Clinical Chemistry and Laboratory Medicine (CCLM)

Published by De Gruyter

Volume 51 Issue 3 - Special issue: Advances and controversies in B vitamins and choline / Editors: Rima Obeid, Wolfgang Herrmann

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Contents
Journal Overview

Masthead

Publicly Available March 1, 2013

Masthead

Page range: i-iv

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Editorial

Publicly Available March 1, 2013

Special issue on advances and controversies in B vitamins and choline

Wolfgang Herrmann, Rima Obeid

Page range: 455-456

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Reviews

Publicly Available August 24, 2012

B-Vitamin dependent methionine metabolism and alcoholic liver disease

Charles H. Halsted

Page range: 457-465

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Abstract

Convincing evidence links aberrant B-vitamin dependent hepatic methionine metabolism to the pathogenesis of alcoholic liver disease (ALD). This review focuses on the essential roles of folate and vitamins B6 and B12 in hepatic methionine metabolism, the causes of their deficiencies among chronic alcoholic persons, and how their deficiencies together with chronic alcohol exposure impact on aberrant methionine metabolism in the pathogenesis of ALD. Folate is the dietary transmethylation donor for the production of S-adenosylmethionine (SAM), which is the substrate for all methyltransferases that regulate gene expressions in pathways of liver injury, as well as a regulator of the transsulfuration pathway that is essential for production of glutathione (GSH), the principal antioxidant for defense against oxidative liver injury. Vitamin B12 regulates transmethylation reactions for SAM production and vitamin B6 regulates transsulfuration reactions for GSH production. Folate deficiency accelerates the experimental development of ALD in ethanol-fed animals while reducing liver SAM levels with resultant abnormal gene expression and decreased production of antioxidant GSH. Through its effects on folate metabolism, reduced SAM also impairs nucleotide balance with resultant increased DNA strand breaks, oxidation, hepatocellular apoptosis, and risk of carcinogenesis. The review encompasses referenced studies on mechanisms for perturbations of methionine metabolism in ALD, evidence for altered gene expressions and their epigenetic regulation in the pathogenesis of ALD, and clinical studies on potential prevention and treatment of ALD by correction of methionine metabolism with SAM.

Publicly Available October 16, 2012

Metabolic crosstalk between choline/1-carbon metabolism and energy homeostasis

Steven H. Zeisel

Page range: 467-475

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Abstract

There are multiple identified mechanisms involved in energy metabolism, insulin resistance and adiposity, but there are here-to-fore unsuspected metabolic factors that also influence these processes. Studies in animal models suggest important links between choline/1-carbon metabolism and energy homeostasis. Rodents fed choline deficient diets become hypermetabolic. Mice with deletions in one of several different genes of choline metabolism have phenotypes that include increased metabolic rate, decreased body fat/lean mass ratio, increased insulin sensitivity, decreased ATP production by mitochondria, or decreased weight gain on a high fat diet. In addition, farmers have recognized that the addition of a metabolite of choline (betaine) to cattle and swine feed reduces body fat/lean mass ratio. Choline dietary intake in humans varies over a >three-fold range, and genetic variation exists that modifies individual requirements for this nutrient. Although there are some epidemiologic studies in humans suggesting a link between choline/1-carbon metabolism and energy metabolism, there have been no controlled studies in humans that were specifically designed to examine this relationship.

Publicly Available December 12, 2012

Proteomics of vitamin B₁₂ processing

Luciana Hannibal, Patricia M. DiBello, Donald W. Jacobsen

Page range: 477-488

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Abstract

The causes of cobalamin (B 12 , Cbl) deficiency are multifactorial. Whether nutritional due to poor dietary intake, or functional due to impairments in absorption or intracellular processing and trafficking events, the major symptoms of Cbl deficiency include megaloblastic anemia, neurological deterioration and in extreme cases, failure to thrive and death. The common biomarkers of Cbl deficiency (hyperhomocysteinemia and methylmalonic acidemia) are extremely valuable diagnostic indicators of the condition, but little is known about the changes that occur at the protein level. A mechanistic explanation bridging the physiological changes associated with functional B 12 deficiency with its intracellular processers and carriers is lacking. In this article, we will cover the effects of B 12 deficiency in a cblC -disrupted background (also referred to as MMACHC) as a model of functional Cbl deficiency. As will be shown, major protein changes involve the cytoskeleton, the neurological system as well as signaling and detoxification pathways. Supplementation of cultured MMACHC-mutant cells with hydroxocobalamin (HOCbl) failed to restore these variants to the normal phenotype, suggesting that a defective Cbl processing pathway produces irreversible changes at the protein level.

Publicly Available December 8, 2012

Unexpected high plasma cobalamin/Proposal for a diagnostic strategy

Johan F.B. Arendt, Ebba Nexo

Page range: 489-496

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Abstract

It is well-established that more than 8% of patients examined for vitamin B12 deficiency unexpectedly have increased plasma levels of the vitamin, but so far there are no guidelines for the clinical interpretation of such findings. In this review, we summarise known associations between high plasma cobalamin and diseases. We report associations mainly with cancer, liver and kidney diseases, but also with a number of other diagnostic entities. The pathogenic background is poorly understood and is likely to be multi-factorial, involving increased concentrations of one or both of the circulating cobalamin binding proteins, transcobalamin and haptocorrin. Based on current knowledge, we suggest a strategy for the clinical interpretation of unexpected high plasma cobalamin. Since a number of the associated diseases are critical and life-threatening, the strategy promotes the concept of ‘think the worst first’. It is important to realise that high cobalamin levels can be an unspecific marker for cancer. If this can be ruled out, diseases of the liver and kidney should be considered.

