Intracellular Vitamin Research Articles

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.

The vitamin D receptor gene ApaI polymorphism is associated with increased risk of renal cell carcinoma in Chinese population.

Molecular epidemiologic studies previously reported that 1,25-dihydroxy vitamin D3 (1,25(OH)2 D3) appears to influence cancer risk. It exerts its activity through the intracellular vitamin D receptor (VDR), which regulates the transcription of genes. This study aimed to investigate the genetic association of VDR polymorphisms with renal cell carcinoma (RCC) risk in the Chinese population. The genotypes of five VDR polymorphisms (TaqI, BsmI, Cdx-2, ApaI, and FokI) were studied using polymerase chain reaction in 302 RCC patients and 302 healthy controls. ApaI variant AA and AC genotypes were found to be associated with a significantly increased risk of RCC compared with the CC genotype (OR = 2.60, 95% CI = 1.39–4.85 for AA vs. CC, and OR = 1.52, 95% CI = 1.08–2.13 for AC vs. CC). The AA genotype was also associated with a higher Fuhrman grade (OR = 2.87, 95% CI = 1.15–7.16 for AA vs. CC). No significant difference was found between the other four VDR polymorphisms and RCC risk. Our study suggests that VDR ApaI genotypes may be involved in the increased risk and progression of RCC in the Chinese Han population.

MP04-14 PHASE IIA, RANDOMIZED PLACEBO-CONTROLLED TRIAL OF SINGLE HIGH DOSE CHOLECALCIFEROL (VITAMIN D3) AND DAILY GENISTEIN (G-2535) VERSUS PLACEBO IN MEN WITH EARLY STAGE PROSTATE CANCER UNDERGOING PROSTATECTOMY

You have accessJournal of UrologyProstate Cancer: Epidemiology & Natural History I1 Apr 2016MP04-14 PHASE IIA, RANDOMIZED PLACEBO-CONTROLLED TRIAL OF SINGLE HIGH DOSE CHOLECALCIFEROL (VITAMIN D3) AND DAILY GENISTEIN (G-2535) VERSUS PLACEBO IN MEN WITH EARLY STAGE PROSTATE CANCER UNDERGOING PROSTATECTOMY David F. Jarrard, Badrinath R. Konety, Joel Slaton, Wei Huang, Tracy Downs, Jill Kolesar, KyungMann Kim, Tom Havighurst, Margaret G. House, Howard L. Parnes, and Howard H. Bailey David F. JarrardDavid F. Jarrard More articles by this author , Badrinath R. KonetyBadrinath R. Konety More articles by this author , Joel SlatonJoel Slaton More articles by this author , Wei HuangWei Huang More articles by this author , Tracy DownsTracy Downs More articles by this author , Jill KolesarJill Kolesar More articles by this author , KyungMann KimKyungMann Kim More articles by this author , Tom HavighurstTom Havighurst More articles by this author , Margaret G. HouseMargaret G. House More articles by this author , Howard L. ParnesHoward L. Parnes More articles by this author , and Howard H. BaileyHoward H. Bailey More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1949AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) represents an important target for chemoprevention given its prolonged natural history and predilection for afflicting an older population. Epidemiologic and laboratory data suggest that vitamin D and genistein (soy) in decreasing PCa progression. The effect of vitamin D on prostate epithelial cell proliferation and differentiation is well documented and genistein may augment this affect through inhibition of the CYP24 enzyme, which is responsible for intracellular vitamin D metabolism. In addition, both genistein and vitamin D inhibit the intraprostatic synthesis of prostaglandin E2, an important mediator of inflammation. In the current prospective, multicenter trial in patients undergoing radical prostatectomy for localized prostate cancer, the safety and effect of adding genistein to patients receiving a single, large dose of cholecalciferol was assessed. METHODS Men undergoing radical prostatectomy were randomly assigned to one of two treatment groups: (1) cholecalciferol: 200,000 IU as one dose at study entry plus genistein: 600 mg daily or (2) placebo cholecalciferol day 1 and placebo genistein PO daily for 21-28 days prior to radical prostatectomy. Serum and tissue analyses were performed and side-effects recorded. RESULTS A total of 15 patients were enrolled, 8 in the placebo arm and 7 in the vitamin D + genistein (VD+G) arm. All patients were compliant and completed the study. No significant differences in side effect profiles were noted. Utilization of the VD+G showed a trend toward increased calcitriol serum concentrations when compared to placebo (0.104 +/- 0.1 vs. 0.0013 +/-0.07; p=0.08); however, prostate tissue levels did not increase. Immunohistochemistry for marker analyses using VECTRA automated quantitation revealed a significant increase in AR expression (p=0.04) and a trend toward increased TUNEL staining (p=0.1) in prostate cancer tissues in men randomized to receive VD+G compared to placebo. CONCLUSIONS In this first study testing the combination of a single, large dose of cholecalciferol (200,000IU) and daily genistein (G-2535) the agents were well tolerated. While serum levels of calcitriol were increased on the intervention arm, prostate tissue levels were not. Increased prostatic AR expression (suggesting increased differentiation) and a trend toward increased TUNEL staining (increased apoptosis) were observed among men randomized to the combination. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e33-e34 Advertisement Copyright & Permissions© 2016MetricsAuthor Information David F. Jarrard More articles by this author Badrinath R. Konety More articles by this author Joel Slaton More articles by this author Wei Huang More articles by this author Tracy Downs More articles by this author Jill Kolesar More articles by this author KyungMann Kim More articles by this author Tom Havighurst More articles by this author Margaret G. House More articles by this author Howard L. Parnes More articles by this author Howard H. Bailey More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Impact of vitamin D receptor polymorphisms in centenarians.

