Vitamin B12, homocysteine and carotid plaque in the era of folic acid fortification of enriched cereal grain products

Background: Carotid plaque area is a strong predictor of cardiovascular events. High homocysteine levels, which are associated with plaque formation, can result from inadequate intake of folate and vitamin B12. Now that folic acid fortification is widespread in North America, vitamin B12 has become an important determinant of homocysteine levels. We sought to determine the prevalence of low serum levels of vitamin B12, and their relation to homocysteine levels and carotid plaque area among patients referred for treatment of vascular disease since folic acid fortification of enriched grain products. Methods: We evaluated 421 consecutive new patients with complete data whom we saw in our vascular disease prevention clinics between January 1998 and January 2002. We measured total carotid plaque area by ultrasound and determined homocysteine and serum vitamin B12 levels in all patients. Results: The patients, 215 men and 206 women, ranged in age from 37 to 90 years (mean 66 years). Most were taking medications for hypertension (67%) and dyslipidemia (62%). Seventy-three patients (17%) had vitamin B12 deficiency (vitamin B12 level \u3c 258 pmol/L with homocysteine level \u3e 14 μmol/L or methylmalonic acid level \u3e 271 nmol/L). The mean area of carotid plaque was significantly larger among the group of patients whose vitamin B12 level was below the median of 253 pmol/L than among those whose vitamin B12 level was above the median: 1.36 (standard deviation [SD] 1.27) cm2 v. 1.09 (SD 1.0) cm2; p = 0.016. Conclusions: Vitamin B12 deficiency is surprisingly common among patients with vascular disease, and, in the setting of folic acid fortification, low serum vitamin B12 levels are a major determinant of elevated homocysteine levels and increased carotid plaque area. © 2005 CMA Media Inc. or its licensors

Evidence Favoring a Positive Feedback Loop for Physiologic Auto Upregulation of hnRNP-E1 during Prolonged Folate Deficiency in Human Placental Cells

Background: Previously, we determined that heterogeneous nuclear ribonucleoprotein E1 (hnRNP-E1) functions as an intracellular physiologic sensor of folate deficiency. In this model, l-homocysteine, which accumulates intracellularly in proportion to the extent of folate deficiency, covalently binds to and thereby activates homocysteinylated hnRNP-E1 to interact with folate receptor-α mRNA; this high-affinity interaction triggers the translational upregulation of cell surface folate receptors, which enables cells to optimize folate uptake from the external milieu. However, integral to this model is the need for ongoing generation of hnRNP-E1 to replenish homocysteinylated hnRNP-E1 that is degraded.Objective: We searched for an interrelated physiologic mechanism that could also maintain the steady-state concentration of hnRNP-E1 during prolonged folate deficiency.Methods: A novel RNA-protein interaction was functionally characterized by using molecular and biochemical approaches in vitro and in vivo.Results: l-homocysteine triggered a dose-dependent high-affinity interaction between hnRNP-E1 and a 25-nucleotide cis element within the 5'-untranslated region of hnRNP-E1 mRNA; this led to a proportionate increase in these RNA-protein complexes, and translation of hnRNP-E1 both in vitro and within placental cells. Targeted perturbation of this RNA-protein interaction either by specific 25-nucleotide antisense oligonucleotides or mutation within this cis element or by small interfering RNA to hnRNP-E1 mRNA significantly reduced cellular biosynthesis of hnRNP-E1. Conversely, transfection of hnRNP-E1 mutant proteins that mimicked homocysteinylated hnRNP-E1 stimulated both cellular hnRNP-E1 and folate receptor biosynthesis. In addition, ferrous sulfate heptahydrate [iron(II)], which also binds hnRNP-E1, significantly perturbed this l-homocysteine-triggered RNA-protein interaction in a dose-dependent manner. Finally, folate deficiency induced dual upregulation of hnRNP-E1 and folate receptors in cultured human cells and tumor xenografts, and more selectively in various fetal tissues of folate-deficient dams.Conclusions: This novel positive feedback loop amplifies hnRNP-E1 during prolonged folate deficiency and thereby maximizes upregulation of folate receptors in order to restore folate homeostasis toward normalcy in placental cells. It will also functionally impact several other mRNAs of the nutrition-sensitive, folate-responsive posttranscriptional RNA operon that is orchestrated by homocysteinylated hnRNP-E1

Metabolic phenotype of methylmalonic acidemia in mice and humans: the role of skeletal muscle

