Role of aminothiols as a component of the plasma antioxidant system and relevance to homocysteine-mediated vascular disease

Hyperhomocysteinaemia is considered to be an independent risk factor for vascular disease. Elevated plasma homocysteine may pose an oxidative stress, leading to the development of vascular damage. A component of this effect may be a disturbance of the extracellular aminothiol redox state. The relative contributions of plasma total homocysteine (tHcy) and plasma total cysteine (tCys) to the total antioxidant capacity (TAOC) of plasma was established in subjects with normal and elevated plasma tHcy. A total of 10 subjects with severe hyperhomocysteinaemia (due to inherited metabolic defects), 13 of their heterozygous parents and 72 normal healthy subjects were recruited to the study. The mean plasma tHcy in the patients was 91.8 micromol/l, compared with 13.2 micromol/l in the parents and 14.7 micromol/l in healthy control subjects. Plasma tCys and plasma TAOC were significantly lower in the subjects with severe hyperhomocysteinaemia compared with the parents and healthy control subjects (P<0.05). In blood samples from subjects with a normal tHcy, a positive correlation was observed between tCys and tHcy (P=0.0001). In contrast, in blood samples with tHcy >or=20 micromol/l, plasma tCys was negatively correlated with tHcy (P=0.0001). In samples with tHcy >or=20 micromol/l, tHcy was inversely correlated with TAOC (P=0.0001), whereas tCys was positively associated with TAOC (P=0.0001). Multiple regression analysis revealed that tCys was the most important independent determinant of TAOC in the patient and control groups when the effects of tHcy and several factors known to influence TAOC, such as urate, were taken into account. Thus hyperhomocysteinaemia may pose an oxidative stress not only through the direct cytotoxicity of homocysteine, but also from an associated fall in plasma cysteine.     

Cigarette smoking and plasma total homocysteine levels in young adults with type 1 diabetes

OBJECTIVE: The purposes of this study were to compare plasma total homocysteine (tHcy) levels, a recognized cardiovascular risk factor, in nondiabetic subjects and type 1 diabetic patients, and to evaluate whether chronic cigarette smoking had a deleterious effect on plasma tHcy levels in type 1 diabetic patients. RESEARCH DESIGN AND METHODS: Plasma tHcy concentrations were measured in 60 young type 1 diabetic patients without clinical evidence of macroangiopathy and in 30 healthy control subjects who were matched for age, sex, BMI, and smoking habit. RESULTS: Plasma tHcy levels were significantly higher in type 1 diabetic patients than in control subjects (12.5 +/- 4.8 vs. 10.3 +/- 2.2 micromol/l, P = 0.01). After stratification by smoking status, diabetic smokers had values for age, sex, BMI, lipids, creatinine, blood pressure, glycometabolic control, diabetes duration, and microvascular complications that were superimposable on their nonsmoking counterparts. Nevertheless, plasma tHcy levels were markedly elevated in diabetic smokers versus nonsmokers (15.5 +/- 5.7 vs. 10.6 +/- 3 pmol/l, P < 0.0001) in a dose-dependent fashion (P < 0.0001, by analysis of variance when subjects were categorized for the number of cigarettes smoked daily). CONCLUSIONS: Chronic cigarette smoking seems to adversely affect plasma tHcy levels in young adults with type 1 diabetes.     

Automated assay for the determination of methylmalonic acid, total homocysteine, and related amino acids in human serum or plasma by means of methylchloroformate derivatization and gas chromatography-mass spectrometry

BACKGROUND: The combined measurement of methylmalonic acid (MMA) and total homocysteine (tHcy) in serum or plasma is useful in diagnosing and distinguishing between cobalamin and folate deficiencies. We developed and validated an isotope-dilution gas chromatography-mass spectrometry (GC-MS) method with automated sample workup for the determination of MMA, tHcy, and the related amino acids Met, total cysteine (tCys), Ser, and Gly in serum or plasma. METHODS: Serum or plasma samples (100 microL) were treated with a reductant (dithioerythritol), deproteinized with ethanol, and derivatized and extracted in a single step by the addition of methylchloroformate and toluene. All liquid handling was performed in 96-well (1 mL) microtiter plates by a robotic workstation. The N(S)-methoxycarbonyl ethyl ester derivatives were analyzed by GC-MS in the selected-ion monitoring mode. RESULTS: Detection limits (signal-to-noise ratio, 5:1) were between 0.03 micromol/L (MMA) and 10 micromol/L (Ser, tCys). The assay was linear to 100 micromol/L for MMA and tHcy and to 1000 micromol/L for Met, tCys, Ser, and Gly. The within-day CVs ranged from 0.7% to 3.6% (n = 20), and the between-day CVs from 2.1% to 8.1% (n = 20). The recovery was between 79% and 99% for the different analytes. CONCLUSION: This assay combines a simple and automated sample preparation with selective and sensitive GC-MS analysis and is well suited for the combined measurement of MMA, tHcy, and the related amino acids.     

