High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500 mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4 mg/100 g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2 mg/100 g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4 µmol/L versus placebo 60.7±5.3 to 59.2±4.8 µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.     

Metabolic and cellular stress responses of catfish,Horabagrus brachysoma (Günther) acclimated to increasing temperatures

We investigated the metabolic and cellular stress responses in an endemic catfish Horabagrus brachysoma acclimated to ambient (26 °C), 31, 33 and 36 °C for 30 days. After acclimation, fish were sampled to investigate changes in the levels of blood glucose, tissue glycogen and ascorbic acid, activities of enzymes involved in glycolysis (LDH), citric acid cycle (MDH), gluconeogenesis (FBPase and G6Pase), pentose phosphate pathway (G6PDH), protein metabolism (AST and ALT), phosphate metabolism (ACP and ALP) and energy metabolism (ATPase), and HSP70 levels in various tissues. Acclimation to higher temperatures (33 and 36 °C) significantly increased activities of LDH, MDH, ALP, ACP, AST, ALT and ATPase and blood glucose levels, whereas decreased the G6PDH enzyme activity and, tissue glycogen and ascorbic acid. Results indicated an overall increase in the carbohydrate, protein and lipid metabolism implying increased metabolic demands for maintaining homeostasis in fish acclimated to higher temperatures (33 and 36 °C). We observed tissue specific response of HSP70 in H. brachysoma, with significant increase in gill and liver at 33 and 36 °C, and in brain and muscle at 36 °C, enabling cellular protection at higher acclimation temperatures. In conclusion, H. brachysoma adjusted metabolic and cellular responses to withstand increased temperatures, however, these responses suggest that the fish was under stress at 33 °C or higher temperature.     

Mitochondrial ascorbic acid transport is mediated by a low-affinity form of the sodium-coupled ascorbic acid transporter-2

Despite the fundamental importance of the redox metabolism of mitochondria under normal and pathological conditions, our knowledge regarding the transport of vitamin C across mitochondrial membranes remains far from complete. We report here that human HEK-293 cells express a mitochondrial low-affinity ascorbic acid transporter that molecularly corresponds to SVCT2, a member of the sodium-coupled ascorbic acid transporter family 2. The transporter SVCT1 is absent from HEK-293 cells. Confocal colocalization experiments with anti-SVCT2 and anti-organelle protein markers revealed that most of the SVCT2 immunoreactivity was associated with mitochondria, with minor colocalization at the endoplasmic reticulum and very low immunoreactivity at the plasma membrane. Immunoblotting of proteins extracted from highly purified mitochondrial fractions confirmed that SVCT2 protein was associated with mitochondria, and transport analysis revealed a sigmoidal ascorbic acid concentration curve with an apparent ascorbic acid transport K_m of 0.6 mM. Use of SVCT2 siRNA for silencing SVCT2 expression produced a major decrease in mitochondrial SVCT2 immunoreactivity, and immunoblotting revealed decreased SVCT2 protein expression by approximately 75%. Most importantly, the decreased protein expression was accompanied by a concomitant decrease in the mitochondrial ascorbic acid transport rate. Further studies using HEK-293 cells overexpressing SVCT2 at the plasma membrane revealed that the altered kinetic properties of mitochondrial SVCT2 are due to the ionic intracellular microenvironment (low in sodium and high in potassium), with potassium acting as a concentration-dependent inhibitor of SVCT2. We discarded the participation of two glucose transporters previously described as mitochondrial dehydroascorbic acid transporters; GLUT1 is absent from mitochondria and GLUT10 is not expressed in HEK-293 cells. Overall, our data indicate that intracellular SVCT2 is localized in mitochondria, is sensitive to an intracellular microenvironment low in sodium and high in potassium, and functions as a low-affinity ascorbic acid transporter. We propose that the mitochondrial localization of SVCT2 is a property shared across cells, tissues, and species.     

