A quantitative method for the assessment of homocysteine thiolactonase enzyme activity.

This assay elucidates an accurate, simple, and precise protocol to quantify the activity of homocysteine thiolactonase (HTase). To establish HTase activity, the enzyme samples were incubated with a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, which contained suitable concentrations of the homocysteine thiolactone as a substrate. To stop the enzyme's reaction, the CUPRAC reagent (Cu(Nc)22+) was added after a suitable incubation time. The reduction of Cu(II)-neocuproine complex (Cu(Nc)22+) to highly coloured Cu(I)-neocuproine complex (Cu(Nc)2+) by the produced homocysteine was quantified spectrophotometrically at 450 nm (CUPRAC method). The increase in the absorbance of the coloured Cu(I)-neocuproine complex (Cu(Nc)2+) was correlated directly to the activity of HTase. ANOVA analysis was utilised to validate the new method against homocysteine thiolactonase activity using the H+ ions liberating method in matched samples. In conclusion, according to the obtained correlation coefficient (0.9995) from the comparison of the current method with the reference method, the current method is effective in assay HTase activity with high reliability.

High-throughput method for the quantitation of metabolites and co-factors from homocysteine-methionine cycle for nutritional status assessment.

AIM: There is increasing interest in the profiling and quantitation of methionine pathway metabolites for health management research. Currently, several analytical approaches are required to cover metabolites and co-factors. RESULTS: We report the development and the validation of a method for the simultaneous detection and quantitation of 13 metabolites in red blood cells. The method, validated in a cohort of healthy human volunteers, shows a high level of accuracy and reproducibility. CONCLUSION: This high-throughput protocol provides a robust coverage of central metabolites and co-factors in one single analysis and in a high-throughput fashion. In large-scale clinical settings, the use of such an approach will significantly advance the field of nutritional research in health and disease.

Simultaneous assessment of endogenous thiol compounds by LC-MS/MS.

Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC-MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC-MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1-400µM. The lowest limits of detection (S/N=3) in a range from 0.31fmol (for NAC) to 4.98fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.

Combined cobalamin and iron deficiency anemia: a diagnostic approach using a model based on age and homocysteine assessment.

Macrocytosis, the hallmark of cobalamin/folate deficiency anemia, is frequently absent. Clinicians have to be aware of coexisting conditions that can mask the macrocytosis expression of megaloblastic anemia, especially iron deficiency. The objective of this work was to investigate the degree of overlap between iron deficiency anemia (IDA) and cobalamin deficiency and to develop a predictive model for differentiating IDA from combined deficiency. A prospective case and control study was carried out to investigate vitamin B12 and folate status in iron deficiency anemia. A total of 658 patients were recruited, 41 of whom (6.2 %) were excluded. The remaining 617 subjects consisted of 130 controls and 487 with IDA. Low vitamin B12 (LB12) was considered when serum vitamin B12 was ≤200 pmol/L. High serum homocysteine (Hcy) was defined by Hcy >17 µM/L. A multivariate analysis (including a logistic regression) was performed to develop a diagnostic model. Low vitamin B12 levels were found in 17.8 % of IDA subjects. Ten out of 11 subjects (91 %) with IDA and serum vitamin B12 (B12) ≤100 pmol/L showed vitamin B12 deficiency. Moreover, vitamin B12 deficiency was demonstrated in 48 % of cases with IDA and B12 between 101 and 150 pmol/L and in 40 % with IDA and B12 between 151 and 200 pmol/, respectively. As a result of multivariate logistic analysis, neutrophil counts and age predicted subjects with vitamin B12 ≤200 and Hcy >17 µmol/L, [Formula: see text]. Using the age of 60 as a cutoff, sensitivity was 91 % (39 out of the 43 patients with vitamin B12 deficiency and IDA were identified). In summary, low vitamin B12 was found in 18 % of patients with IDA. Vitamin B12 deficiency was demonstrated in many patients with LB12 and IDA. Age over 60 years was used to separate patients with combined deficiency (sensitivity 91 %). Therefore, for a diagnostic purpose, serum vitamin B12 should be evaluated in IDA patients over 60 years. This diagnostic model needs to be validated in a different population.

[Is bone biopsy necessary for the diagnosis of metabolic bone diseases? Non- invasive assessment of bone turn over markers could define the cause of metabolic bone diseases].

Recent advances of the measurement of bone turn over markers contribute to non-invasive assessment of bone-metabolic disorders. We can detect the cause of the metabolic disorders with bone turn over markers and hormonal profiles more easily than before. Today, we can diagnose and treat metabolic bone diseases without invasive procedure such as bone biopsy.

Enhancing recruitment of healthy African American volunteers in a city with a small African American community: results from a dietary supplement crossover trial.