血浆同型半胱氨酸及其相关硫醇物在全血中的稳定性研究

目的 观察含EDTA的全血样本放置时间、保存温度以及不同稳定剂对血浆同型半胱氨酸(homocysteine,Hcy)及其相关硫醇物水平的影响.方法 用含EDTA、EDTA-氟化钠(EDTA-NaF)、EDTA-3-Deazaadenosine(EDTA-3DA)的试管收集17名健康成人静脉血,置于碎冰上(0～4 ℃)或室温中(25 ℃)保存,放置0、3、6、24、48 h后分离血浆.用HPLC法测定血浆总同型半胱氨酸(tHcy)、总半胱氨酸(total cysteine,tCys)、总半胱氨酰甘氨酸(total cysteinylglycine,tCysGly)、总谷胱甘肽(total glutathione,tGSH)浓度.设定全血样本0 h分离血浆所测硫醇物浓度为基础值.结果 EDTA管在室温中放置3、6、24、48 h,tHcy分别增加38.5%、64.2%、141.9%、225.4%;tCysGly、tGSH在3 h分别增加20.0%、37.9%,tCys则降低3.5%.EDTA管在碎冰上保存,各硫醇物浓度6 h内增加不超过5%.EDTA-3DA和EDTA-NaF管在室温放置3 h,与各自基础值相比,血浆tHcy、tCys、tCysGly、tGSH浓度差异无统计学意义(EDTA-3DA管:F值分别为0.01、0.94、0.09、0.01,P值均>0.05;EDTA-NaF管:F值分别为0.85、0.04、0.03、0.02,P值均>0.05).结论 EDTA抗凝血浆,所测tHcy及其相关硫醇物浓度呈时间、温度依赖性增加.血浆tHcy等硫醇物测定的分析前处理条件必须标准化.EDTA-3DA和EDTA-NaF管可使血浆tHcy、tCys、tCysGly、tGSH在室温中至少稳定3 h.

Abstract：

Objective To investigate the effects of different stabilizers, time and temperature before centrifugation on the stability of homocysteine (Hcy) and other related thiols levels involving EDTA-containing whole blood.Methods Blood was drawn from 17 healthy adults and collected into tubes containing EDTA, EDTA plus NaF and EDTA plus 3-deazaadenosine(3DA),then stored on crush ice(0-4 ℃) immediately or at room temperature(25 ℃).Plasma was separated at 0, 3, 6, 24 and 48 hours, respectively.The levels of plasma total Hcy (tHcy), total cysteine (tCys), tatal cysteinylglycine (tCysGly) and tatal glutathione (tGSH) were measured by HPLC.The plasma immediately separated was used as baseline sample. Results In EDTA tubes stored at room temperature, tHcy levels increased by 38.5%, 64.2%, 141.9%, 225.4% for 3, 6, 24, and 48 h, respectively.The levels of tCysGly and tGSH increased by 20.0% and 37.9% within 3 h, however, tCys decreased by 3.5%.The levels of the thiols increase by less than 5% up to 6 h in EDTA tubes stored on crush ice.In EDTA-3DA and EDTA-NaF tubes, no statistical differences were observed in the plasma levels of tHcy, tCys,tCysGly and tGSH compared with their respective baseline values at room temperature for 3 h(EDTA-3DA tubes:F=0.01,0.94,0.09,0.01,all P>0.05;EDTA-NaF tubes:F=0.85,0.04,0.03,0.02,all P>0.05).Conclusions The EDTA-plasma levels of tHcy and other related thiols are time and temperature-dependent. There is a strong need for standardization of blood sample collection and processing in tHcy and other thiols assays. The plasma concentrations of tHcy, tCys, tCysGly and tGSH were stable for 3 h at least in the EDTA-3DA and EDTA-NaF tubes kept at room temperature.     