<p>Abstract</p> <p>Background</p> <p>Mutations in methylmalonyl-CoA mutase cause methylmalonic acidemia, a common organic aciduria. Current treatment regimens rely on dietary management and, in severely affected patients, liver or combined liver-kidney transplantation. For undetermined reasons, transplantation does not correct the biochemical phenotype.</p> <p>Methods</p> <p>To study the metabolic disturbances seen in this disorder, we have created a murine model with a null allele at the methylmalonyl-CoA mutase locus and correlated the results observed in the knock-out mice to patient data. To gain insight into the origin and magnitude of methylmalonic acid (MMA) production in humans with methylmalonyl-CoA mutase deficiency, we evaluated two methylmalonic acidemia patients who had received different variants of combined liver-kidney transplants, one with a complete liver replacement-kidney transplant and the other with an auxiliary liver graft-kidney transplant, and compared their metabolite production to four untransplanted patients with intact renal function.</p> <p>Results</p> <p>Enzymatic, Western and Northern analyses demonstrated that the targeted allele was null and correctable by lentiviral complementation. Metabolite studies defined the magnitude and tempo of plasma MMA concentrations in the mice. Before a fatal metabolic crisis developed in the first 24–48 hours, the methylmalonic acid content per gram wet-weight was massively elevated in the skeletal muscle as well as the kidneys, liver and brain. Near the end of life, extreme elevations in tissue MMA were present primarily in the liver. The transplant patients studied when well and on dietary therapy, displayed massive elevations of MMA in the plasma and urine, comparable to the levels seen in the untransplanted patients with similar enzymatic phenotypes and dietary regimens.</p> <p>Conclusion</p> <p>The combined observations from the murine metabolite studies and patient investigations indicate that during homeostasis, a large portion of circulating MMA has an extra-heptorenal origin and likely derives from the skeletal muscle. Our studies suggest that modulating skeletal muscle metabolism may represent a strategy to increase metabolic capacity in methylmalonic acidemia as well as other organic acidurias. This mouse model will be useful for further investigations exploring disease mechanisms and therapeutic interventions in methylmalonic acidemia, a devastating disorder of intermediary metabolism.</p

Elevated serum S-adenosylhomocysteine in cobalamin-deficient elderly and response to treatment

Elevated serum S-adenosylhomocysteine in cobalamin-deficient elderly and response to treatment1,2,3 Sally P Stabler1, Robert H Allen1, Evi T Dolce1 and Mary Ann Johnson1 1 From the Department of Medicine, University of Colorado Health Sciences Center, Denver, CO (RHA and SPS), and the Department of Foods and Nutrition, University of Georgia, Athens, GA (MAJ and ETD) Background: S-Adenosylmethionine (SAM)\u96dependent methylation reactions produce S-adenosylhomocysteine (SAH), the precursor of homocysteine, which has been associated with adverse events when it is elevated. Objective: We studied a cohort of elderly with a high prevalence of cobalamin deficiency to determine whether SAH, SAM, or their ratio was abnormal; whether they correlated with other markers of vitamin deficiency; and whether they changed with cobalamin therapy. Design: A convenience sample of elderly attending nutrition centers was enrolled for baseline demographic, biochemical, and nutritional assessments. Methylmalonic acid (MMA), total homocysteine, and other metabolites were measured by using gas chromatography\u96mass spectrometry. Serum SAM and SAH were measured by using stable-isotope-dilution liquid chromatography\u96mass spectrometry. Subjects found to have elevated serum MMA were treated with oral cyanocobalamin tablets (1000 \ub5g/d) for 3 mo. Subjects with normal MMA were randomly assigned to 1 of 3 dosage groups: 0, 25, or 100 \ub5g cyanocobalamin/d. Results: The 149 elderly subjects had a mean age of 76.3 y; 81% were female, and 30% were African American. Serum MMA concentrations were elevated in 30% and SAH concentrations were elevated in 64% of the cohort. Those with elevated MMA concentrations had higher SAH and SAM concentrations. High-dose oral cobalamin lowered SAH, MMA, and total homocysteine concentrations significantly, although subjects with creatinine concentrations >109 umol/L had higher posttreatment SAH than did those with lower creatinine. Conclusions: Elevated serum SAH concentrations are common in elderly and are strongly influenced by both renal status and cobalamin deficiency. These elevated concentrations can be lowered with high-dose oral cobalamin therapy. Key Words: Methylmalonic acid \u95 vitamin B-12 \u95 total homocysteine \u95 folate \u95 S-adenosylmethionin