Plasma interleukin-1β concentration is associated with stroke in sickle cell disease

The pathogenesis of sickle cell disease (HbSS), which has numerous complications including stroke, involves inflammation resulting in alteration of plasma inflammatory protein concentration. We investigated HbSS children with abnormal cerebral blood flow detected by trans-cranial Doppler ultrasound (TCD) who participated in the multi-center stroke prevention (STOP) study, to determine if plasma inflammatory protein concentration is associated with the outcome of stroke. Thirty-nine plasma samples from HbSS participants with elevated TCD who had no stroke, HbSS-NS (n = 13) or had stroke, HbSS-S (n = 13), HbSS steady-state controls (n = 7) and controls with normal hemoglobin, HbAA (n = 6), were analyzed simultaneously for 27 circulating inflammatory proteins. Logistic regression and receiver operating characteristics curve analysis of stroke on plasma inflammatory mediator concentration, adjusted for age and gender, demonstrated that interleukin-1β (IL-1β) was protective against stroke development (HbSS-NS = 19, 17–23, HbSS-S = 17, 16–19 pg/mL, median and 25th–75th percentile; odds ratio = 0.59, C.I. = 0.36–0.96) and was a good predictor of stroke (area under curve = 0.852). This result demonstrates a strong association of systemic inflammation with stroke development in HbSS via moderately increased plasma IL-1β concentration, which is furthermore associated with a decreased likelihood of stroke in HbSS.     

Meta-analysis of longitudinal studies: Serum vitamin D and prostate cancer risk

Aim: To review and summarize evidence from longitudinal studies on the association between serum 25-hydroxyvitamin D (25(OH)D) and the risk of prostate cancer (PC). Methods: Relevant prospective cohort studies and nested case-control studies published until July 2009 were identified by systematically searching Ovid Medline, EMBASE, and ISI Web of Knowledge databases and by cross-referencing. The following data were extracted in a standardized manner from eligible studies: first author, publication year, country, study design, characteristics of the study population, duration of follow-up, PC incidence/PC mortality according to serum vitamin D status and the respective risk ratios, and covariates adjusted for in the analysis. Due to the heterogeneity of studies in categorizing serum vitamin D levels, all results were recalculated for an increase in serum 25(OH)D by 10 ng/ml. Summary odds ratios (ORs) were calculated using meta-analysis methods. Results: Overall, eleven original articles were included, ten of which reported on the association between serum vitamin D levels and PC incidence and one article reported on the association with PC mortality. Meta-analysis of studies on PC incidence resulted in a summary OR (95% confidence interval, CI) of 1.03 (0.96–1.11) associated with an increase of 25(OH)D by 10 ng/ml (P = 0.362). No indication for heterogeneity and publication bias was found. Conclusions: According to available evidence from longitudinal studies, serum 25(OH)D is not associated with PC incidence.     

Impairment of the proteasome is crucial for glucose-induced lifespan reduction in the mev-1 mutant of Caenorhabditis elegans

Hyperglycemia is a hallmark of diabetes that is associated with diabetic complications and a reduction of lifespan. Using the mev-1 mutant of the nematode Caenorhabditis elegans we here tried to identify molecular mechanisms underlying the lifespan reducing effects of glucose. The lowest glucose concentration tested (10 mM) caused a significant lifespan reduction at 37 °C and was used to assess effects on mitochondrial efficiency, formation of protein carbonyls and levels of methylglyoxal, a precursor of advanced glycation end products (AGEs). RNA-interference (RNAi) served the identification of targets for glucose-induced damage. Levels of protein carbonyls and AGEs remained unaffected by 10 mM glucose. Levels of reactive oxygen species inside mitochondria were increased but their scavenging by ascorbic acid did not influence lifespan reduction by glucose. Mitochondrial efficiency was reduced by glucose as concluded from a lowered P/O-ratio. A reduced lifespan of mev-1 that was unaffected by the addition of glucose resulted from RNAi of key players of mitochondrial unfolded protein response. Besides increased accumulation of misfolded proteins, reduced proteasomal degradation caused the same phenotype as was evidenced by RNAi for UBQ-1 or UBA-1. Accumulation of functionally impaired proteins, e.g. in mitochondria, underlies the lifespan reducing effects of glucose. Our study provides evidence for a crucial importance of the proteostasis network for lifespan regulation which is impaired by glucose.     