终末期肾病患者血浆同型半胱氨酸及其他氨基硫醇物水平变化

目的 观察终末期肾病(end-stage renal disease,ESRD)患者血液透析前后血浆同型半胱氨酸(homocysteine,Hcy)及其他氨基硫醇物的水平变化.方法 选择慢性维持血液透析患者26例,健康对照组54名.用HPLC-荧光检测法测定血浆总Hey(tHcy)、总半胱氨酸(total cysteine,tCys)、总半胱氨酰甘氨酸(total cysteinylglycine,tCysGly)、总谷胱甘肽(total Glutathione,tGSH)浓度.同时测定受检者的血脂水平和肾功能.结果 ESRD患者血液透析前血浆tHcy、tCys、tCysGly浓度分别为(16.70±3.51)μmol/L、(341.87±70.65)μmol/L和(41.33±32.95)μmol/L,明显高于健康对照组的(10.95±3.07)μmol/L、(249.76±13.18)μmol/L和(31.3±11.78)μmol/L,差异有统计学意义(t值分别为3.625、6.219和3.530,P均<0.01);ESRD患者血液透析前血浆tGSH浓度为(5.91±0.08)μmol/L,低于健康对照组的(9.33 ±2.62)μmol/L,差异有统计学意义(t=-5.404,P<0.01);血液透析后,tHcy、tCys浓度分别为(11.74±3.42)μmol/L、(272.67±64.18)μmol/L,较透析前显著降低,但不能恢复至正常水平;血液透析前后tCysGly浓度分别为(41.33±32.95)μmol/L与(44.93±13.88)μmol/L,差异无统计学意义(t=-0.758,P>0.05);tGSH浓度分别为(5.91±0.08)μmol/L与(5.93±0.38)μmol/L,差异无统计学意义(t=-0.068,P>0.05).患者血浆tHcy与tCys和Cr呈正相关(r值分别为0.753 6、0.458 2,P均<0.01),与tGSH呈负相关(r值为-0.6099,P=0.000 9),与tCysGly和血脂参数无关.结论 ESRD患者普遍存在氨基硫醇物代谢紊乱.     

Reference interval and subject variation in excretion of urinary metabolites of nicotine from non-smoking healthy subjects in Denmark

BACKGROUND: Passive smoking has been found to be a respiratory health hazard in humans. The present study describes the calculation of a reference interval for urinary nicotine metabolites calculated as cotinine equivalents on the basis of 72 non-smokers exposed to tobacco smoke less than 25% of the day. METHODS: Twenty subjects (passive smokers) exposed to tobacco smoke more than 25% of the day (subjectively assessed) and 32 smokers were used to validate the estimated reference interval. Urine samples were collected three times during the day approximately at 06.30, 17.00 and 22.45 h. RESULTS: Within-subject variation was found to be 89.4, 72.6, and 79.2% and between-subject variation was found to be 64.5, 64.2, and 36.1%. No gender difference could be demonstrated. In general all subjects showed increased concentrations in the afternoon and evening samples compared to the morning samples. Parametric reference interval for excretion of nicotine metabolites in urine from non-smokers was established according to International Union of Pure and Applied Chemistry (IUPAC) and International Federation for Clinical Chemistry (IFCC) for use of risk assessment of exposure to tobacco smoke. The reference interval for urinary cotinine was estimated to be 1.1-90.0 micromol/mol creatinine in morning samples from non-smokers. An intercomparison between the radioimmunoassay (RIA) method used for determination of nicotine metabolites and a gas chromatography-mass spectrometry (GC-MS) method for determination of cotinine was carried out on 27 samples from non-smokers and smokers. Results obtained from the RIA method showed 2.84 [confidence interval (CI): 2.50; 3.18] times higher results compared to the GC-MS method. A linear correlation between the two methods was demonstrated (rho=0.96). CONCLUSION: The RIA method is rapid and adequate for clinical use in the assessment of exposure to tobacco smoke, i.e. ratio between CV(a)/CV(ti) was<0.50.     

Association of hyperhomocysteinemia and <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> infection with carotid atherosclerosis and coronary artery disease in Japanese patients

An elevated plasma level of homocysteine (Hcy) and infection by Chlamydia pneumoniae (C. pneumoniae) have been suggested as independent risk factors for carotid atherosclerosis (CA) and coronary artery disease (CAD), but the mechanisms involved are unclear. We investigated the correlation between positivity for antibody to C. pneumonia (anti-C. pneumoniae) and the Hcy level in patients with CA and CAD. The total plasma homocysteine (tHcy) concentration was determined in 99 patients with CA and 31 patients with CAD, as well as 119 controls with matched risk factors for atherosclerosis. The tHcy level was measured with a Bio-Rad microplate enzyme immunoassay. In the CAD group, the tHcy level (13.67 micromol/l) was significantly higher than that in other groups (CA group, 10.96 micromol/l; control group, 9.95 micromol/l; ANOVA, P = 0.0006). Positivity for anti-C. pneumoniae IgG was significantly more common in the CAD group (77.4%) than in the other groups (CA group, 53.5%; control group, 54.6%; ANOVA, P = 0.0490). There was no association between anti-C. pneumoniae IgA positivity or tHcy and conventional risk factors. However, anti-C. pneumoniae IgG positivity was significantly more common in subjects with higher tHcy levels than in those with low tHcy levels from each of the 3 groups. The CAD group had significantly higher tHcy levels, and tHcy was significantly associated with anti-C. pneumoniae IgG positivity. These findings indicate that elevation of tHcy is related to positivity for anti-C. pneumoniae IgG in patients with CAD.     