Publicly Available December 20, 2012

Clinical recognition and aspects of the cerebral folate deficiency syndromes

Vincent Ramaekers, Jeffrey M. Sequeira, Edward V. Quadros

Page range: 497-511

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Abstract

We characterized cerebral folate deficiency (CFD) as any neuro-psychiatric condition associated with low spinal fluid (CSF) N 5 -methyltetrahydrofolate (MTHF) but normal folate status outside the central nervous system (CNS). The commonest cause underlying CFD syndromes is the presence of serum autoantibodies of the blocking type directed against folate receptor-α (FRα) attached to the plasma-side of choroid plexus epithelial cells. Blocking FR antibodies inhibit MTHF transport across the choroid plexus. Serum titers of FR antibodies may fluctuate significantly over time. Less frequent causes of CFD are FOLR-1 mutations, mitochondrial disorders and inborn errors affecting folate metabolism. Maternal FR antibodies have been associated with neural tube defects while the presence of FR antibodies in either one or both parents increases the risk of an offspring with infantile autism. Recognizable CFD syndromes attributed to FR-antibodies in childhood are infantile-onset CFD presenting 4–6 months after birth, infantile autism with neurological deficits, and a spastic ataxic syndrome from the age of 1 year, while progressive dystonic or schizophrenic syndromes develop during adolescence. FR autoantibodies are frequently found in autism spectrum disorders, in an Aicardi-Goutières variant and in Rett syndrome. The heterogeneous phenotype of CFD syndromes might be determined by different ages of onset and periods when FR autoantibodies are generated with consequent CNS folate deficiency. Folate deficiency during various critical stages of fetal and infantile development affects structural and functional refinement of the brain. Awareness of CFD syndromes should lead to early detection, diagnosis and improved prognosis of these potentially treatable group of autoimmune and genetically determined conditions.

Publicly Available January 9, 2013

Choline-containing phospholipids: relevance to brain functional pathways

Seyed Khosrow Tayebati, Francesco Amenta

Page range: 513-521

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Abstract

Choline participates in several relevant neurochemical processes. It is the precursor and metabolite of acetylcholine (ACh), plays a role in single-carbon metabolism and is an essential component of different membrane phospholipids (PLs). PLs are structural components of cell membranes involved in intraneuronal signal transduction. This paper reviews the roles of choline and of choline-containing phospholipids (CCPLs) on brain metabolism in health and disease followed by an analysis of the effects of exogenously administered CCPLs on the brain, a topic extensively investigated by literature. Based on the observation of decreased cholinergic neurotransmission in brain disorders characterized by cognitive impairment, cholinergic precursor loading therapy with CCPLs was the first approach used to attempt for relieving the cognitive symptoms of Alzheimer’s disease. This therapeutic strategy was discontinued due to the negative clinical results obtained with choline or lecithin. Negative results obtained with some compounds cannot be generalized for all CCPLs, as CDP-choline (citicoline) and to a greater extent choline alphoscerate (GPC) displayed interesting effects documented in preclinical studies and limited clinical trials. We provide evidence in favor of CDP-choline and GPC activity in cerebrovascular or neurodegenerative disorders characterized by cholinergic neurotransmission impairment. Based on the results of the controlled clinical trials available, we suggest that due to the lack of novel therapeutic strategies, safe compounds developed a long time ago such as effective CCPLs could have still a place in pharmacotherapy. Therefore selected compounds of this class should be further investigated by new appropriate clinical studies.

Publicly Available November 21, 2012

The ‘golden age’ of DNA methylation in neurodegenerative diseases

Andrea Fuso

Page range: 523-534

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Abstract

DNA methylation reactions are regulated, in the first instance, by enzymes and the intermediates that constitute the ‘so called’ one-carbon metabolism. This is a complex biochemical pathway, also known as the homocysteine cycle, regulated by the presence of B vitamins (folate, B6, B12) and choline, among other metabolites. One of the intermediates of this metabolism is S-adenosylmethionine, which represent the methyl donor in all the DNA methyltransferase reactions in eukaryotes. The one-carbon metabolism therefore produces the substrate necessary for the transferring of a methyl group on the cytosine residues of DNA; S-adenosylmethionine also regulates the activity of the enzymes that catalyze this reaction, namely the DNA methyltransferases (DNMTs). Alterations of this metabolic cycle can therefore be responsible for aberrant DNA methylation processes possibly leading to several human diseases. As a matter of fact, increasing evidences indicate that a number of human diseases with multifactorial origin may have an epigenetic basis. This is also due to the great technical advances in the field of epigenetic research. Among the human diseases associated with epigenetic factors, aging-related and neurodegenerative diseases are probably the object of most intense research. This review will present the main evidences linking several human diseases to DNA methylation, with particular focus on neurodegenerative diseases, together with a short description of the state-of-the-art of methylation assays.