Vitamin D is a seco-sterol produced endogenously in the skin or obtained from certain foods. It exerts its action through binding to intracellular vitamin D receptor (VDR). Lately, the role of vitamin D has been revised regarding its potential advantage on delaying the process of aging. The aim of this study was to assess the contribution of VDR gene polymorphisms in healthy aging and longevity. We evaluated the frequency of four polymorphisms of the VDR gene (FokI, BsmI, ApaI, and TaqI) in centenarians (102 subjects, mean age: 102.3±0.3years), compared to septuagenarians (163 subjects, mean age: 73.0±0.6years) and we analyzed a variety of pathophysiologically relevant functions in centenarians. BsmI and ApaI provided a significant association with longevity: there was a highly significant difference in the frequency of BsmI genotypes (p=0.037), ApaI genotypes (p=0.022), and ApaI alleles (p=0.050) in centenarians versus septuagenarians. Furthermore, we found a significant correlation of all the VDR gene polymorphisms in centenarians with some measured variables such as hand grip strength, body mass index, blood pressure, HDL cholesterol, and mini-mental state examination. We also found a correlation with the prevalence of medical history of hypertension, acute myocardial infarction, angina, venous insufficiency, dementia, chronic obstructive pulmonary disease, and arthrosis. In conclusion, this study proposes a new scenario in which the variability of the VDR gene is relevant in the aging process and emphasizes the role of VDR genetic background in determining healthy aging.

Ophthalmic Manifestations and Long-Term Visual Outcomes in Patients with Cobalamin C Deficiency