The relationship among homocysteine, creatinine clearance, and albuminuria in patients with type 2 diabetes

OBJECTIVE: Although it is accepted that elevated plasma homocysteine (tHcy) levels occur in end-stage renal disease and type 2 diabetes, the changes with milder renal dysfunction (e.g., microalbuminuria) are less clearly established. This study explores the relationship among tHcy, creatinine clearance (Ccr), and albumin excretion rate (AER) in a population with type 2 diabetes. RESEARCH DESIGN AND METHODS: A total of 260 patients with type 2 diabetes were screened in our outpatient clinic during 10 months. Fasting blood samples were collected, and AER was calculated from an overnight timed urine sample. Ccr was calculated using the Cockroft-Gault formula. RESULTS: A total of 198 subjects (76%) had normoalbuminuria (<20 microg/min), 50 subjects (19%) had microalbuminuria (20-200 microg/min), and 12 subjects (5%) had macroalbuminuria (>or=200 microg/min). Those with microalbuminuria had higher levels of tHcy than those with normoalbuminuria (13.2 +/- 7.8 vs. 11.3 +/- 4.6 micromol/l, P < 0.05). Patients were then subdivided based on low Ccr (<80 ml x min(-1) x 1.73 m(-2)) and normal Ccr (>or=80 x min(-1) x 1.73 m(-2)). None of the patients with macroalbuminuria had normal Ccr. In those with normoalbuminuria, tHcy levels were higher than in those with low Ccr than in those with normal Ccr (12.0 +/- 4.6 vs. 10.0 +/- 4.4 micromol/l, P < 0.01). The same was found for those with microalbuminuria (low Ccr versus normal Ccr: 14.6 +/- 9.0 vs. 10.2 +/- 2.8 micromol/l, P < 0.02). For normal Ccr, tHcy was similar irrespective of AER (normoalbuminuria versus microalbuminuria: 10.0 +/- 4.4 vs. 10.2 +/- 2.8 micromol/l, NS). For low Ccr, tHcy was higher in those with microalbuminuria versus normoalbuminuria (14.6 +/- 9.0 vs. 12.0 +/- 4.6 micromol/l, P = 0.01). Using multivariate regression, Ccr, but neither AER nor the presence of albuminuria, was an independent predictor of tHcy. CONCLUSIONS: These data strongly suggest that in patients with type 2 diabetes, the relationship between plasma tHcy and AER is largely due to associated changes in renal function, as defined by Ccr.     

Plasma, salivary and urinary cotinine in non-smoker Italian women exposed and unexposed to environmental tobacco smoking (SEASD study).

BACKGROUND: The aim of this study was to compare cotinine determinations in three biological fluids for assessing environmental tobacco smoke (ETS) exposure in female non-smokers (n=1605) in Italy. METHODS: Information about ETS exposure at home, in the workplace, and in other places within the previous week was collected via questionnaire. Plasma, salivary and urinary cotinine levels were measured. Cotinine levels of > or =0.1 ng/mL for plasma, > or =0.2 ng/mL for saliva, and > or =0.5 ng/mL for urine were used to determine biochemical exposure. RESULTS: Median cotinine levels were significantly higher in exposed than in unexposed women (0.21 vs. 0.05 ng/mL in plasma, 0.80 vs. 0.41 ng/mL in saliva, and 9.74 vs. 5.30 ng/mL in urine). Self-reported ETS exposure was significantly related to biochemical exposure [odds ratio 2.99, (95% CI 2.40-3.72) for plasma; 1.90 (1.51-2.39) for saliva; and 2.67 (2.14-3.34) for urine]. Cotinine significantly increased with increasing exposure level, regardless of the exposure source. Among self-reported exposed subjects, higher percentages of cotinine level above the cut-off, i.e., indicating exposure, were found in saliva (76%) and urine (75%) than in plasma (52%). CONCLUSIONS: In general, women correctly reported their ETS exposure status. Both non-invasive salivary and urinary cotinine determinations seem preferable in epidemiological studies, in view of their higher sensitivity, when compared to plasma cotinine.     