Publicly Available January 3, 2013

Mechanisms of the beneficial effects of vitamin B6 and pyridoxal 5-phosphate on cardiac performance in ischemic heart disease

Naranjan S. Dhalla, Satoshi Takeda, Vijayan Elimban

Page range: 535-543

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Abstract

Although vitamin B6 and its metabolite, pyridoxal 5′-phosphate (PLP), have been shown to exert beneficial effects in ischemic heart disease, the mechanisms of their action are not fully understood. Some studies have shown that ventricular arrhythmias and mortality upon the occlusion of coronary artery were attenuated by pretreatment of animals with PLP. Furthermore, ischemia-reperfusion-induced abnormalities in cardiac performance and defects in sarcoplasmic reticular Ca 2+ -transport activities were decreased by PLP. The increase in cardiac contractile activity of isolated heart by ATP was reduced by PLP, unlike propranolol, whereas that by isoproterenol was not depressed by PLP. ATP-induced increase in [Ca 2+ ] i , unlike KCl-induced increase in [Ca 2+ ] i in cardiomyocytes was depressed by PLP. Both high- and low-affinity sites for ATP binding in sarcolemmal membranes were also decreased by PLP. These observations support the view that PLP may produce cardioprotective effects in ischemic heart disease by attenuating the occurrence of intracellular Ca 2+ overload due to the blockade of purinergic receptors.

Publicly Available December 25, 2012

The diagnostic utility of folate receptor autoantibodies in blood

Jeffrey M. Sequeira, Vincent Th. Ramaekers, Edward V. Quadros

Page range: 545-554

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Abstract

Folate supplementation reduces the risk of neural tube defect (NTD) pregnancy, and folinic acid has been used to correct cerebral folate deficiency (CFD) in children with developmental disorders. In the absence of systemic folate deficiency, the discovery of autoantibodies (AuAbs) to folate receptor α (FRα) that block the uptake of folate offers one mechanism to explain the response to folate in these disorders. The association of FRα AuAbs with pregnancy-related complications, CFD syndrome, and autism spectrum disorders and response to folate therapy is highly suggestive of the involvement of these AuAbs in the disruption of brain development and function via folate pathways. The two types of antibodies identified in the serum of patients are blocking antibody and binding antibody. The two antibodies can be measured by the specific assays described and exert their pathological effects either by functional blocking of folate transport as previously shown or hypothetically by disrupting the FR by an antigen-antibody-mediated inflammatory response. We have identified both IgG and IgM AuAbs in these conditions. The predominant antibodies in women with NTD pregnancy belong to the IgG1 and IgG2 isotype and in CFD children, the IgG1 and IgG4 isotype. This review describes the methods used to measure these AuAbs, their binding characteristics, affinity, cross-reactivity, and potential mechanisms by which folate therapy could work. Because these AuAbs are associated with various pathologies during fetal and neonatal development, early detection and intervention could prevent or reverse the consequences of exposure to these AuAbs.

Publicly Available March 1, 2013

Red cell or serum folate: what to do in clinical practice?

Christopher-John L. Farrell, Susanne H. Kirsch, Markus Herrmann

Page range: 555-569

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Abstract

Folate deficiency has been linked to diverse clinical manifestations and despite the importance of accurate assessment of folate status, the best test for routine use is uncertain. Both serum and red cell folate assays are widely available in clinical laboratories; however, red cell folate is the more time-consuming and costly test. This review sought to evaluate whether the red cell assay demonstrated superior performance characteristics to justify these disadvantages. Red cell folate, but not serum folate, measurements demonstrated analytical variation due to sample pre-treatment parameters, oxygen saturation of haemoglobin and haematocrit. Neither marker was clearly superior in characterising deficiency but serum folate more frequently showed the higher correlation with homocysteine, a sensitive marker of deficiency. Similarly, both serum and red cell folate were shown to increase in response to folic acid supplementation. However, serum folate generally gave the greater response and was able to distinguish different supplementation doses. The C677T polymorphism of methylenetetrahydrofolate reductase alters the distribution of folate forms in red cells and may thereby cause further analytical variability in routine red cell folate assays. Overall, serum folate is cheaper and faster to perform than red cell folate, is influenced by fewer analytical variables and provides an assessment of folate status that may be superior to red cell folate.

Publicly Available December 14, 2012

Formate: an essential metabolite, a biomarker, or more?

Simon G. Lamarre, Gregory Morrow, Luke Macmillan, Margaret E. Brosnan, John T. Brosnan

Page range: 571-578

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Abstract

Plasma and urinary formate concentrations were recently found to be elevated during vitamin B12 and folate deficiencies. It was proposed that formate may be a valuable biomarker of impaired one-carbon metabolism. Formate is an essential intermediary metabolite in folate-mediated one-carbon metabolism and, despite its importance, our knowledge of its metabolism is limited. Formate can be produced from several substrates (e.g., methanol, branched chain fatty acids, amino acids), some reactions being folate-dependent while others are not. Formate removal proceeds via two pathways; the major one being folate-dependent. Formate is a potentially toxic molecule and we suggest that formate may play a role in some of the pathologies associated with defective one-carbon metabolism.