Vitamin D-Binding Protein in Healthy Pre- and Postmenopausal Women: Relationship with Estradiol Concentrations

Objective: To examine the relationship between endogenous serum estradiol and vitamin D–binding protein (DBP) and total, free, and bioavailable 25-hydroxyvitamin D (25OHD) concentrations in pre- and postmenopausal women.Methods: In 165 healthy women (ages, 26 to 75 years) not taking any form of exogenous estrogen, the serum concentrations of estradiol, 25OHD, DBP, parathyroid hormone, and albumin were measured. Free and bioavailable 25OHD (free + albumin-bound) levels were calculated from total 25OHD, DBP, and serum albumin levels.Results: Premenopausal women had higher serum 25OHD (31.5 ± 7.9 ng/mL), DBP (45.3 ± 6.2 mg/dL), and estradiol (52.8 ± 35.0 pg/mL) levels than postmenopausal women (26.5 ± 4.9 ng/mL, 41.7 ± 5.7 mg/dL, and 12.9 ± 4.9 pg/mL), respectively. In addition, the calculated free and bioavailable 25OHD levels were higher in prethan postmenopausal women (P<.05). Serum estradiol correlated with DBP (r = 0.22; P<.01) and total 25OHD (r = 0.27; P<.01). In multivariate regression models (with or without serum 25OHD), estradiol was independently associated with DBP (P<.05).Conclusion: Lower estradiol level is one of the factors that contribute to lower DBP levels in older women. Our data indicate that besides well-known factors such as age, gender, and race, serum estradiol concentrations are also a physiologic predictor of DBP concentration.Abbreviations: 25OHD = 25-hydroxyvitamin D BMI = body mass index CV = coefficient of variation DBP = vitamin D–binding protein PTH = parathyroid hormone SHBG = sex hormone–binding globulin     

Elevated Fructosamine Levelsin Acquired Immunodeficiency Syndrome

ObjectiveTo investigate whether the mechanism of increased glycation in acquired immunodeficiency syndrome (AIDS) is due to an alteration in a circulatory plasma enhancer.MethodsWe assessed glycation of serum protein and hemoglobin in patients with AIDS without altered carbohydrate metabolism. Fasting concentrations of glucose, ethanol, vitamin E, fructosamine, hemoglobin, hemoglobin A1c (A1C), and partial pressure of alveolar oxygen (Pao₂) were determined in 50 men with AIDS and in 25 age-matched healthy men in whom normal glucose tolerance was established by oral glucose tolerance tests.ResultsFasting serum glucose was not significantly different between the men with AIDS (87 ± 4 mg/dL) and the healthy male volunteers (84 ± 6 mg/dL); however, A1C (6.9 ± 0.2%) and serum fructosamine levels (288 ± 15 μmol/L) were significantly higher (P < .01) in the patients with AIDS than in the normal subjects (A1C, 5.6 ± 0.1%; fructosamine, 204 ± 14 μmol/L). Moreover, both A1C and fructosamine concentrations were significantly higher (P < .01) in the patients with AIDS than in the normal subjects divided into subgroups on the basis of fasting plasma glucose concentrations (70 to 79 mg/dL, 80 to 89 mg/dL, and 90 to 99 mg/dL). None of the study participants had anemia (hemoglobin < 12 g/dL) or hypoxia (Pao₂ < 95 mm Hg), and serum ethanol was undetectable. Furthermore, vitamin E concentrations were not significantly different between the patients with AIDS (25 ± 3 mg/L) and the normal subjects (22 ± 4 mg/L).ConclusionOn the basis of this study, glycation of some circulating proteins appears to be enhanced in AIDS and may be induced by an undetermined plasma enhancer, inasmuch as known circulating factors promoting glycation were absent. (Endocr Pract. 2008;14:686-690)     

Chronic vitamin E deficiency promotes vitamin C deficiency in zebrafish leading to degenerative myopathy and impaired swimming behavior

We hypothesized that zebrafish (Danio rerio) undergoing long-term vitamin E deficiency with marginal vitamin C status would develop myopathy resulting in impaired swimming. Zebrafish were fed for 1 y a defined diet without (E ?) and with (E +) vitamin E (500 mg α-tocopherol/kg diet). For the last 150 days, dietary ascorbic acid concentrations were decreased from 3500 to 50 mg/kg diet and the fish sampled periodically to assess ascorbic acid concentrations. The ascorbic acid depletion curves were faster in the E ? compared with E + fish (P < 0.0001); the estimated half-life of depletion in the E ? fish was 34 days, while in it was 55 days in the E + fish. To assess swimming behavior, zebrafish were monitored individually following a “startle-response” stimulus, using computer and video technology. Muscle histopathology was assessed using hematoxylin and eosin staining on paramedian sections of fixed zebrafish. At study end, E ? fish contained 300-fold less α-tocopherol (p < 0.0001), half the ascorbic acid (p = 0.0001) and 3-fold more malondialdehyde (p = 0.0005) than did E + fish. During the first minute following a tap stimulus (p < 0.05), E + fish swam twice as far as did E ? fish. In the E ? fish, the sluggish behavior was associated with a multifocal, polyphasic, degenerative myopathy of the skeletal muscle. The myopathy severity ranged from scattered acute necrosis to widespread fibrosis and was accompanied by increased anti-hydroxynonenal staining. Thus, vitamin E deficiency in zebrafish causes increased oxidative stress and a secondary depletion of ascorbic acid, resulting in severe damage to muscle tissue and impaired muscle function.     