Folate Deficiency Facilitates Genomic Integration of Human Papillomavirus Type 16 DNA In Vivo in a Novel Mouse Model for Rapid Oncogenic Transformation of Human Keratinocytes

Background Epidemiologic and in vitro studies suggest independent linkages between poor folate and/or vitamin B-12 nutrition, genomic human papillomavirus (HPV) type 16 viral integration, and cancer. However, there is no direct evidence in vivo to support the causative role of poor folate nutrition in HPV16 integration into the cellular genome. Objective We tested the hypothesis that folate deficiency enables the integration of HPV16 into the genome of HPV16-harboring keratinocytes, and could thereby influence earlier transformation of these cells to cancer in an animal model. Methods HPV16-harboring human keratinocytes [(HPV16)BC-1-Ep/SL] were differentiated into 3-dimensional HPV16-organotypic rafts under either folate-replete or folate-deficient conditions in vitro. These were then subcutaneously implanted in severely immunocompromised female Beige Nude XID (Hsd: NIHS-LystbgFoxn1nuBtkxid) mice (4–6 wk old, 16–18 g) fed either a folate-replete diet (1200 nmol folate/kg diet) or a progressively folate-deficient diet (600 or 400 nmol folate/kg diet) for 2 mo prior to raft-implantation surgery, and indefinitely thereafter. The tumors that subsequently developed were characterized for onset, pattern of growth, morphology, HPV16 oncogene expression, and HPV16-genomic integration. Results All HPV16-organotypic rafts developed in either folate-replete or physiologic low-folate media in vitro and subsequently implanted in folate-replete mice eventually transformed into aggressive malignancies within weeks. When compared to HPV16-high folate-organotypic raft-derived tumors from mice fed either a 1200 or 600 nmol folate/kg diet, those raft-derived cancers that developed in mice fed a 400 nmol folate/kg diet expressed significantly more HPV16 E6 (1.8-fold more) and E7 (2.8-fold more) oncogenic proteins (P = 0.001), and revealed significantly more HPV16-integration sites in genomic DNA (2-fold more), either directly into, or in the vicinity of, cellular genes (P < 0.05). Conclusions This unprecedented animal model for the consistent rapid transformation of differentiated (HPV16)BC-1-Ep/SL-derived organotypic raft-keratinocytes to cancer in Beige Nude XID mice confirms that dietary folate deficiency can profoundly influence and modulate events leading to HPV16-induced carcinogenesis, and facilitates genomic integration of HPV16 DNA in vivo

Lack of Anemia Despite Marked Elevation of Serum Methylmalonic Acid and Total Homocysteine in a Multiethnic Cohort

Abstract It is controversial whether asymptomatic patients with metabolic evidence of cobalamin (Cbl) deficiency need vitamin treatment. Patients with clinical abnormalities due to Cbl deficiency which are proven to correct with treatment invariably have elevated serum methylmalonic acid(MMA) and total homocysteine(tHcy). However, many subjects with elevated MMA are found in screening studies of seniors and they often have no classical clinical abnormalities. We correlated hematologic and neurologic findings with elevated MMA and tHcy in subjects enrolled in the Northern Manhattan Study. Eligible subjects were over 40 yr, had not had a stroke and lived in Manhattan. Approximately 3000 subjects had serum MMA, tHcy and other relevant data available. The samples were collected from 1993 to 2001 thus spanning food folate fortification in the United States. We analyzed those in the top 1% MMA and/or tHcy since these subjects had values seen also in clinical Cbl deficiency. There were 30 subjects with MMA 975 to 179,900 nM (normal100fL. The male subject with the highest MCV (109.8 fL), had a hematocrit of 42.7 %, MMA 179,900 nM, tHcy 86.3 uM and Cbl 34 pg/mL with no neurologic findings. Neurologic abnormalities were present in 5 of 30. One subject had high grade spastic paraparesis, 3 had peripheral nervous system deficits, and one had central nervous system defects.Serum tHcy and MMA were not higher in those with neurologic abnormalities. The serum creatinine was 975 nM in 1% of a randomly selected urban multiethnic cohort. 2. Hematologic abnormalities were absent or mild in this group with high MMA. 3. Neurologic abnormalities were found consistent with Cbl deficiency in 17 % of those with high MMA. 4. Both MMA and tHcy are usually elevated. 5. Even though the elevations of MMA and tHcy overlapped those found in clinical studies of Cbl deficiciency these randomly discovered subjects were generally asymptomatic, suggesting that the pathophysiology of megaloblastic anemia and combined systems disease still needs to be investigated