Publicly Available March 1, 2013

The role of homocysteine in bone remodeling

Thomas P. Vacek, Anuradha Kalani, Michael J. Voor, Suresh C. Tyagi, Neetu Tyagi

Page range: 579-590

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Abstract

Bone remodeling is a very complex process. Homocysteine (Hcy) is known to modulate this process via several known mechanisms such as increase in osteoclast activity, decrease in osteoblast activity and direct action of Hcy on bone matrix. Evidence from previous studies further support a detrimental effect on bone via decrease in bone blood flow and an increase in matrix metalloproteinases (MMPs) that degrade extracellular bone matrix. Hcy binds directly to extracellular matrix and reduces bone strength. There are several bone markers that can be used as parameters to determine how high levels of plasma Hcy (hyperhomocysteinemia, HHcy) affect bone such as: hydroxyproline, N-terminal collagen 1 telopeptides. Mitochondrion serves an important role in generating reactive oxygen species (ROS). Mitochondrial abnormalities have been identified during HHcy. The mechanism of Hcy-induced bone remodeling via the mitochondrial pathway is largely unknown. Therefore, we propose a mitochondrial mechanism by which Hcy can contribute to alter bone properties. This may occur both through generations of ROS that activate MMPs and could be extruded into matrix to degrade bone matrix. However, there are contrasting reports on whether Hcy affects bone density, with some reports in favour and others not. Earlier studies also found an alteration in bone biomechanical properties with deficiencies of vitamin B 12 , folate and HHcy conditions. Moreover, existing data opens speculation that folate and vitamin therapy act not only via Hcy-dependent pathways but also via Hcy-independent pathways. However, more studies are needed to clarify the mechanistic role of Hcy during bone diseases.

Mini Reviews

January 3, 2013

Neuroprotective actions of perinatal choline nutrition

Jan Krzysztof Blusztajn, Tiffany J. Mellott

Page range: 591-599

Abstract

Choline is an essential nutrient for humans. Studies in rats and mice have shown that high choline intake during gestation or the perinatal period improves cognitive function in adulthood, prevents memory decline of old age, and protects the brain from damage and cognitive and neurological deterioration associated with epilepsy and hereditary conditions such as Down’s and Rett syndromes. These behavioral changes are accompanied by modified patterns of expression of hundreds of cortical and hippocampal genes including those encoding proteins central for learning and memory processing. The effects of choline correlate with cerebral cortical changes in DNA and histone methylation, thus suggesting an epigenomic mechanism of action of perinatal choline.

November 21, 2012

Normal prions as a new target of cobalamin (vitamin B₁₂) in rat central nervous system

Giuseppe Scalabrino, Daniela Veber

Page range: 601-606

Abstract

The pathogenesis of cobalamin (Cbl)-deficient (Cbl-D) neuropathy and the role of normal prions (PrP c s) in myelin maintenance are both subjects of debate. We have demonstrated that Cbl deficiency damages myelin by increasing tumor necrosis factor (TNF)-α, and decreasing epidermal growth factor (EGF) levels in the rat central nervous system (CNS). It is known that TNF-α and EGF regulate PrP c expression in vitro, and that myelin vacuolation, reactive astrocytosis and microglial activation are common to rat Cbl-D neuropathy and some prion diseases. We have shown that Cbl deficiency leads to high levels of PrP c s [particularly the octapeptide repeat (OR) domains] in the rat CNS thereby damaging the spinal cord (SC) myelin, and that chronic intra-cerebroventricular treatment with anti-OR antibodies normalizes SC myelin morphology. We have also found that PrP c levels are increased in the SC of Cbl-D rats by the time the myelin lesions appear, and that this increase is mediated by excess myelinotoxic TNF-α and prevented by EGF treatment, which has proved to be as effective as Cbl in preventing Cbl deficiency-induced lesions. Cbl stimulates PrP c mRNA-related synthesis in Cbl-D SC and duodenum, two rat tissues that are severely affected by Cbl deficiency. New PrP c synthesis is a common effect of various myelinotrophic agents, two of which (EGF and anti-TNF-α antibodies) also stimulate PrP c mRNA-related synthesis in the SC of Cbl-D rats.

December 12, 2012

Molecular mechanisms underlying the potentially adverse effects of folate

Kyle C. Strickland, Natalia I. Krupenko, Sergey A. Krupenko

Page range: 607-616

Abstract

The importance of proper consumption of dietary folate for human health has been highlighted by an extensive number of publications over several decades. Fortification of grain products with folic acid was initiated with the specific intent to prevent neural tube defects, and the scope of this endeavor is unique in that its target population (women of the periconceptional period) is many times smaller than the population it affects (everyone who ingests fortified grain products). Folate fortification has been wildly successful in terms of its goal; since its inception, the incidence of neural tube defects has markedly decreased. In the wake of this public health triumph, it is important to catalog both the serendipitous benefits and potential side effects of folic acid supplementation. The vitamin is generally regarded as a harmless nutrient based on studies evaluating the safe upper limits of folate intake. In recent years, however, a concern has been raised with respect to a potential downside to folate supplementation; namely, its proposed ability to enhance proliferation of malignant tumors. The current review summarizes the available literature on the effects of folate supplementation and the molecular mechanisms by which high doses of folate may have negative consequences on human health, especially with regard to cancer.