Hyperhomocysteinemia in type 2 diabetes: relationship to macroangiopathy, nephropathy, and insulin resistance

OBJECTIVE: The aim of this study was to determine the distribution of plasma total homocysteine (tHcy) concentrations in type 2 diabetic patients and to assess whether high tHcy values were related to chronic complications (particularly macroangiopathy and nephropathy) and/or the degree of insulin resistance. RESEARCH DESIGN AND METHODS: Fasting tHcy levels were measured in 122 type 2 diabetic patients in whom the presence of chronic complications (e.g., macroangiopathy, microalbuminuria, macroproteinuria, decreased creatinine clearance, hypertension, retinopathy, and neuropathy) was recorded alongside an assessment of insulin resistance by the homeostasis model assessment (HOMA). RESULTS: We found that 31% of the cohort (group 1) had raised tHcy (mean +/- 1 SD) values (20.8 +/- 5.1 micromol/l), whereas 69% (group 2) had normal values (10.2 +/- 2.0 micromol/l). The prevalence of macroangiopathy was higher in group 1 than in group 2 subjects (70 vs. 42%, P < 0.01); the prevalence of coronary artery disease was particularly higher in group 1 (46 vs. 21%, P < 0.02). The prevalence of impaired renal function, evidenced by decreased creatinine clearance, was higher in group 1 (32 vs. 10%, P < 0.005). Other clinical and biological characteristics of both groups were comparable, although group 1 had lower levels of folic acid than group 2 (5.2 +/- 2.9 vs. 7.0 +/- 3.4 ng/ml, P < 0.01). No differences were found for microalbuminuria (33 vs. 31%), retinopathy (45 vs. 42%), or neuropathy (70 vs. 59%) between groups 1 and 2, respectively The degree of insulin resistance was similar in groups 1 and 2 (46 +/- 21 and 42 +/- 20% of HOMA-insulin sensitivity) as was the assessment of beta-cell function (63 +/- 28 and 65 +/- 46%, respectively). No differences in tHcy levels were found between subjects receiving metformin and those not receiving metformin. In contrast, the plasma tHcy level was higher in diabetic patients treated with fibrates (P = 0.0016). CONCLUSIONS: Elevated plasma tHcy levels in type 2 diabetes is associated with a higher prevalence of macroangiopathy and nephropathy when assessed from creatinine clearance indexes and is not associated with different degrees of insulin resistance.     

Hyperhomocysteinemia and end-stage renal disease: determinants and association with cardiovascular disease in Tunisian patients.

The study reports on plasma total homocysteine (tHcy) levels in Tunisian patients with chronic renal failure (CRF) and those treated with hemodialysis (HD) and renal transplant (RT). The aims of the study were to identify the determinants of tHcy concentration and to test the association between hyperhomocysteinemia and atherothrombotic disease in end-stage renal disease (ESRD). A total of 35 CRF patients on conservative treatment, 50 HD patients, and 30 RT recipients, and 31 age- and sex-matched healthy subjects were included. Plasma tHcy was assessed by a fluorescent-polarizing immunoassay method. Multivariate analysis was applied to identify the main determinants of tHcy concentration and to assess the relationship between hyperhomocysteinemia and cardiovascular disease. Plasma mean tHcy concentration was significantly increased (p < 0.001) in CRF patients (mean +/- SD) (28.9 +/- 9.8 micromol/l), in HD patients (29.4 +/- 11.1 micromol/l), and in RT (19.3 +/- 6.3 micromol/l) patients compared to controls (11.9 +/- 4.1 micromol/l). Multivariate analysis using GLM ANOVA modeling demonstrated that tHcy was significantly higher in males (p = 0.02), and was related to age (p = 0.008), albumin (p = 0.005), vitamin B12 (p = 0.002), folate (p = 0.00001), and creatinine clearance (p = 0.0008). However, tHcy was not associated with C-reactive protein and did not significantly differ between CRF, HD, or RT patients. The upper quartile of tHcy concentration was significantly associated with atherothrombotic cardiovascular disease (unadjusted odds ratio (OR) = 3.09; 95% CI, 1.11-8.61; p = 0.01). This association remained significant after adjusting for sex, age, hypertension, and smoking (multi-adjusted OR = 4.78; 95% CI, 1.92-11.9; p = 0.0008). The mean tHcy concentration was 2 to 3 times higher in ESRD patients than in subjects with normal renal function. This increase could be related to glomerular filtration rate reduction and functional B vitamins deficiency, but was not associated with inflammation. The upper quartile of tHcy concentrations confers 4.78-fold increased independent risk for atherothrombotic events in ESRD patients.     

Analytical performance and method comparison study of the total homocysteine fluorescence polarization immunoassay (FPIA) on the AxSYM analyzer.