To explore the ocular manifestations of cobalamin C (cblC) deficiency, an inborn error of intracellular vitamin B12 metabolism. Retrospective, observational case series. Twenty-five cblC patients underwent clinical and ophthalmic examination at the National Institutes of Health between August 2004 and September 2012. Patient ages ranged from 2 to 27 years at last ophthalmic visit, and follow-up ranged from 0 to 83 months (median, 37 months; range, 13-83 months) over a total of 69 visits. Best-corrected visual acuity, slit-lamp biomicroscopy, dilated fundus examination, wide-field photography, fundus autofluorescence imaging, sedated electroretinography, optical coherence tomography, genetics and metabolite assessment. Visual acuity and presence and degree of retinal degeneration and optic nerve pallor. Nystagmus (64%), strabismus (52%), macular degeneration (72%), optic nerve pallor (68%), and vascular changes (64%) were present. c.271dupA (p.R91KfsX14) homozygous patients (n = 14) showed early and extensive macular degeneration. Electroretinography showed that scotopic and photopic responses were reduced and delayed, but were preserved remarkably in some patients despite severe degeneration. Optical coherence tomography images through the central macular lesion of a patient with severe retinal degeneration showed extreme thinning, some preservation of retinal lamination, and nearly complete loss of the outer nuclear layer. Despite hyperhomocysteinemia, no patients exhibited lens dislocation. This longitudinal study reports ocular outcomes in the largest group of patients with cblC deficiency systematically examined at a single center over an extended period. Differences in progression and severity of macular degeneration, optic nerve pallor, and vascular attenuation between homozygous c.271dupA (p.R91KfsX14) patients and compound heterozygotes were noted. The pace and chronicity of ophthalmic manifestations lacked strict correlation to metabolic status as measured during visits. Prenatal or early treatment, or both, may have mitigated ocular disease, leading to better functional acuity, but patients still progressed to severe macular degeneration. The effects of prenatal or early treatment, or both, in siblings; the manifestation of severe disease in infancy; the presence of comorbid developmental abnormalities; and the possible laminar structural defect noted in many patients are findings showing that cblC deficiency displays a developmental as well as a degenerative ocular phenotype.

Vitamin A Transport and Cell Signaling by the Retinol-Binding Protein Receptor STRA6.

Vitamin A, retinol, circulates in blood bound to retinol binding protein (RBP). In some tissues, the retinol-RBP complex (holo-RBP) is recognized by a membrane receptor, termed STRA6, which mediates uptake of retinol into cells. Recent studies have revealed that, in addition to serving as a retinol transporter, STRA6 is a ligand-activated cell surface signaling receptor that, upon binding of holo-RBP activates JAK/STAT signaling, culminating in the induction of STAT target genes. It has further been shown that retinol transport and cell signaling by STRA6 are critically interdependent and that both are coupled to intracellular vitamin A metabolism. The molecular mechanism of action of STRA6 and its associated machinery is beginning to be revealed, but further work is needed to identify and characterize the complete range of genes and associated signaling cascades that are regulated by STRA6 in different tissues. An understanding of STRA6 is clinically relevant, as for example, it has been shown to be hyper- activated in obese animals, leading to insulin resistance. A potential role for STRA6 in other pathologies, including cancer, awaits further investigation.

Improving Bariatric Patient Aftercare Outcome by Improved Detection of a Functional Vitamin B12 Deficiency.

Vitamin B12 deficiency is common after bariatric surgery. Vitamin B12 is a poor predictor of functional vitamin B12 status, since deficiencies might even occur within the reference limits. Therefore, vitamin B12 deficiencies with serum vitamin B12 levels are between 140 and 200pmol/L remain undetected. Methylmalonic acid (MMA), however, will detect these deficiencies as accumulates due to functional intracellular vitamin B12 deficiencies. MMA is a relative expensive analysis and is therefore not generally available. To lower the costs, we only request MMA when vitamin B12 levels are between these levels. As a result, more biochemical deficiencies are found. However, it was not known whether bariatric patients with vitamin B12 levels between 140 and 200pmol/L would benefit from supplementation. Bariatric patients with vitamin B12 levels between 140 and 200pmol/L with (n = 45) and without (n = 45) intramuscular hydroxocobalamin injections were compared. Treated patients showed a significant increase of vitamin B12 levels (P < 0.001) and a significant decrease in MMA levels (P < 0.001). Biochemical improvement occurs in both patients with and without clinical symptoms. The control group showed a significant increase of MMA levels (P < 0.001). To examine whether biochemical benefits of vitamin B12 supplementation are correlated with clinical improvement, patient records were checked for complaints. Complaints were disappeared after treatment, while no improvement was seen in untreated patients. This study shows that all bariatric patients with vitamin B12 levels between 140 and 200pmol/L benefit clinical and biochemical from vitamin B12 supplementation, regardless the MMA levels.

