microangiopathy. Etiologic analyses, which included ADAMTS13 activity, stool culture, complement factor proteins (C3, C4, factor H, factor I, and MCP [membrane cofactor protein]), anti-factor H antibodies, HIV (human immunodeficiency virus) serology, and antinuclear and antiphospholipid antibodies, returned normal results. Malignant hypertension was diagnosed. Ten months later, we observed a relapse of acute kidney injury and mechanical hemolysis. Considering a diagnosis of complement dysregulation-related atypical hemolytic uremic syndrome (HUS), we began treatment with eculizumab. Despite the efficient complement blockade, the patient's kidney function continued to decline. We performed additional analyses and found that the patient's homocysteine levels were dramatically increased, with no vitamin B12 (cobalamin) or folate deficiencies. We observed very low plasma methionine levels associated with methylmalonic aciduria, which suggested cobalamin C disease. We stopped the eculizumab infusions and initiated specific treatment, which resulted in complete cessation of hemolysis. MMACHC (methylmalonic aciduria and homocystinuria type C protein) sequencing revealed compound heterozygosity for 2 causative mutations. To our knowledge, this is the first report of adult-onset cobalamin C-related HUS. Considering the wide availability and low cost of the homocysteine assay, we suggest that it be included in the diagnostic algorithm for adult patients who present with HUS.

Determination of cysteine, homocysteine, cystine, and homocystine in biological fluids by HPLC using fluorosurfactant-capped gold nanoparticles as postcolumn colorimetric reagents.

We have demonstrated for the first time the suitability of fluorosurfactant-capped spherical gold nanoparticles as HPLC postcolumn colorimetric reagents for the direct assay of cysteine, homocysteine, cystine, and homocystine. The success of this work was based on the use of an on-line tris(2-carboxyethyl)phosphine reduction column for cystine and homocystine. Several parameters affecting the separation efficiency and the postcolumn colorimetric detection were thoroughly investigated. Under the optimized conditions, cysteine, homocysteine, cystine, and homocystine in human urine and plasma samples were determined. Detection limits for cysteine, homocysteine, cystine, and homocystine ranged from 0.16-0.49 µM. The accuracy in terms of recoveries ranged between 94.0-102.1%. This proposed method was rapid, inexpensive, and simple.

Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase.

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 µM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.

Inborn errors of cobalamin absorption and metabolism.

Derivatives of cobalamin (vitamin B(12)) are required for activity of two enzymes in humans. Adenosylcobalamin is required for activity of mitochondrial methylmalonylCoA mutase and methylcobalamin is required for activity of cytoplasmic methionine synthase. Deficiency in cobalamin, or inability to absorb cobalamin normally, can result in accumulation of methylmalonic acid and homocysteine in blood and urine. Methylmalonic acidemia can result in metabolic acidosis which in severe cases may be fatal. Hyperhomocysteinemia along with hypomethioninemia can result in hematologic (megaloblastic anemia, neutropenia, thrombocytopenia) and neurologic (subacute combined degeneration of the cord, dementia, psychosis) defects. Inborn errors affecting cobalamin absorption (inherited intrinsic factor deficiency, Imerslund­Gra¨ sbeck syndrome) and transport (transcobalamin deficiency) have been described. A series of inborn errors of intracellular cobalamin metabolism, designated cblA-cblG, have been differentiated by complementation analysis. These can give rise to isolated methylmalonic acidemia (cblA, cblB, cblD variant 2), isolated hyperhomocysteinemia (cblD variant 1, cblE, cblG) or combined methylmalonic acidemia and hyperhomocysteinemia (cblC, classic cblD, cblF). All these disorders are inherited as autosomal recessive traits. The genes underlying each of these disorders have been identified. Two other disorders, haptocorrin deficiency and transcobalamin receptor deficiency, have been described, but it is not clear that they have any consistent clinical phenotype.

Use of isotope differential derivatization for simultaneous determination of thiols and oxidized thiols by liquid chromatography tandem mass spectrometry.

Here we report a new isotopic pair of derivatization reagents, ω-bromoacetonylquinolinium bromide (BQB) and d(7)-ω-bromoacetonylquinolinium bromide (d(7)-BQB). BQB and d(7)-BQB both rapidly and selectively reacted with thiols in acidic medium within 3min with the aid of a microwave. Reduced thiols and total thiols in urine were labeled with BQB and d(7)-BQB, respectively. The BQB- and d(7)-BQB-labeled urine samples were then mixed and separated on a HILIC (hydrophilic interaction chromatography) column followed by electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection. The new strategy, which we have named isotope differential derivatization, allows us to simultaneously determine thiols and oxidized thiols in a single run. Compared with positive mode ESI detection of unlabeled thiols, the positive mode ESI-MS signal intensities of BQB-labeled thiols were found to increase by 10-, 20-, and 40-fold for cysteine (Cys), homocysteine (HCys), and glutathione (GSH), respectively (unlabeled N-acetylcysteine (Nac) is difficult to detect by ESI-MS in positive mode due to its low ionization efficiency). The detection limits calculated at a signal-to-noise ratio of 3 were found to be 8.02, 1.56, 0.833, and 3.27nmol/L for Cys, HCys, Nac, and GSH, respectively. Recoveries of thiols and disulfides from spiked urine samples were between 80% and 105%. The method was successfully used to determine thiols and oxidized thiols in urine samples of 25 healthy volunteers.