Serum cytokines and muscle strength after anterior cruciate ligament surgery are not modulated by high-doses of vitamins E (α- and γ-tocopherol’s) and C

The purpose of this investigation was to identify if supplemental vitamin E (consisting of α- and γ-tocopherol’s) and C modulate serum cytokine and muscle strength following an ACL injury and surgery. Subjects were randomly assigned to one of two groups: (1) placebo (n = 14) or (2) vitamins E (α-[600 mg RRR-α-tocopherol, αT] and γ-[600 mg of RRR-γT]) and C (1000 mg ascorbic acid, AA) (EC; n = 15). Supplements were taken daily starting ∼2-wk prior to and concluding 16-wk after surgery. Fasting blood samples were obtained and single-leg peak isometric force measurements were performed at baseline (prior to supplementation), before surgery (∼120-min – blood draw only), and 8-wk, 12-wk, and 16-wk after surgery. αT, γT, AA, and cytokines were measured in each blood sample, and peak isometric force was measured on the injured and non-injured legs separately at each testing session. An exercise protocol consisting of repetitive knee and hip extension and flexion contractions to exhaustion was performed on the injured limb at 16-wk. Vitamin E and C supplementation significantly (all p < 0.05) increased plasma αT (∼40%), γT (∼160%), and AA (∼50%) concentrations. Serum cytokine concentrations, peak isometric force, and time to exhaustion during the exercise protocol were not significantly different between groups. Based on these findings, we conclude that vitamin E and C supplementation increases their endogenous levels without minimizing muscular weakness or modulating serum cytokine concentrations after ACL surgery.     

In vivo effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression in striped catfish (Pangasianodon hypophthalmus)

Lipolysaccharide (LPS), a component of outer membrane protein of gram-negative bacteria, reportedly stimulates fish immune system. However, mechanisms driving this immunomodulatory effect are yet unknown. To determine effects of Escherichia coli lipopolysaccharide on regulation of immune response and protein expression of striped catfish (Pangasianodon hypophthalmus), juvenile fish (20–25 g) were injected with 3, 15 or 45 mg E.coli LPS/kg and challenged with Edwardsiella ictaluri. Plasma cortisol and glucose were rather low and did not differ (p < 0.05) among treatments. All LPS treatments differed regarding blood cell count and immune variables such as plasma and spleen lysozyme, complement activity and antibody titer, 3 mg LPS/kg yielding best results; red blood cell count was not affected by LPS treatment. Accumulated mortalities after bacterial challenge were 23.4, 32.8, 37.7 and 52.5% for treatment 3, 15, 45 mg LPS/kg fish and control respectively. Proteomic analysis of peripheral blood mononuclear cells (PBMC) confirmed that LPS induced differentially over-expressed immune proteins such as complement component C3 and lysozyme C2 precursor. Regulation of other proteins such as Wap65, alpha-2 macroglobulin-3 and transferrin precursor was also demonstrated. Striped catfish injected with E.coli LPS enhanced innate immune responses.     

Modulation of inflammation by vitamin E and C supplementation prior to anterior cruciate ligament surgery

Muscle atrophy commonly follows anterior cruciate ligament (ACL) injury and surgery. Proinflammatory cytokines can induce and exacerbate oxidative stress, potentiating muscle atrophy. The purpose of this study was to evaluate the influence of prior antioxidant (AO) supplementation on circulating cytokines following ACL surgery. A randomized, double-blind, placebo-controlled trial was conducted in men undergoing ACL surgery, who were randomly assigned to either: (1) AO (200 IU of vitamin E (50% d-alpha-tocopheryl acetate and 50% d-α-tocopherol) and 500 mg ascorbic acid), or (2) matching placebos (PL). Subjects took supplements twice daily for 2 weeks prior to and up to 12 weeks after surgery. Each subject provided five blood samples: (1) baseline (Bsl, prior to supplementation and ～2 weeks prior to surgery), (2) presurgery (Pre), (3) 90 min, (4) 72 h, and (5) 7 days postsurgery. Following surgery, inflammation and muscle damage increased in both groups, as assessed by increased circulating IL-6, C-reactive protein, and creatine kinase. During AO supplementation, plasma α-T and AA increased while γ-T concentrations decreased significantly (P <  0.05). At 90 min the AO group displayed a significant decrease in AA, an inverse correlation between AA and (interleukin) IL-8 (r² =  0.50, P <  0.05), and a significantly lower IL-10 response than that of the PL group. IL-10 was significantly elevated at 90 min and 72 h in the PL group. In summary, our findings show that circulating inflammatory cytokines increase and AO supplementation attenuated the increase in IL-10 in patients post-ACL surgery.     