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Prenatal diagnosis using genetic sequencing and identification of a novel mutation in MMACHC.

BackgroundCombined methylmalonic aciduria and homocystinuria, cobalamin(cbl)C deficiency, is a rare disorder of intracellular vitamin B12(cbl) metabolism caused by mutations in the MMACHC gene. Both genetic and biochemical approach have been established to diagnose children and fetuses with cblC deficiency, while in China there is no report of prenatal genetic diagnosis of cblC deficiency. The aim of the present study was to characterize the mutational spectrum of cblC deficiency and investigate the feasibility of genetic-sequencing-based prenatal diagnosis for cblC deficiency.Methods10 pedigrees were recruited in this study with the probands clinically and biochemically confirmed combined methymalonic aciduria and homocystinuria. Peripheral blood samples were collected for MMACHC genetic test from the probands and their parents (4 probands had already dead) and 50 control subjects. The entire coding region and adjacent splice sites of MMACHC were sequenced. After the genotypes of the pedigrees were identified, chorionic villi sampling were performed for 3 high-risk pregnant women for prenatal genetic diagnosis.ResultsA total of 7 mutations were identified: c.217C > T (R73X), c.394C > T (R132X), c.463G > C (G155R), c.609G > A (W203X), c.616C > T (R206W), c.658-660delAAG (220delK), and c.567dupT (I190YfsX13), as well as 2 polymophsims: c.321G > A(V107V), c.-302G > T. And G155R is a novel mutation that haven’t been reported in the literatures. All the 6 probands identified with compound heterozygous mutations or homozygous mutations of MMACHC gene, and all the parents of the probands were found to have one MMACHC mutation at a heterozygous level. Prenatal diagnosis of fetuses from 3 families with a child affected cblC deficiency showed that one fetus had the same compound heterozygous mutations as the proband, one did not have MMACHC mutation, and the third fetus had a mutation at a heterozygous level of MMACHC gene. Results from the follow-ups were consistent with the prenatal diagnosis.ConclusionA novel mutation p.G155R of the MMACHC gene is identified. Genetic diagonsis is an accurate and convenient method for prenatal diagnosis and early intervention of combined methylmalonic aciduria and homocystinuria.

Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations.

We used multiple sets of simulations both at the atomistic and coarse-grained level of resolution to investigate interaction and binding of α-tochoperol transfer protein (α-TTP) to phosphatidylinositol phosphate lipids (PIPs). Our calculations indicate that enrichment of membranes with such lipids facilitate membrane anchoring. Atomistic models suggest that PIP can be incorporated into the binding cavity of α-TTP and therefore confirm that such protein can work as lipid exchanger between the endosome and the plasma membrane. Comparison of the atomistic models of the α-TTP-PIPs complex with membrane-bound α-TTP revealed different roles for the various basic residues composing the basic patch that is key for the protein/ligand interaction. Such residues are of critical importance as several point mutations at their position lead to severe forms of ataxia with vitamin E deficiency (AVED) phenotypes. Specifically, R221 is main residue responsible for the stabilization of the complex. R68 and R192 exchange strong interactions in the protein or in the membrane complex only, suggesting that the two residues alternate contact formation, thus facilitating lipid flipping from the membrane into the protein cavity during the lipid exchange process. Finally, R59 shows weaker interactions with PIPs anyway with a clear preference for specific phosphorylation positions, hinting a role in early membrane selectivity for the protein. Altogether, our simulations reveal significant aspects at the atomistic scale of interactions of α-TTP with the plasma membrane and with PIP, providing clarifications on the mechanism of intracellular vitamin E trafficking and helping establishing the role of key residue for the functionality of α-TTP.