March 1, 2013

Betaine homocysteine methyltransferase (BHMT)-dependent remethylation pathway in human healthy and tumoral liver

Hélène Pellanda

Page range: 617-621

Abstract

Carcinogenesis is a multi-step and multifactorial process. It includes genetic, epigenetic, nutritional and environmental factors, which are closely interconnected. Human hepatocellular carcinoma (HCC) is among the most frequent and lethal cancers. Imbalance in the S-adenosylmethionine (SAM) concentration, the main methyl group donor, strongly influences the development of HCC. Key enzymes of carbon metabolism are greatly reduced in patients with cirrhosis and HCC. These alterations play a role in genetic instability and epigenetic modifications (DNA methylation, and histone modifications), however, the molecular underlying mechanisms are still poorly understood. We aimed to investigate betaine homocysteine methyltransferase (BHMT) expression in HepG2 cells and human hepatocarcinoma tissues. Tumor and surrounding healthy tissue were compared. HepG2 cells and tumor samples showed a strong decrease in BHMT transcripts resulting from the transcription of a splicing variant that contained a frameshift mutation generating a premature termination codon and gene loss of function. This splicing variant, not detected in normal adult and fetal liver, cannot be explained by any mechanism involving the known splicing consensus sequences. BHMT activity was abolished in HepG2 cells and protein expression was detected neither in HepG2 cells nor in five of the six tumor samples investigated. Further investigation is needed to elucidate whether this abnormal BHMT transcription is part of cause or consequence of liver carcinogenesis.

November 30, 2012

Hydrogen sulfide as an oxygen sensor

Kenneth R. Olson

Page range: 623-632

Abstract

The ability to monitor oxygen (O 2 ) availability and delivery is crucial to an animal’s survival. Vertebrates have a number of O 2 ‘sensing’ cells that monitor environmental oxygen and ensure adequate delivery to the tissues. While there is little doubt that these cells perform important homeostatic functions, there is little consensus on how a change in O 2 concentration, or partial pressure (pO 2 ), is transduced into a physiological response. We recently proposed that the metabolism of hydrogen sulfide (H 2 S) functions as the O 2 sensor in a variety of tissues. In this mechanism, the concentration of biologically active H 2 S is regulated by the simple balance between constitutive H 2 S production and its oxidation by mitochondria. This hypothesis is supported by a number of experimental observations in a wide range of O 2 sensing tissues: 1) exogenous H 2 S produces the same physiological response as hypoxia; 2) cellular H 2 S production is inversely related to pO 2 at physiologically relevant pO 2 s; 3) agonists and antagonists of H 2 S biosynthesis augment and inhibit hypoxic responses, respectively; and 4) H 2 S and hypoxia appear to act via common effector pathways. The reciprocal relationship between H 2 S and O 2 also has a long evolutionary history suggesting these gases have been inexorably intertwined throughout evolution. The intent of this review is to elaborate on the mechanism of H 2 S-mediated O 2 sensing.

Opinion Paper

March 1, 2013

B vitamin therapy for homocysteine: renal function and vitamin B12 determine cardiovascular outcomes

John David Spence

Page range: 633-637

Abstract

Therapy to lower homocysteine with B vitamins does reduce the risk of stroke, if not myocardial infarction. The apparent lack of efficacy of vitamin therapy in most of the large clinical trials was probably determined by the failure to take account of the metabolic deficiency of vitamin B12, which is very common and often missed, and by the failure to take account of impaired renal function. Metabolic B12 deficiency is present in 20% of people over 65 years of age, and in 30% of vascular patients above 70 years, so higher doses of B12 are needed in elderly patients. However, high-dose cyanocobalamin leads to accumulation of cyanide in patients with renal failure. B vitamin therapy is beneficial in patients with good renal function, but harmful in patients with significantly impaired renal function (a glomerular filtration rate <50). It seems likely that in patients with renal impairment, methylcobalamin should be used instead cyanocobalamin.

Research Articles

November 21, 2012

One year B and D vitamins supplementation improves metabolic bone markers

Wolfgang Herrmann, Susanne H. Kirsch, Vera Kruse, Rudolf Eckert, Stefan Gräber, Jürgen Geisel, Rima Obeid