Effect of Metformin Therapy on Vitamin D and Vitamin B12 Levels in Patients with Type 2 Diabetes Mellitus

ObjectiveTo determine the effect of metformin on 25-hydroxyvitamin D [25(OH)D] and vitamin B₁₂ levels in patients with type 2 diabetes mellitus.MethodsWe performed a retrospective review of medical records of patients treated between 2003 and 2009 at Loyola University Medical Center, Maywood, Illinois, in both ambulatory primary care and endocrinology clinics. The study cohort consisted of 706 patients with type 2 diabetes mellitus who were 20 to 93 years old (mean age, 63 ± 13) and had a mean body mass index of 33.1 kg/m². Of these patients, 42% were treated with metformin, and 34% had been diagnosed with osteoporosis or osteopenia.ResultsPatients taking metformin had statistically significant lower vitamin B₁₂ levels than those not receiving metformin (P < .0001; 95% confidence interval [CI] = − 220 to − 84 pg/mL). No statistically significant difference was found between users and nonusers of metformin in regard to 25(OH)D levels when adjusted for variables (P = .297; 95% CI for mean difference = − 0.7 to 2.2 ng/mL). Metformin use did not adversely affect successful treatment of vitamin D deficiency in this patient population as a whole, nor did it affect the subgroup with osteoporosis (P = .956). The patients with osteoporosis had statistically significant lower baseline 25(OH)D levels in comparison with those without osteoporosis, when adjustments were made for all variables (P = .003; 95% CI = 0.7 to 3.5 ng/ mL).ConclusionThis study confirms the higher prevalence of vitamin B₁₂ deficiency in metformin-treated patients with type 2 diabetes than in those not treated with metformin. This study also suggests that vitamin D deficiency is not a clinical concern among metformin-treated patients with type 2 diabetes and that metformin does not negatively affect treatment of vitamin D deficiency in these patients. (Endocr Pract. 2012;18:179–184)     

Vitamin D,Parathyroid Hormone,and Heart Failure in A Chinese Elderly Population

ObjectiveHeart failure (HF) is a major cause of morbidity and mortality worldwide. Low vitamin D status has been shown to be associated with increased risk of developing cardiovascular disease. In this study, we examined the association between vitamin D and parathyroid hormone (PTH) levels and HF in and elderly population in China.MethodsA population-based cross-sectional study was conducted in the spring of 2013 among 2,047 community-dwelling healthy individuals, aged 60 to 101 years. 25-Hydroxyvitamin D (25[OH]D) was measured using a chemiluminescence assay. PTH levels were measured with an electrochemiluminescence immunoassay.ResultsA total of 2,047 participants, including 1,121 women (54.7%), were evaluated in 2013. The median concentrations of serum 25(OH)D and PTH for the entire group were 16.1 ng/mL and 41.5 pg/mL, respectively. Serum 25(OH)D and PTH levels were associated with serum N-terminal pro-brain natriuretic peptide levels and left ventricular ejection fraction in a multivariate adjusted linear regression analysis (P < .05). In logistic regression analyses, serum 25(OH)D and PTH levels were associated with a risk of HF in single and multiple regression models (P < .05). Compared with patients with 25(OH)D levels between 30.0 and 44.9 ng/mL, patients with 25(OH)D levels less than 10 ng/mL had a higher mean hazard ratio for HF (2.88; 95% confidence interval, 1.59 to 4.38).ConclusionSerum 25(OH)D and PTH levels are independently associated with risk of HF in a Chinese elderly population. (Endocr Pract. 2014;21:30-40)     

Plasma Ascorbic Acid,A Priori Diet Quality Score,and Incident Hypertension: A Prospective Cohort Study