Methionine synthase reductase deficiency (CblE): A report of two patients and a novel mutation

Importance: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment.Clinical presentation: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them.Intervention: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677–1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency.Conclusion: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme.

Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Vitamin C Deficiency in the Brain Impairs Cognition, Increases Amyloid Accumulation and Deposition, and Oxidative Stress in APP/PSEN1 and Normally Aging Mice

Subclinical vitamin C deficiency is widespread in many populations, but its role in both Alzheimer's disease and normal aging is understudied. In the present study, we decreased brain vitamin C in the APPSWE/PSEN1deltaE9 mouse model of Alzheimer's disease by crossing APP/PSEN1(+) bigenic mice with SVCT2(+/-) heterozygous knockout mice, which have lower numbers of the sodium-dependent vitamin C transporter required for neuronal vitamin C transport. SVCT2(+/-) mice performed less well on the rotarod task at both 5 and 12 months of age compared to littermates. SVCT2(+/-) and APP/PSEN1(+) mice and the combination genotype SVCT2(+/-)APP/PSEN1(+) were also impaired on multiple tests of cognitive ability (olfactory memory task, Y-maze alternation, conditioned fear, Morris water maze). In younger mice, both low vitamin C (SVCT2(+/-)) and APP/PSEN1 mutations increased brain cortex oxidative stress (malondialdehyde, protein carbonyls, F2-isoprostanes) and decreased total glutathione compared to wild-type controls. SVCT2(+/-) mice also had increased amounts of both soluble and insoluble Aβ1-42 and a higher Aβ1-42/1-40 ratio. By 14 months of age, oxidative stress levels were similar among groups, but there were more amyloid-β plaque deposits in both hippocampus and cortex of SVCT2(+/-)APP/PSEN1(+) mice compared to APP/PSEN1(+) mice with normal brain vitamin C. These data suggest that even moderate intracellular vitamin C deficiency plays an important role in accelerating amyloid pathogenesis, particularly during early stages of disease development, and that these effects are likely modulated by oxidative stress pathways.

Ascorbic acid transport in brain microvascular pericytes

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 μM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.

The authors' reply.

