New Insight on the Relationship between LDL Composition,Associated Proteins,Oxidative Resistance and Preparation Procedure

Oxidized low density lipoprotein (LDL) plays an important role in atherogenesis. It is generally thought that LDL is mainly oxidized in the intima of vessel walls, surrounded by hydrophilic antioxidants and proteins such as albumin. The aim of this study was to investigate the possible interrelationships between oxidation resistance of LDL and its protein and lipid moieties. Proteins and to a lesser extent lipids, appeared to be the major determinants in the LDL Cu 2+ -oxidation resistance, which in turn depend on the ultracentrifugation (UC) procedure used. Comparing high speed/short time (HS/ST, 4 &#117 h), high speed/long time (HS/LT, 6-16 &#117 h) and low speed/long time (LS/LT, 24 &#117 h) conditions of UC, HS with the shortest time (4 &#117 h) led to prepare LDL (named LDL·HS-4 &#117 h) with higher total protein and triglyceride contents, unchanged total cholesterol, phospholipids and Vitamin E, and higher Cu 2+ -oxidation resistance. Among proteins, only albumin allows to explain changes. PAF acetyl hydrolase appeared to be unaffected, whereas its pro-oxidant role was established and found only in the absence of albumin. In contrast the pro-oxidant role of caeruloplasmin took place regardless of the albumin content of LDL. The antioxidant effect of albumin (the oxidation lag time was doubled for 20 &#117 mol/mol albumin per LDL) is assumed to be due to its capacity at decreasing LDL affinity for Cu 2+ . Interestingly, the LDL·HS-4 &#117 h albumin content mirrored the intrinsic characteristics of LDL in the plasma and was not affected by added free albumin. Moreover, it has been verified that in 121 healthy subjects albumin was the best resistance predictor of the Cu 2+ -oxidation of LDL·HS-4 &#117 h, with a multiple regression equation: lag time (min)=62.1+0.67(HSA/apoB)+0.02 (TG/apoB) &#109 0.01(TC/apoB); r =0.54, P <0.0001. Accounted for by lag time, the oxidation resistance did not correlate with &#102 -tocopherol and ubiquinol contents of LDL. The mean albumin content was about 10 &#117 mol/mol, and highly variable (0-58 &#117 mol/mol) with subjects. The LDL·HS-4 &#117 h may account for the status of LDL in its natural environment more adequately than LDL resulting from other conditions of UC.     

γ-Tocopherol biokinetics and transformation in humans

Background: The uptake and biotransformation of γ-tocopherol (γ-T) in humans is largely unknown. Using a stable isotope method we investigated these aspects of γ-T biology in healthy volunteers and their response to γ-T supplementation.

Methods: A single bolus of 100 mg of deuterium labeled γ-T acetate (d²-γ-TAC, 94% isotopic purity) was administered with a standard meal to 21 healthy subjects. Blood and urine (first morning void) were collected at baseline and a range of time points between 6 and 240 h post-supplemetation. The concentrations of d² and d⁰-γ-T in plasma and its major metabolite 2,7,8-trimethyl-2-(b-carboxyethyl)-6-hydroxychroman (-γ-CEHC) in plasma and urine were measured by GC-MS. In two subjects, the total urine volume was collected for 72 h post-supplementation. The effects of γ-T supplementation on α-T concentrations in plasma and α-T and γ-T metabolite formation were also assessed by HPLC or GC-MS analysis.

Results: At baseline, mean plasma α-T concentration was approximately 15 times higher than γ-T (28.3 vs. 1.9 µmol/l). In contrast, plasma γ-CEHC concentration (0.191 µmol/l) was 12 fold greater than α-CEHC (0.016 µmol/l) while in urine it was 3.5 fold lower (0.82 and 2.87 µmol, respectively) suggesting that the clearance of α-CEHC from plasma was more than 40 times that of γ-CEHC. After d²-γ-TAC administration, the d² forms of γ-T and γ-CEHC in plasma and urine increased, but with marked inter-individual variability, while the d⁰ species were hardly affected. Mean total concentrations of γ-T and γ-CEHC in plasma and urine peaked, respectively, between 0–9, 6–12 and 9–24 h post-supplementation with increases over baseline levels of 6–14 fold. All these parameters returned to baseline by 72 h. Following challenge, the total urinary excretion of d²-γ-T equivalents was approximately 7 mg. Baseline levels of γ-T correlated positively with the post-supplementation rise of (d⁰ + d²) – γ – T and γ-CEHC levels in plasma, but correlated negatively with urinary levels of (d⁰ + d²)-γ-CEHC. Supplementation with 100 mg γ-TAC had minimal influence on plasma concentrations of α-T and α-T-related metabolite formation and excretion.