Vitamin C may reduce risk of hypertension, either in itself or by marking a healthy diet pattern. We assessed whether plasma ascorbic acid and the a priori diet quality score relate to incident hypertension and whether they explain each other’s predictive abilities. Data were from 2884 black and white adults (43% black, mean age 35 years) initially hypertension-free in the Coronary Artery Risk Development in Young Adults Study (study year 10, 1995–1996). Plasma ascorbic acid was assessed at year 10 and the diet quality score at year 7. Eight-hundred-and-forty cases of hypertension were documented between years 10 and 25. After multiple adjustments, each 12-point (1 SD) higher diet quality score at year 7 related to mean 3.7 μmol/L (95% CI 2.9 to 4.6) higher plasma ascorbic acid at year 10. In separate multiple-adjusted Cox regression models, the hazard ratio of hypertension per 19.6-μmol/L (1 SD) higher ascorbic acid was 0.85 (95% CI 0.79–0.92) and per 12-points higher diet score 0.86 (95% CI 0.79–0.94). These hazard ratios changed little with mutual adjustment of ascorbic acid and diet quality score for each other, or when adjusted for anthropometric variables, diabetes, and systolic blood pressure at year 10. Intake of dietary vitamin C and several food groups high in vitamin C content were inversely related to hypertension, whereas supplemental vitamin C was not. In conclusion, plasma ascorbic acid and the a priori diet quality score independently predict hypertension. This suggests that hypertension risk is reduced by improving overall diet quality and/or vitamin C status. The inverse association seen for dietary but not for supplemental vitamin C suggests that vitamin C status is preferably improved by eating foods rich in vitamin C, in addition to not smoking and other dietary habits that prevent ascorbic acid from depletion.     

Duffy antigen receptor genetic variant and the association with Interleukin 8 levels

The aim of this study is to identify loci associated with circulating levels of Interleukin 8 (IL8). We investigated the associations of 121,445 single nucleotide polymorphisms (SNPs) from the Illumina 200 K CardioMetabochip with IL8 levels in 1077 controls from the Stockholm Heart Epidemiology Program (SHEEP) study, using linear regression under an additive model of inheritance.Five SNPs (rs12075A/G, rs13179413C/T, rs6907989T/A, rs9352745A/C, rs1779553T/C) reached the pre-defined threshold of genome-wide significance (p < 1.0 × 10⁻⁵) and were tested for in silico replication in three independent populations, derived from the PIVUS, MDC-CC and SCARF studies. IL8 was measured in serum (SHEEP, PIVUS) and plasma (MDC-CC, SCARF). The strongest association was found with the SNP rs12075 A/G, Asp42Gly (p = 1.6 × 10⁻⁶), mapping to the Duffy antigen receptor for chemokines (DARC) gene on chromosome 1. The minor allele G was associated with 15.6% and 10.4% reduction in serum IL8 per copy of the allele in SHEEP and PIVUS studies respectively. No association was observed between rs12075 and plasma IL8.Conclusion: rs12075 was associated with serum levels but not with plasma levels of IL8. It is likely that serum IL8 represents the combination of levels of circulating plasma IL8 and additional chemokine liberated from the erythrocyte DARC reservoir due to clotting. These findings highlight the importance of understanding IL8 as a biomarker in cardiometabolic diseases.     

Improvement in Pancreatic β-Cell Function with Vitamin D and Calcium Supplementation in Vitamin D-Deficient Nondiabetic Subjects