We thank Dr. Kayadibi and colleagues (1) for their comments regarding our paper on hyperhomocysteinemia. (2) As it is known, vitamin B9, also called folate or folic acid, is one of the eighth forms of vitamin B. (3, 4) So, in the current study by Monfared et al. (2), folic acid is obviously considered as one of the forms of vitamin B, so the authors did not separately mention folic acid, and it is considered as an exclusion criteria. In addition, if despite adequate vitamin supplementation, adequately lowering plasma homocysteine has failed, the patients should be evaluated for vitamin deficiency, renal impairment, and thyroid dysfunction. It should be remembered that a (low) “normal” vitamin B12 level does not exclude intracellular vitamin B12 deficiency. Serum methylmalonic acid (MMA) and holotranscobalamin II levels are more reliable markers of vitamin B12 deficiency compared to the serum vitamin B12 concentration (5). Although Solomon demonstrated that high serum folate levels (>20 ng/mL) are associated with higher MMA, at the same time, homocysteine levels and cobalamin levels (201–300 pg/mL) are low normal. It should be mentioned that its effect is age dependent (≥60 years). In the case of having a normal range of serum folate, the effects would not progress (suggesting a threshold effect), but it would be reversed by cobalamin therapy (6). In our study, hyperhomocysteinemic patients had a mean folate level of 16.26±4.06 ng/mL (normal levels) with mid-normal cobalamin levels (405.56±169.28 pg/mL). It seems that, as Kayadibi et al. (1) proposed, evaluation of the functional deficiency of vitamin B12 by means of MMA, as an early functional marker of tissue vitamin B12 deficiency, could provide more accurate results. Further studies should consider this point. It is recommended by Kayadibi and colleagues (1) that multiple linear regression analysis should be performed with sex adjustment to eliminate tHcy differences that may be because of sex differences, so homogeneous distribution of sex would be achieved. As it is shown, in the univariate analysis, a significant association was found between tHcy and age (P=0.02), male sex (P=0001), serum creatinine (P=0.0001), blood urea nitrogen (P=0.0001), estimated creatinine clearance (P=0.001), the history of glomerulonephritis before transplantation (P=0.05), body mass index (P=0.02), serum uric acid (P=0.003), serum folate (P=0.0001), cyclosporine A dose (P=0.01), and CsA trough level (C0; P=0.02) (2). Then, in the multivariate analysis, multiple linear regression models were used by stepwise (entry=0.05, removal=0.1) method for determining associated factors of Hcy levels after adjusting all the demographic (including sex) and clinical variables (2). In response to tHcy analysis, fasting blood sample was collected into an ethylenediaminetetraacetic acid tube which was used for measuring plasma homocysteine of each patient. To separate the plasma, the blood sample was centrifuged immediately after collecting. Then the separated plasma was deeply frozen (−20°C). By this method, we tried to minimize the production and release of tHcy by red blood cells after obtaining the blood sample. (2) Congruent to the conclusion of Kayadibi and colleagues, we considered that measuring MMA, in addition to tHCY, folate, and vitamin B12, could reveal more accurate results. Ali Monfared 1 Seyyede Zeinab Azimi1 Ehsan Kazemnezhad1 Masoud Khosravi1 Mohammadkazem Lebadi1 Ebrahim Mirzajani2 Mohammad Najafi Ashtiani2 1 Urology Research Center School of Medicine Guilan University of Medical Sciences Rasht, Iran 2 School of Medicine Guilan University of Medical Sciences Rasht, Iran

The beneficial impact of vitamin D treatment in CKD patients: what's next?