A fluorescence polarization immunoassay (FPIA) has been commercially released for routine large-scale testing of total homocysteine (tHcy) on the AxSYM analyzer. We evaluated the analytical performance of the AxSYM tHcy FPIA and compared it with the well established high-performance liquid chromatography (HPLC) and IMx tHcy FPIA methods. Homocysteine concentrations were measured by AxSYM and IMx tHcy FPIA and by a rapid isocratic HPLC method with fluorescence detection. Coefficient of variation (CV) of total imprecision for AxSYM tHcy was < or = 5%, mean dilution recovery 102%, analytical sensitivity 0.70 micromol/l and linearity was good up to 1:8 dilution. Spearman rank correlations, rho, were 0.83 (p < 0.0001) for AxSYM vs. HPLC, 0.97 (p < 0.0001) for AxSYM vs. IMx and 0.83 (p < 0.0001) for IMx vs. HPLC. Passing and Bablok regression Y-intercepts and slopes were: 2.944/0.937 (AxSYM vs. HPLC), -0.367/ 1.142 (AxSYM vs. IMx) and 2.632/0.805 (IMx vs. HPLC). Corresponding mean differences (AxSYM-Comparison Assay) recorded over a 5-50 micromol/l measured range were 1.80, -0.73 and 2.53 micromol/l. AxSYM tHcy FPIA's first rate precision, supported by the complete automation of the AxSYM analyzer, makes it fit for routine use and suitable for laboratories requiring homocysteine high-throughput testing capabilities.     

Impact of insulin resistance and nephropathy on homocysteine in type 2 diabetes

OBJECTIVE: To assess the impacts of insulin resistance and renal function on plasma total homocysteine (tHcy) levels in patients with type 2 diabetes with a wide range of nephropathy. RESEARCH DESIGN AND METHODS: Plasma tHcy levels were measured using the enzyme immunoassay method in 75 patients with type 2 diabetes and compared with those in 54 healthy control subjects. Insulin sensitivity indexes were assessed in patients with type 2 diabetes by hyperinsulinemic-euglycemic clamp using artificial pancreas. RESULTS: Plasma tHcy levels and their log-translormed values (log tHcy) were significantly higher in all patients with diabetes than in control subjects (tHcy, 12.0 +/- 0.7 [SE] vs. 8.7 +/- 0.3 micromol/l, P < 0.0001; log tHcy, 1.040 +/- 0.021 vs. 0.920 +/- 0.016 micromol/l, P < 0.0001). Plasma tHcy levels in patients with diabetes were significantly increased according to degree of nephropathy (P < 0.0001). On simple regression analyses, log tHcy correlated with insulin sensitivity indexes (r = -0.319, P = 0.005) as well as creatinine clearance (r = 0.634, P < 0.0001) in all patients with diabetes. Multiple regression analyses showed that insulin sensitivity indexes (beta = -0.245) as well as creatinine clearance were independent contributors to log tHcy in all patients with diabetes (R2 = 0.750, P < 0.0001). For the 59 patients with diabetes with creatinine clearance >60 ml/min, insulin sensitivity indexes were also shown to be a significant contributor to log tHcy (beta = -0.438, R2 = 0.561, P < 0.001). CONCLUSION: Insulin resistance and renal function are independent determinants of tHcy levels in patients with type 2 diabetes.     

Effects of passive smoking on theophylline clearance

Theophylline disposition was examined in seven passive smokers, defined as nonsmokers with long-term exposure to cigarette smoke, and seven age-matched nonsmokers with minimal smoke exposure. Subjects were given an intravenous infusion of aminophylline (6 mg/kg) and blood samples were drawn before and during the 48-hour postinfusion period. Clearance for passive smokers was 6.01 x 10(-2) L/hr.kg and for nonsmokers, clearance was 4.09 x 10(-2) L/hr.kg (p less than 0.025). Terminal elimination half-life for passive smokers was 6.93 hours versus 8.69 hours for nonsmokers (p less than 0.05). The mean residence time for passive smokers was 9.89 hours. For nonsmokers, the mean residence time was 13.11 hours (p less than 0.05). These measurements were statistically different, whereas there was no difference in volume of distribution between the groups, suggesting that passive smokers metabolize theophylline more rapidly than nonsmokers. Plasma and urine cotinine and nicotine concentrations were measured in all subjects. There was a significant difference between the subject groups in plasma (p less than 0.004) and urine (p less than 0.002) cotinine concentrations. Theophylline clearance correlated with both plasma (r = 0.73, p less than 0.01) and urine (r = 0.79, p less than 0.01) cotinine concentrations. Additional studies should be conducted to further define the pharmacokinetic characteristics of passive smokers and to assess the effects of passive smoking on drugs metabolized by the mixed function oxidase system.     

Relationship between thiolactonase activity and hyperhomocysteinemia according to MTHFR gene polymorphism in Tunisian Beh?et's disease patients.