ObjectiveThere are varied reports on the effect of vitamin D supplementation on β-cell function and plasma glucose levels. The objective of this study was to examine the effect of vitamin D and calcium supplementation on β-cell function and plasma glucose levels in subjects with vitamin D deficiency.MethodsNondiabetic subjects (N = 48) were screened for their serum 25-hydroxyvitamin D (25-OHD), albumin, creatinine, calcium, phosphorus, alkaline phosphatase, and intact parathyroid hormone (PTH) status. Subjects with 25-OHD deficiency underwent a 2-hour oral glucose tolerance test. Cholecalciferol (9,570 international units [IU]/day; tolerable upper intake level, 10,000 IU/day; according to the Endocrine Society guidelines for vitamin D supplementation) and calcium (1 g/day) were supplemented.ResultsThirty-seven patients with 25-OHD deficiency participated in the study. The baseline and postvitamin D/calcium supplementation and the difference (corrected) were: serum calcium, 9 ± 0.33 and 8.33 ± 1.09 mg/dL (− 0.66 ± 1.11 mg/dL); 25-OHD, 8.75 ± 4.75 and 36.83 ± 18.68 ng/mL (28.00 ± 18.33 ng/mL); PTH, 57.9 ± 29.3 and 36.33 ± 22.48 pg/mL (− 20.25 ± 22.45 pg/mL); fasting plasma glucose, 78.23 ± 7.60 and 73.47 ± 9.82 mg/dL (− 4.88 ± 10.65 mg/dL); and homeostasis model assessment-2–percent β-cell function C-peptide secretion (HOMA-2–%B C-PEP), 183.17 ± 88.74 and 194.67 ± 54.71 (11.38 ± 94.27). Significant differences were observed between baseline and post-vitamin D/calcium supplementation serum levels of corrected calcium (Z, − 3.751; P < .0001), 25-OHD (Z, − 4.9; P < .0001), intact PTH (Z, − 4.04; P < .0001), fasting plasma glucose (Z, − 2.7; P < .007), and HOMA-2–%B C-PEP (Z, − 1.923; P < .05) as determined by Wilcoxon signed rank test. Insulin resistance as measured by HOMA was unchanged.ConclusionOptimizing serum 25-OHD concentrations and supplementation with calcium improves fasting plasma glucose levels and β-cell secretory reserve. Larger randomized control studies are needed to determine if correction of 25-OHD deficiency will improve insulin secretion and prevent abnormalities of glucose homeostasis. (Endocr Pract. 2014;20:129-138)     

Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay

The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D₃ and 25-OH vitamin D₂ in human serum. Hexadeuterium labelled 25-OH vitamin D₃ internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's ≤6% for both 25-OH vitamin D₃ and 25-OH vitamin D₂. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D₃ and 82.7 and 100.3% for 25-OH vitamin D₂. Our results for the DEQAS serum pools averaged ?7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r² = 0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r² = 0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D₃ and 25-OH vitamin D₂ in human serum.     

Vitamin D sufficiency associates with an increase in anti-inflammatory cytokines after intense exercise in humans

The purpose of this study was to identify the influence of vitamin D status (insufficient vs. sufficient) on circulating cytokines and skeletal muscle strength after muscular injury. To induce muscular injury, one randomly selected leg (SSC) performed exercise consisting of repetitive eccentric–concentric contractions. The other leg served as the control. An averaged serum 25(OH)D concentration from two blood samples collected before exercise and on separate occasions was used to establish vitamin D insufficiency (<30 ng/mL, n = 6) and sufficiency (>30 ng/mL, n = 7) in young, adult males. Serum cytokine concentrations, single-leg peak isometric force, and single-leg peak power output were measured before and during the days following the exercise protocol. The serum IL-10 and IL-13 responses to muscular injury were significantly (both p < 0.05) increased in the vitamin D sufficient group. The immediate and persistent (days) peak isometric force (p < 0.05) and peak power output (p < 0.05) deficits in the SSC leg after the exercise protocol were not ameliorated with vitamin D sufficiency. We conclude that vitamin D sufficiency increases the anti-inflammatory cytokine response to muscular injury.     

Histopathology and cytotoxicity as biomarkers in treated rats with cadmium and some therapeutic agents

The present study aimed to investigate the protective role of ascorbic acid (vitamin C) and zinc (Zn) against cadmium (Cd) induced histopathological changes in tissues of liver, kidney, lung and testis of rats as well as chromosomal aberrations. For this purpose, 60 male albino rats were divided into six groups; each group contained 10 animals. The first group served as control and was given only distilled water. The second and third groups received distilled water supplemented with 2 g ascorbic acid/l and 500 mg Zn/l, respectively. The fourth group received a daily oral dose containing 3 mg Cd/kg b.w. (1/30 LD₅₀). The fifth group received Cd + ascorbic acid (3 mg Cd/kg b.w. + 2 g ascorbic acid/l), while the sixth group received Cd + Zn (3 mg Cd/kg b.w. +500 mg Zn/l). The treatment in all groups lasted for 90 consecutive days. Rats exposed to cadmium showed severe histopathological changes in the liver, kidney, lung and testicular tissues as well as chromosomal aberrations such as: break, ring, centromeric separation and polyploidy. Co-treatment with zinc partially improved the histopathological changes and chromosomal aberrations while co-treatment with vitamin C exhibited a more protective role and markedly reduced tissues damage induced by Cd.