Patients with chronic kidney disease (CKD) have markedly higher rates of severe vitamin D deficiency and reduced ability to convert 25-(OH)vitamin D into the active form, 1,25-dihydroxyvitamin D [1]. In the setting of CKD, secondary hyperparathyroidism develops as a consequence of reduced renal production of active vitamin D and phosphate retention resulting in hypocalcaemia and hyperphosphataemia. This is a process that is dangerously linked with metabolic bone disease, arterial calcifications and cardiovascular mortality [2]. Therefore, the conventional rationale for vitamin D treatment in CKD is to slow the progression of secondary hyperparathyroidism. In addition to the classical pathway for activation of 25-(OH)vitamin D to 1,25-(OH)2 vitamin D, a peripheral autocrine pathway exists and results in calcitriol synthesis in a variety of peripheral extra-renal tissues [3]. By binding with its intracellular vitamin D receptor (VDR) in these tissues, calcitriol can regulate cellular proliferation and differentiation, inflammation, the immune system and the endocrine system, including insulin resistance, lipid metabolism and renin–angiotensin system (RAS) [4]. Interestingly, active vitamin D analogues have shown demonstrably favourable effects on proteinuria, likely through interference with RAS [5, 6]. The discovery of this non-classical pathway has brought new significance to the importance of addressing nutritional vitamin D deficiency [7]. Vitamin D deficiency has been associated with all-cause and cardiovascular mortality in patients with CKD, whereas therapies with vitamin D and analogues have been associated with reduced mortality, recently also in meta-analysis of observational studies (Table 1). However, evidence from randomized controlled trials (RCTs) supporting a survival benefit from active and/or pre-active vitamin D administration in CKD patients is still lacking. Moreover, it is not even known whether different types of active vitamin D, selective or non-selective VDR activators, or precursors have a diversified effect on mortality in the CKD population. Table 1. Systematic literature reviews on vitamin D in patients with CKD In the present issue of the Clinical Kidney Journal, Mann et al. [8] present a meta-analysis of RCTs to investigate the effect of oral vitamin D therapy versus placebo on mortality and cardiovascular outcomes among adults with CKD, whereas vitamin D supplementation was not found to exert any significant effect on these hard outcomes. Analysis of pooled data displayed a substantial overlap in confidence intervals and homogeneity between study results. Stratification of trials by CKD stage, weekly vitamin D dose, proportion of diabetic subjects and vitamin D compound displayed similar results. In detail, 13 trials that, overall, enrolled 1469 patients with CKD stage 1–5D were selected for analysis and none of them had mortality as a primary outcome. These studies were mainly designed to test biochemical or bone histological end points and consequently had a rather short follow-up. On the whole 41 all-cause deaths (2.8%) were recorded during a follow-up ranging from 3 to 104 weeks (mean 41 weeks). Of note, about two-thirds of the patients (n= 1087) had been followed for <1 year (mean 21, range 3–48 weeks), registering 17 all-cause deaths (41%), 8 cardiovascular deaths (62%) and 18 cardiovascular events (86%). Only two trials (total patient number = 233) had a follow-up time up to 2 years, but they registered only 11 deaths of which, 5 had a cardiovascular cause. Taken together, these observations could indicate that the duration of follow-up may have been insufficient to capture possible differences in mortality, as correctly stated from the authors in the limitation section and as well as suggested by the relatively low number of events displayed. Moreover, not negligible differences are also present in patient populations (End Stage Renal Disease in 5 of 13 trials) and in interventions, above all considering the heterogeneity in administered vitamin D compounds and dosages. In conclusion, it is not the time to say that interventions based on vitamin D may reduce mortality in patients with CKD, but the opposite cannot be said yet beyond all reasonable doubt. In fact, given the paucity of good quality data, the reliability of the pooled results is still uncertain.

Vitamin D receptor and cathelicidin expressions in children with atopic dermatitis

Background Atopic dermatitis (AD) is a common chronic inflammatory type of eczema. Vitamin D may enhance the innate immune response by induction of cathelicidin (LL-37), an endogenous antimicrobial peptide. Several recent reports suggest a connection between vitamin D3 and the expression of antimicrobial peptides in keratinocytes. 1,25-dihydroxyvitamin D (1,25(OH) 2 D 3 ) is the active form of vitamin D and exerts its actions through a specific intracellular vitamin D receptor (VDR). Aim To evaluate the expression of VDR and cathelicidin in children with AD in Egypt. Methods The study was carried out on 15 patients with AD and 15 healthy controls. Patients were divided into mild, moderate, and severe groups according to the SCORAD score. All individuals were subjected to skin biopsy. VDR and cathelicidin expressions were estimated using real-time PCR in patients and controls. Results A highly significant difference was detected between patients and controls in VDR and cathelicidin expression ( P =0.001). A statistically significant correlation was observed between VDR and cathelicidin expressions. Conclusions Influencing cathelicidin expression by targeting the vitamin D3 pathway might present a novel therapeutic approach for the treatment of AD.