BACKGROUND: Beh?et's disease (BD) is a multisystemic immuno-inflammatory disorder. Inflammatory processes may cause lipid peroxidation, alteration of lipid profile and increase the risk of atherosclerosis. The aim of this study was to evaluate the association between thiolactonase (HTLase) activity and plasma homocysteine levels (tHcy) in a BD population and to investigate their association with methylenetetrahydrofolate reductase (MTHFR) 677C-->T genotype. METHOD: A total of 35 BD patients were compared to 39 healthy volunteers. RESULTS: Significantly higher tHcy levels associated with lower HTLase activities were found in BD patients as compared to healthy controls (p<0.001). These patients also exhibited lower values of triglycerides and high-density lipoprotein cholesterol (HDL-C). Homozygosity for the T allele of the MTHFR gene was more frequent in BD patients (14.3% vs. 7.7%). It was associated with significantly higher tHcy levels (16.9 micromol/L for n=17 vs. 13.1 micromol/L for n=18; p<0.05) and markedly lower HTLase activity (362.6+/-156.7 U/L vs. 414.2+/-180.2 U/L) for the (TT+CT) and CC genotypes, respectively. Moreover, HDL-C levels were inversely correlated with tHcy (r=-0.5; p=0.004) but positively associated with HTLase activity (r=0.374; p=0.038). These correlations were also present in several clinical manifestations, such as ocular, neurological involvement or thrombosis. CONCLUSIONS: Homozygosity of the T allele of the MTHFR gene is prevalent in BD patients. High levels of tHcy associated with low HTLase activities may be one of the causes leading to thrombosis in BD patients.     

HMG-CoA reductase inhibitors are associated with decreased serum neopterin levels in stable coronary artery disease.

Neopterin, a marker of stimulated cellular immune response, is increased in unstable angina, acute myocardial infarction and possibly stable coronary artery disease. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) have anti-inflammatory properties, but their effect on neopterin is largely unknown. Neopterin was measured in 232 patients undergoing elective coronary angiography and compared to the degree of atherosclerosis, use of concomitant medications and demographics. Neopterin was lower in subjects using statins (n = 66) compared to those not taking statins (median (range): 6.65 (4.1-18.3) vs. 7.70 (3.6-29.1) nmol/l, p < 0.0001). This association was also found in the subgroup of patients with coronary artery disease (1-3-vessel disease, n = 164; 6.60 (4.1-18.3) vs. 7.80 (3.6-29.1) nmol/l, p = 0.0012), whereas only a slight tendency toward lower neopterin levels was found in the group without atherosclerosis (6.90 (5.1-16.0) vs. 7.60 (4.0-18.5) nmol/l, p = 0.17). In patients with coronary atherosclerosis, neopterin concentrations were lower in smokers (n = 105) compared to non-smokers (7.20 (3.6-29.1) vs. 7.90 (4.4-18.6) nmol/l, p < 0.02), confirming previous observations. However, use of statins was associated with lower neopterin levels in both non-smokers and smokers (6.70 (4.1-18.3) vs. 7.60 (3.6-29.1) nmol/l, p < 0.05, and 6.20 (5.2-16.0) vs. 7.80 (4.4-18.6) nmol/l, p < 0.05, respectively). Overall, similar serum neopterin concentrations were found in patients with coronary atherosclerosis and those without. In accordance with their anti-inflammatory effects, the use of statins is associated with lower neopterin levels in patients undergoing elective coronary angiography.     

Relationship between urinary cotinine and serum vitamin A levels in Korean adults: the Korea National Health and Nutrition Examination Survey (KNHANES), 2016–2018

ObjectiveTo examine the relationship between urinary cotinine and serum vitamin A levels in Korean adults.MethodsA total of 4445 adults (age ≥19 years) participating in the Korea National Health and Nutrition Examination Survey (KNHANES) from 2016 to 2018 were classified by sex and as smokers/electronic cigarette users (SE) or non-smokers (NS). Data were analyzed using complex sample general linear models.ResultsThere were no differences in dietary intake of vitamin A, carotene, or retinol between the SE and NS groups. Adjusted mean serum vitamin A levels were higher in the SE group compared with those in the NS group (0.63 mg/L vs 0.60 mg/L among men; 0.55 mg/L vs 0.51 mg/L among women). Among all participants, urinary cotinine and serum vitamin A levels were positively correlated (R² = 0.037). However, no correlation was observed in either the SE or NS groups individually. In a model adjusted for age, body mass index, sex, frequency of binge drinking, and dyslipidemia, a stronger correlation was observed (R² = 0.244).ConclusionIn Korean adults, urinary cotinine levels were positively associated with serum vitamin A levels. Mean serum vitamin A levels were significantly higher in the SE group compared with the NS group.     