Page range: 639-647

Abstract

Background: Vitamin D and vitamin B deficiency are common in elderly subjects and are important risk factors for osteoporosis and age-related diseases. Supplementation with these vitamins is a promising preventative strategy. The objective of this study was to evaluate the effects of vitamins D3 and B supplementation on bone turnover and metabolism in elderly people. Methods: Healthy subjects (n=93; >54 years) were randomly assigned to receive either daily vitamin D3 (1200 IU), folic acid (0.5 mg), vitamin B12 (0.5 mg), vitamin B6 (50 mg), and calcium carbonate (456 mg) (group A) or only vitamin D3 plus calcium carbonate (group B) in a double blind trial. We measured at baseline and after 6 and 12 months of supplementation vitamins, metabolites, and bone turnover markers. Results: At baseline mean plasma 25-hydroxy vitamin D [25(OH)D] was low (40 or 30 nmol/L) and parathormone was high (63.7 or 77.9 pg/mL). 25(OH)D and parathormone correlated inversely. S-Adenosyl homocysteine and S-adenosyl methionine correlated with bone alkaline phosphatase, sclerostin, and parathormone. One year vitamin D3 or D3 and B supplementation increased plasma 25(OH)D by median 87.6% (group A) and 133.3% (group B). Parathormone was lowered by median 28.3% (A) and 41.2% (B), bone alkaline phosphatase decreased by 2.8% (A) and 16.2% (B), osteocalin by 37.5% (A) and 49.4% (B), and tartrate-resistant-acid-phosphatase 5b by 6.1% (A) and 36.0% (B). Median total homocysteine (tHcy) was high at baseline (group A: 12.6, group B: 12.3 µmol/L) and decreased by B vitamins (group A) to 8.9 µmol/L (29.4%). tHcy lowering had no additional effect on bone turnover. Conclusions: One year vitamin D3 supplementation with or without B vitamins decreased the bone turnover significantly. Vitamin D3 lowered parathormone. The additional application of B vitamins did not further improve bone turnover. The marked tHcy lowering by B vitamins may modulate the osteoporotic risk.

January 11, 2013

Effect of 1 year B and D vitamin supplementation on LINE-1 repetitive element methylation in older subjects

Ulrich Hübner, Jürgen Geisel, Susanne H. Kirsch, Vera Kruse, Marion Bodis, Cosima Klein, Wolfgang Herrmann, Rima Obeid

Page range: 649-655

Abstract

Background: Disturbed DNA methylation is causally related to chronic diseases like cancer and atherosclerosis. B vitamins are cofactors required for methyl group synthesis and may therefore affect DNA methylation. Vitamin D has epigenetic effects. We tested if B and D vitamin supplementation has an effect on genomic long interspersed nuclear element-1 (LINE-1) methylation and the metabolites S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). Methods: Fifty subjects (median age 68.0 years) were supplemented with a daily oral dose of B vitamins (500 µg folic acid, 500 µg vitamin B12 and 50 mg vitamin B6), 1200 IU vitamin D and 456 mg calcium. Fasting blood samples were collected before and after 1 year of supplementation. LINE-1 methylation was determined in genomic DNA from blood cells as a surrogate for whole genome methylation. In addition, SAM, SAH and total homocysteine (tHcy) were measured in plasma samples. Results: Plasma homocysteine decreased significantly after supplementation (12.8 vs. 9.1 µmol/L; p<0.05), whereas SAM, SAH, the SAM/SAH ratio and LINE-1 methylation did not change significantly. LINE-1 methylation was not significantly correlated with SAH, homocysteine or B vitamins. Conclusions: Long-term vitamin B supplementation had no effect on LINE-1 methylation in blood cells nor on plasma levels of SAM and SAH. Vitamin B and D supplementation seems to have no effect on DNA methylation, especially in cases where no severe deficiency exists.

November 12, 2012

Aqueous humor glycation marker and plasma homocysteine in macular degeneration

Rima Obeid, Kouris Ninios, Ursula Loew, Zisis Gatzioufas, Stephan Hoffmann, Berthold Seitz, Jurgen Geisel, Wolfgang Herrmann

Page range: 657-663

Abstract

Background: We investigated concentrations of total homocysteine (tHcy) in elderly people without and those with age-related macular degeneration (AMD). In addition, we tested the association between plasma tHcy and one glycation marker in aqueous humor. Methods: People with cataract only (n=48), patients with dry AMD (n=38) and those with wet AMD (n=31) were studied. Blood concentrations of tHcy, and methylation and vitamin markers were measured in 116 blood samples. The concentrations of the extracellular soluble receptor for advanced glycated end products (esRAGE) were measured in 77 aqueous humor samples. Results: Mean aqueous humor concentration of esRAGE and that of plasma tHcy did not differ significantly between the groups. Arterial hypertension but not eye disease explained the tHcy elevation in plasma in this study. In the cataract group, a significant negative correlation was found between plasma tHcy and that of esRAGE in aqueous humor (r=–0.483, p=0.006). In patients with dry AMD, the concentration of esRAGE in aqueous humor correlated negatively to tHcy and positively to serum folate. Conclusions: Plasma tHcy levels were positively associated with hypertension, but not with AMD in this study. Higher esRAGE in aqueous humor was related to higher folate and lower tHcy in blood. Following studies may assess whether B-vitamins can protect against age-related ocular diseases by reducing glycation.