OBJECTIVE: To describe strategies for enhancing recruitment of African Americans to a longterm intervention study requiring frequent blood draws and follow-up visits, in a city with relatively few African Americans. DESIGN: The intervention study was a 14-month, double-blind, crossover study evaluating the effects of three oral folic acid doses on blood homocysteine levels. The goal was to have 40 African Americans complete the study, in addition to 160 participants from other races and ethnicities. RESULTS: Of 707 healthy, adult men and women recruited, 57 were African Americans. Recruitment advice was sought from African American community leaders interested in health research and the advice can be attributable to the success of recruitment. As suggested by the community leaders, our female African American project manager made oral presentations to select community groups. Word-of-mouth support from community leaders and study participants helped recruitment. Although the adult Seattle population is 7.4% African American, the group completing the study comprised 15% African Americans. Retention in the dietary intervention was 74% (31 out of 42) among African Americans, 81% (158 out of 196) among non-African Americans--a statistically non-significant difference. CONCLUSIONS: Advice from African American community leaders about targeting appropriate civic/professional groups, churches, and community organizations can lead to effective recruitment of African Americans. Advice should be sought before beginning recruitment and endorsement for the study should be obtained. Effective retention of African American participants is possible for intervention studies requiring multiple blood draws and follow-up visits.

Serum homocysteine is related to food intake in adolescents: the Child and Adolescent Trial for Cardiovascular Health.

BACKGROUND: An understanding of the relation in adolescents between serum homocysteine and foods rich in vitamin B-6, vitamin B-12, and folate is important because high homocysteine concentrations in childhood and adolescence may be a risk factor for later cardiovascular disease. However, little is known about the relation between food intake and homocysteine in adolescents. OBJECTIVE: Five years after national folic acid fortification of enriched grain products, cross-sectional relations between food intake and serum homocysteine concentrations were examined in 2695 adolescents [x age: 18.3 (range: 15-20) y] enrolled in the Child and Adolescent Trial for Cardiovascular Health. DESIGN: A nonfasting blood specimen was analyzed for serum homocysteine, folate, and vitamins B-6 and B-12. Dietary intake was assessed by using a food-frequency questionnaire. Multiple regression analyses were used to evaluate the relation of intakes of whole grains, refined grains, fruit, vegetables, dairy products, red and processed meats, and poultry with serum homocysteine concentrations after adjustment for demographic characteristics, lifestyle factors, and food intake. RESULTS: Serum homocysteine concentrations were lower with greater intakes of whole grains (P for trend = 0.002), refined grains (P for trend = 0.02), and dairy foods (P for trend <0.001); were higher with greater intake of poultry (P for trend = 0.004); and were not related to intakes of fruit, vegetables, or red or processed meat. After additional adjustment for serum B vitamins, the relations of serum homocysteine with most food groups were attenuated. CONCLUSION: These observational findings suggest a beneficial effect of whole-grain, refined-grain, and dairy products on serum homocysteine concentrations in an adolescent population.

High-sensitivity C-reactive protein: potential adjunct for global risk assessment in the primary prevention of cardiovascular disease.

Inflammation plays a major role in atherothrombosis, and measurement of inflammatory markers such as high-sensitivity C-reactive protein (HSCRP) may provide a novel method for detecting individuals at high risk of plaque rupture. Several large-scale prospective studies demonstrate that HSCRP is a strong independent predictor of future myocardial infarction and stroke among apparently healthy men and women and that the addition of HSCRP to standard lipid screening may improve global risk prediction among those with high as well as low cholesterol levels. Because agents such as aspirin and statins seem to attenuate inflammatory risk, HSCRP may also have utility in targeting proven therapies for primary prevention. Inexpensive commercial assays for HSCRP are now available; they have shown variability and classification accuracy similar to that of cholesterol screening. Risk prediction algorithms using a simple quintile approach to HSCRP evaluation have been developed for outpatient use. Thus, although limitations inherent to inflammatory screening remain, available data suggest that HSCRP has the potential to play an important role as an adjunct for global risk assessment in the primary prevention of cardiovascular disease.

Energy cost of proofreading in vivo: the charging of methionine tRNAs in Escherichia coli.

Previous in vitro work has shown that Escherichia coli methionyl-tRNA synthetase has a limited ability to discriminate against cognate methionine in the editing site designed for noncognate homocysteine. As a result, a small fraction of the correct product Met-tRNA is deacylated with the formation of a cyclic sulfonium compound, S-methyl-homocysteine thiolactone. This is exploited here to estimate energy costs associated with the destruction of a correct product by methionyl-tRNA synthetase in bacterial cells. In vivo measurements of S-methyl-homocysteine thiolactone indicate that in Escherichia coli 3.3 molecules of Met-tRNA are destroyed by deacylation per 10,000 molecules of Met-tRNA successfully transferring methionine to protein. This number of destroyed molecules of a correct product, Met-tRNA, is 30 times lower than the number of destroyed molecules of an incorrect product, homocysteinyl adenylate. Thus, most of the energy cost of proofreading in vivo is due to editing of the noncognate amino acid.