Conclusions: Ingestion of 100mg of γ-TAC transiently increases plasma concentrations of γ-T as it undergoes sustained catabolism to CEHC without markedly influencing the pre-existing plasma pool of γ-T nor the concentration and metabolism of α-T. These pathways appear tightly regulated, most probably to keep high steady-state blood ratios α-T to γ-T and γ-CEHC to α-CEHC.     

Vitamin K, 2,3-Epoxide And Quinone Reduction: Mechanism And Inhibition

The chemical and enzymatic pathways of vitamin K₁ epoxide and quinone reduction have been investigated. The reduction of the epoxide by thiols is known to involve a thiol-adduct and a hydroxy vitamin K enolate intermediate which eliminates water to yield the quinone. Sodium borohydride treatment resulted in carbonyl reduction generating relatively stable compounds that did not proceed to quinone in the presence of base. NAD(P)H:quinone oxidoreductase (DT-diaphorase. E.C. I.6.99.2) reduction of vitamin K to the hydroquinone was a significant process in intact microsomes. but 1/5th the rate of the dithiothreitol (DTT)-dependent reduction. No evidence was found for DT-diaphorase catalyzed reduction of vitamin K₁ epoxide, nor was it capable of mediating transfer of electrons from NADH to the microsomal epoxide reducing enzyme. Purified diaphorase reduced detergent- solubilized vitamin K, 10^?5 as rapidly as it reduced dichlorophenylindophenol(DCPIP). Reduction of 10 μM vitamin K, by200 μM NADH was not inhibited by 10μM dicoumarol. whereas DCPIP reduction was fully inhibited. In contrast to vitamin K, (menadione). vitamin K₁ (phylloquinone) did not stimulate microsomal NADPH consumption in the presence or absence of dicoumarol. DTT-dependent vitamin K epoxide reduction and vitamin K reduction were shown to be mutually inhibitory reactions. suggesting that both occur at the same enzymatic site. On this basis, a mechanism for reduction of the quinone by thiols is proposed. Both the DTT-dependent reduction of vitamin K₁ epoxide and quinone. and the reduction of DCPIP by purified DT-diaphorase were inhibited by dicoumarol, warfarin. lapachol. and sulphaquinoxaline     

ReviewPart of the Series: From Dietary Antioxidants to Regulators in Cellular Signalling and Gene ExpressionReview: When is an antioxidant not an antioxidant? A review of novel actions and reactions of vitamin C

Vitamin C (or ascorbic acid) is regarded as the most important water-soluble antioxidant in human plasma and mammalian cells which have mechanisms to recycle and accumulate it against a concentration gradient, suggesting that the vitamin might also have important intracellular functions. In this review we summarize evidence from human trials that have attempted an association between vitamin C supplementation and an effect on biomarkers of oxidative DNA damage. Most studies reviewed herein showed either a vitamin C-mediated reduction in oxidative DNA damage or a null effect, whereas only a few studies showed an increase in specific base lesions. We also address the possible beneficial effects of vitamin C supplementation for the prevention of cancer and cardiovascular disease. Finally, we discuss the contribution of cell culture studies to our understanding of the mode of action of vitamin C and we review recent evidence that vitamin C is able to modulate gene expression and cellular function, with a particular interest in cell differentiation.     

Severe and uncontrolled adult asthma is associated with vitamin D insufficiency and deficiency

Background

Vitamin D has effects on the innate and adaptive immune system. In asthmatic children low vitamin D levels are associated with poor asthma control, reduced lung function, increased medication intake, and exacerbations. Little is known about vitamin D in adult asthma patients or its association with asthma severity and control.

Methods

Clinical parameters of asthma control and 25-hydroxyvitamin D (25(OH)D) serum concentrations were evaluated in 280 adult asthma patients (mean ± SD: 45.0 ± 13.8 yrs., 40% male, FEV1 74.9 ± 23.4%, 55% severe, 51% uncontrolled).