February 1, 2013

Homocysteine plasma levels in patients treated with antiepileptic drugs depend on folate and vitamin B12 serum levels, but not on genetic variants of homocysteine metabolism

Alexander Semmler, Susanna Moskau-Hartmann, Birgit Stoffel-Wagner, Christian Elger, Michael Linnebank

Page range: 665-669

Abstract

Background: Antiepileptic drugs (AEDs) are commonly used in the treatment of epilepsy, psychiatric diseases and pain disorders. Several of these drugs influence blood levels of folate and vitamin B12 and, consequently, homocysteine. This may be relevant for AED effects and side effects. However, not only folate and vitamin B12, but also genetic variants modify homocysteine metabolism. Here, we aimed to determine whether there is a pharmacogenetic interaction between folate, vitamin B12 and genetic variants and homocysteine plasma level in AED-treated patients. Methods: In this mono-center study, we measured homocysteine, folate and vitamin B12 plasma levels in a population of 498 AED-treated adult patients with epilepsy. In addition, we analyzed the genotypes of seven common genetic variants of homocysteine metabolism: methylenetetrahydrofolate reductase ( MTHFR ) c.677C>T and c.1298A>C, methionine synthase ( MTR ) c.2756A>G, dihydrofolate reductase ( DHFR ) c.594+59del19bp, cystathionine β-synthase ( CBS ) c.844_855ins68, transcobalamin 2 ( TC2 ) c.776C>G and methionine synthase reductase ( MTRR ) c.66G>A. Results: On multivariate logistic regression, folate and vitamin B12 levels, but none of the genetic variants, were predictive for homocysteine levels. Conclusions: These data suggest that, in AED-treated patients, folate and vitamin B12 play important roles in the development of hyperhomocysteinemia, whereas genetic variants of homocysteine metabolism do not and thus do not contribute to the risk of developing hyperhomocysteinemia during AED treatment.

November 8, 2012

Trends in clinical laboratory homocysteine testing from 1997 to 2010: the impact of evidence on clinical practice at a single institution

Corinne M. Klykov, Steven R. Lentz

Page range: 671-675

Abstract

Background: During the past decade, several clinical trials investigating the potential benefits of homocysteine-lowering therapy for the secondary prevention of vascular events were completed and published. The objective of the study was to determine trends in clinical laboratory testing for homocysteine at a single institution over the time period from 1997 to 2010. Methods: A single-center, retrospective analysis was performed at a large, academic, tertiary care medical center in the USA. The database was searched for clinical laboratory measurements of plasma or serum homocysteine between January 1, 1997 and December 31, 2010. Individual medical records were reviewed for a subset of 221 unique patients in the 10-year period from 2001 to 2010 who had homocysteine values ≥20 μmol/L. Results: From 1997 to 2003, there was a 16-fold increase in the annual number of homocysteine assays performed. Testing for homocysteine declined in 2004 and 2006, coinciding with the publication of intervention trials that failed to support a clinical benefit of homocysteine-lowering therapy for the secondary prevention of vascular events. Subgroup analysis suggested that homocysteine testing for the indication of suspected nutritional deficiency or hypercoagulability remained steady despite a decline in testing for the indication of cardiac risk assessment. Conclusions: The frequency of clinical laboratory testing for plasma or serum homocysteine changed bimodally over the time period from 1997 to 2010. These observations suggest that clinical practice was impacted by emerging evidence from association studies and intervention trials investigating homocysteine as a potentially treatable cardiac risk factor.

November 23, 2012

Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320)

Elke Hoffmann-Lücke, Johan F.B. Arendt, Peter H. Nissen, Gustav Mikkelsen, Jan O. Aasly, Ebba Nexo

Page range: 677-682

Abstract

Background: Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin. Methods: We included a patient referred for cobalamin measurement due to neurological symptoms, her son and her daughter. Mother and son both suffered from myotonic dystrophy type II, while the daughter tested negative for this disease. Blood samples were analyzed for cobalamin, haptocorrin, transcobalamin, holoTC, and sCD320. We employed gel filtration and antibody precipitation for further characterization. The protein coding region of the TCN2 gene, encoding transcobalamin, was sequenced. Results: The patient, her {son} and [daughter] all had cobalamin levels above the measurement range of the routine method employed (>1476 pmol/L). Total transcobalamin and (holoTC) were 5980 (1500), {5260 (2410)} and [5630 (1340)] pmol/L, which is well above the upper reference limits of 1500 (160) pmol/L. The sCD320 concentration was also well above the upper reference limit of 97 arb.u.: 1340, {1510} and [1090] arb.u. Haptocorrin levels were within the reference range and no signs of cobalamin deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-transcobalamin antibodies, we precipitated both transcobalamin and part of sCD320. Conclusions: The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation.

September 12, 2012

Plasma choline and betaine correlate with serum folate, plasma S-adenosyl-methionine and S-adenosyl-homocysteine in healthy volunteers

Apolline Imbard, Yvo M. Smulders, Rob Barto, Desiree E.C. Smith, Robert M. Kok, Cornelis Jakobs, Henk J. Blom