Highly sensitive immuno-assays for the determination of cotinine in serum and saliva. Comparison between RIA and an avidin-biotin ELISA

Two immuno-assay methods (RIA and ELISA) have been developed for the accurate and sensitive measurement of cotinine in human body fluids (serum, saliva). RIA uses [3H]cotinine as antigen and charcoal/dextran for separating cotinine-bound antibodies from the free derivative. Another technique (ELISA) was developed to avoid the use of radio-labelled compounds and to determine cotinine in large populations, including passive or non-smokers who usually present very low concentrations. The two techniques were analytically validated. The detection limit was similar (0.1 micrograms/l) and the precision was better than 10% for both techniques. Non-smoker values ranged from 0.1 to 17 micrograms/l by ELISA and 0.1 to 27.5 micrograms/l by RIA, whereas smoker values ranged from 50 to 1000 micrograms/l (ELISA) and from 70 to 800 micrograms/l (RIA). The comparative analysis of cotinine in 96 human sera revealed a good correlation between the two methods (r = 0.97) and a reliable discrimination between the populations of non-smokers and smokers. As usual, the ELISA is more rapid (4 h 30 min) than the RIA (longer than 48 h). ELISA is proposed for use in the epidemiological investigation of the human tobacco risk.     

Determinants and vitamin responsiveness of intermediate hyperhomocysteinemia (> or = 40 micromol/liter). The Hordaland Homocysteine Study.

From 1992-93, we screened 18,043 subjects, aged 40-67 yr, and found 67 cases (0.4%) with total plasma homocysteine (tHcy) > or = 40 micromol/liter. Compared to 329 controls, the cases had lower plasma folate and cobalamin levels, lower intake of vitamin supplements, consumed more coffee, and were more frequently smokers. Homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase gene was observed in 73.1% of the cases and 10.2% of the controls. Only seven cases with cobalamin deficiency and one with homocystinuria received specific therapeutic instructions. 2 yr after the screening, 58 subjects were reinvestigated. 41 still had tHcy > 20 micromol/liter, and in 37 of these, intervention with low dose folic acid (0.2 mg/d) was started. Notably, 34 of 37 (92%) had homozygosity for the C677T mutation. Plasma tHcy was reduced in all but two after 7 wk, and became normal within 7 mo in 21 of 37 subjects. Most of the remaining subjects obtained a normal tHcy level with 5 mg/d of folic acid. We conclude that most subjects with hyperhomocysteinemia > or = 40 micromol/liter in the general population have the C677T mutation combined with low folate status. Daily supplement of low dose folic acid will reduce and often normalize their tHcy level.     

Hyperhomocysteinemia and impaired vasomotor function in type 2 diabetes mellitus

BACKGROUND: Hyperhomocysteinemia has been shown to adversely affect vascular function. The aim of this study was to determine whether hyperhomocysteinemia was independently associated with changes in endothelium-dependent and -independent vasomotor functions in patients with type 2 diabetes mellitus. MATERIALS AND METHODS: Fasting homocysteine (tHcy) was measured in 123 patients with type 2 diabetes and in 61 nondiabetic controls. Endothelium-dependent and -independent vasodilation was measured using high-resolution vascular ultrasound. RESULTS: Plasma tHcy concentration was increased in the diabetic patients (11.1 +/- 3.7 micromol L(-1) vs. 9.8 +/- 2.9, P < 0.05). The prevalence of hyperhomocysteinemia (defined as tHcy > 15 micromol L(-1)) was higher in the diabetic patients (P < 0.05). Within group comparisons showed that both the abnormalities in endothelium-dependent and -independent vasodilation were significantly more severe in diabetic patients with tHcy 10-15 (P < 0.05) and tHcy > 15 micromol L(-1) (P < 0.05) than in those patients with tHcy < 10 micromol L(-1). When compared with nondiabetic controls matched for tHcy levels, impairment of endothelium-dependent and -independent vasodilation were already evident, even in patients with normal tHcy levels (P < 0.01). Despite significant univariate relationships between tHcy and endothelium-dependent (r = -0.24, P < 0.01) and -independent vasodilation (r = -0.33, P < 0.01) in patients with diabetes, only the relationship between tHcy and endothelium-independent vasodilation remained significant after adjusting for other cardiovascular risk factors in multiple regression analysis. CONCLUSIONS: Impairment of endothelium-dependent and -independent vasodilation was already present in diabetic patients with normal tHcy levels, and these abnormalities became more severe with increasing tHcy levels. Only the association between tHcy and endothelium-independent vasodilation was free of other cardiovascular risk factors.