Results

25(OH)D concentrations in adult asthmatics were low (25.6 ±11.8 ng/ml) and vitamin D insufficiency or deficiency (vitamin D <30 ng/ml) was common (67%). 25(OH)D levels were related to asthma severity (intermittent: 31.1 ± 13.0 ng/ml, mild: 27.3 ± 11.9 ng/ml, moderate: 26.5 ± 12.0 ng/ml, severe: 24.0 ± 11.8 ng/ml, p = 0.046) and control (controlled: 29.5 ± 12.5 ng/ml, partly controlled 25.9 ± 10.8 ng/ml, uncontrolled: 24.2 ± 11.8 ng/ml, p = 0.030). The frequency of vitamin D insufficiency or deficiency was significantly higher in patients with severe or uncontrolled asthma and was associated with a lower FEV1 (vitamin D <30 vs. ≥30 ng/ml 2.3 ± 0.9 L vs. 2.7 ± 1.0 L, p = 0.006), higher levels of exhaled NO (45 ± 46 ppb vs. 31 ± 37 ppb, p = 0.023), a higher BMI (28.3 ± 6.2 vs. 25.1 ± 3.9, p < 0.001), and sputum eosinophilia (5.1 ± 11.8% vs. 0.5 ± 1.0%, p = 0.005). The use of oral corticosteroids or sputum eosinophilia was associated with a 20% or 40% higher risk of vitamin D insufficiency or deficiency.

Conclusions

25(OH)D levels below 30 ng/ml are common in adult asthma and most pronounced in patients with severe and/or uncontrolled asthma, supporting the hypothesis that improving suboptimal vitamin D status might be effective in prevention and treatment of asthma.     

Vitamin B12 in neurology and ageing; Clinical and genetic aspects

The classic neurological and psychiatric features associated with vitamin B₁₂ deficiency have been well described and are the subject of many excellent review articles. The advent of sensitive diagnostic tests, including homocysteine and methylmalonic acid assays, has revealed a surprisingly high prevalence of a more subtle ‘subclinical’ form of B₁₂ deficiency, particularly within the elderly. This is often associated with cognitive impairment and dementia, including Alzheimer's disease. Metabolic evidence of B₁₂ deficiency is also reported in association with other neurodegenerative disorders including vascular dementia, Parkinson's disease and multiple sclerosis. These conditions are all associated with chronic neuro-inflammation and oxidative stress. It is possible that these clinical associations reflect compromised vitamin B₁₂ metabolism due to such stress. Physicians are also increasingly aware of considerable inter-individual variation in the clinical response to B₁₂ replacement therapy. Further research is needed to determine to what extent this is attributable to genetic determinants of vitamin B₁₂ absorption, distribution and cellular uptake.     

Metformin increases liver accumulation of vitamin B12 – An experimental study in rats

Aims/hypothesis

Patients treated with metformin exhibit low levels of plasma vitamin B₁₂ (B₁₂), and are considered at risk for developing B₁₂ deficiency. In this study, we investigated the effect of metformin treatment on B₁₂ uptake and distribution in rats.

Methods

Sprague Dawley rats (n = 18) were divided into two groups and given daily subcutaneous injections with metformin or saline (control) for three weeks. Following this, the animals received an oral dose of radio-labeled B₁₂ (⁵⁷[Co]-B₁₂), and urine and feces were collected for 24 h. Plasma, bowel content, liver, and kidneys were collected and analyzed for B₁₂, unsaturated B₁₂-binding capacity, and ⁵⁷[Co]-B₁₂.

Results

Three weeks of metformin treatment reduced plasma B₁₂ by 22% or 289 [47-383] pmol/L (median and [range]) (p = 0.001), while no effect was observed on unsaturated B₁₂-binding capacity. Compared with controls, the amount of B₁₂ in the liver was 36% (p = 0.007) higher in metformin-treated rats, while the B₁₂ content in the kidney was 34% (p = 0.013) lower. No difference in the total amount of absorbed ⁵⁷[Co]-B₁₂ present in the tissues and organs studied was found, suggesting that metformin has no decreasing effect on the B₁₂ absorption.

Conclusions/interpretation

These results show that metformin treatment increases liver accumulation of B₁₂, thereby resulting in decreases in circulating B₁₂ and kidney accumulation of the vitamin. Our data questions whether the low plasma B₁₂ observed in patients treated with metformin reflects impaired B₁₂ status, and rather suggests altered tissue distribution and metabolism of the vitamin.