Page range: 683-692

Abstract

Background: Choline is essential for mammalian cell function. It plays a critical role in cell membrane integrity, neurotransmission, cell signaling and lipid metabolism. Moreover, choline is involved in methylation in two ways: a) its synthesis requires methyl groups donated by S-adenosyl-methionine (AdoMet); and b) choline oxidation product betaine methylates homocysteine (Hcy) to methionine (Met) and produces dimethylglycine. This later donates one carbon units to tetrahydrofolate (THF). Methods: To evaluate the correlations of choline and betaine with folate, AdoMet, S-anenosyl-homocysteine (AdoHcy), total homocysteine (tHcy), and DNA methylation, choline, betaine and dimethylglycine were measured by LC-MS/MS in plasma of 109 healthy volunteers, in whom folate, AdoMet, AdoHcy, tHcy, and DNA methylation have previously been reported. Results: Using a bivariate model, choline and betaine showed strong positive correlations with folate (r=0.346 and r=0.226), AdoHcy (r=0.468 and r=0.296), and correlated negatively with AdoMet/AdoHcy ratio (r=–0.246 and r=–0.379). Only choline was positively correlated with AdoMet (r=0.453). Using a multivariate linear regression model, choline correlated strongly with folate (β=17.416), AdoMet (β=61.272), and AdoHcy (β=9.215). Betaine correlated positively with folate (β=0.133) and negatively with tHcy (β=–0.194) ratio. Choline is an integral part of folate and methylation pathways. Conclusions: Our data highlight the importance of integrating choline in studies concerning addressing pathological conditions related to folate, homocysteine and methylation metabolism.

September 12, 2012

Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: a case-control study in Spain

Montserrat Creus, Ramon Deulofeu, Joana Peñarrubia, Francisco Carmona, Juan Balasch

Page range: 693-699

Abstract

Background: Hyperhomocysteinemia and methylenetetrahydrofolate reductase (MTHFR) gene mutation have been postulated as a possible cause of recurrent miscarriage (RM). There is a wide variation in the prevalence of MTHFR polymorphisms and homocysteine (Hcy) plasma levels among populations around the world. The present study was undertaken to investigate the possible association between hyperhomocysteinemia and its causative genetic or acquired factors and RM in Catalonia, a Mediterranean region in Spain. Methods: Sixty consecutive patients with ≥3 unexplained RM and 30 healthy control women having at least one child but no previous miscarriage were included. Plasma Hcy levels, MTHFR gene mutation, red blood cell (RBC) folate and vitamin B12 serum levels were measured in all subjects. Results: No significant differences were observed neither in plasma Hcy levels, RBC folate and vitamin B12 serum levels nor in the prevalence of homozygous and heterozygous MTHFR gene mutation between the two groups studied. Conclusions: In the present study RM is not associated with hyperhomocysteinemia, and/or the MTHFR gene mutation.

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Volume 51 Issue 3 - Special issue: Advances and controversies in B vitamins and choline / Editors: Rima Obeid, Wolfgang Herrmann

Issue of Clinical Chemistry and Laboratory Medicine (CCLM)

Masthead

Special issue on advances and controversies in B vitamins and choline

B-Vitamin dependent methionine metabolism and alcoholic liver disease

Abstract

Metabolic crosstalk between choline/1-carbon metabolism and energy homeostasis

Abstract

Proteomics of vitamin B12 processing

Abstract

Unexpected high plasma cobalamin/Proposal for a diagnostic strategy

Abstract

Clinical recognition and aspects of the cerebral folate deficiency syndromes

Abstract

Choline-containing phospholipids: relevance to brain functional pathways

Abstract

The ‘golden age’ of DNA methylation in neurodegenerative diseases

Abstract

Mechanisms of the beneficial effects of vitamin B6 and pyridoxal 5-phosphate on cardiac performance in ischemic heart disease

Abstract

The diagnostic utility of folate receptor autoantibodies in blood

Abstract

Red cell or serum folate: what to do in clinical practice?

Abstract

Formate: an essential metabolite, a biomarker, or more?

Abstract

The role of homocysteine in bone remodeling

Abstract

Neuroprotective actions of perinatal choline nutrition

Abstract

Normal prions as a new target of cobalamin (vitamin B12) in rat central nervous system

Abstract

Molecular mechanisms underlying the potentially adverse effects of folate

Abstract

Betaine homocysteine methyltransferase (BHMT)-dependent remethylation pathway in human healthy and tumoral liver

Abstract

Hydrogen sulfide as an oxygen sensor

Abstract

B vitamin therapy for homocysteine: renal function and vitamin B12 determine cardiovascular outcomes

Abstract

One year B and D vitamins supplementation improves metabolic bone markers

Abstract

Effect of 1 year B and D vitamin supplementation on LINE-1 repetitive element methylation in older subjects

Abstract

Aqueous humor glycation marker and plasma homocysteine in macular degeneration

Abstract

Homocysteine plasma levels in patients treated with antiepileptic drugs depend on folate and vitamin B12 serum levels, but not on genetic variants of homocysteine metabolism

Abstract

Trends in clinical laboratory homocysteine testing from 1997 to 2010: the impact of evidence on clinical practice at a single institution

Abstract

Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320)

Abstract

Plasma choline and betaine correlate with serum folate, plasma S-adenosyl-methionine and S-adenosyl-homocysteine in healthy volunteers

Abstract

Plasma homocysteine and vitamin B12 serum levels, red blood cell folate concentrations, C677T methylenetetrahydrofolate reductase gene mutation and risk of recurrent miscarriage: a case-control study in Spain

Abstract

Proteomics of vitamin B₁₂ processing

Normal prions as a new target of cobalamin (vitamin B₁₂) in rat central nervous system