Laboratory Evaluation for Vitamin B12 Deficiency: The Case for Cascade Testing

Potential vitamin B(12) deficiency is a common clinical diagnostic problem, and many providers have a low threshold for initiating therapy. The goal of this study was to systematically evaluate current practice patterns regarding the laboratory evaluation of suspected vitamin B(12) deficiency. This retrospective study reviewed the electronic medical records of 192 patients initiated on intramuscular vitamin B(12) injections. Only 12 patients had objectively documented hematologic responses: decrease of mean corpuscular volume by ≥5 fL with stable or improved hemoglobin. Another 5 patients had equivocal hematologic responses. There was one plausible neurologic response. Thus, only 18 (9.4%) of 192 patients had data supportive of a clinical response. In these 18 patients, the baseline serum B(12) level was ≤107 pg/mL; only 3 patients also had a baseline serum methylmalonic acid level, which was ≥1.29 μmol/L in all 3 patients. Currently, only a small minority of patients initiated on intramuscular vitamin B(12) supplementation derive any meaningful clinical benefit. Furthermore, current testing recommendations for vitamin B(12) deficiency are usually not followed. Up-front ordering of a diagnostic testing cascade is recommended to improve compliance; an example is presented with decision points chosen to improve specificity for clinically evident vitamin B(12) deficiency without loss of sensitivity. Ultimately, a better understanding of vitamin B(12) physiology is needed to develop and evaluate laboratory tests that more accurately reflect true intracellular vitamin B(12) status.

Subcellular location of MMACHC and MMADHC, two human proteins central to intracellular vitamin B12 metabolism

MMACHC and MMADHC are the genes responsible for cblC and cblD defects of vitamin B12 metabolism, respectively. Patients with cblC and cblD defects present with various combinations of methylmalonic aciduria (MMA) and homocystinuria (HC). Those with cblC mutations have both MMA and HC whereas cblD patients can present with one of three distinct biochemical phenotypes: isolated MMA, isolated HC, or combined MMA and HC. Based on the subcellular localization of these enzymatic pathways it is thought that MMACHC functions in the cytoplasm while MMADHC functions downstream of MMACHC in both the cytoplasm and the mitochondrion. In this study we determined the subcellular location of MMACHC and MMADHC by immunofluorescence and subcellular fractionation. We show that MMACHC is cytoplasmic while MMADHC is both mitochondrial and cytoplasmic, consistent with the proposal that MMADHC acts as a branch point for vitamin B12 delivery to the cytoplasm and mitochondria. The factors that determine the distribution of MMADHC between the cytoplasm and mitochondria remain unknown. Functional complementation experiments showed that retroviral expression of the GFP tagged constructs rescued all biochemical defects in cblC and cblD fibroblasts except propionate incorporation in cblD-MMA cells, suggesting that the endogenous mutant protein interferes with the function of the transduced wild type construct.

Serum vitamin B12 not reflecting vitamin B12 status in patients with type 2 diabetes

Contradictory results for concentrations of vitamin B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA) have been reported. We tested the hypothesis that the extracellular vitamin B12 markers are not reflecting the intracellular vitamin B12-dependent biochemical reactions in individuals with type 2 diabetes. The study included 92 patients with diabetes and 72 controls with similar age and sex distribution. We measured vitamin B12 markers [MMA, total serum vitamin B12, holoTC, total homocysteine (tHcy)], red blood cell (RBC)-B12, and the plasma concentrations of the methylation markers [S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)]. In comparison to controls, diabetic patients showed significantly higher concentrations of plasma SAH (median 15.1 vs. 11.8nmol/L; p<0.001) and lower SAM/SAH ratio (9.1 vs. 8.2; p=0.006). Concentrations of total vitamin B12 andholoTC did not differ significantly between the groups, but plasma MMA concentrations were significantly higher in diabetics (250 vs. 206nmol/L). However, RBC-B12 was lower in diabetics compared tocontrols (median 230 vs. 260pmol/L; p=0.001). The inverse correlation between MMA and RBC-B12 was stronger in the controls compared to that in the patients (correlation coefficient in controls R=-0.446, p=0.001; in patients R=-0.289, p=0.022). Metformin treatment was associated with a lower total serum vitamin B12, but a comparable RBC-B12 and a slightly lower MMA and better methylation index. In conclusion, patients with type 2 diabetes showed normal extracellular vitamin B12, but disturbed intracellular B12-dependent biochemical reactions. Metformin treatment was associated with low serum vitamin B12 and improved intracellular vitamin B12 metabolism despite low serum vitamin B12.