Efficacy of short‐term oral cobalamin therapy for the treatment of cobalamin deficiencies related to food‐cobalamin malabsorption: A study of 30 patients

Background: It has been suggested that oral cobalamin (vitamin B₁₂) therapy may be an effective therapy for treating cobalamin deficiencies related to food‐cobalamin malabsorption. However, the duration of this treatment was not determined. Patients and method: In an open‐label, nonplacebo study, we studied 30 patients with established cobalamin deficiency related to food‐cobalamin malabsorption, who received between 250 and 1000 μg of oral crystalline cyanocobalamin per day for at least 1 month. Endpoints: Blood counts, serum cobalamin and homocysteine levels were determined at baseline and during the first month of treatment. Results: During the first month of treatment, 87% of the patients normalized their serum cobalamin levels; 100% increased their serum cobalamin levels (mean increase, +167 pg/dl; P < 0.001 compared with baseline); 100% had evidence of medullary regeneration; 100% corrected their initial macrocytosis; and 54% corrected their anemia. All patients had increased hemoglobin levels (mean increase, +0.6 g/dl) and reticulocyte counts (mean increase, +35 × 10⁶/l) and decreased erythrocyte cell volume (mean decrease, 3 fl; all P < 0.05). Conclusion: Our findings suggest that crystalline cyanocobalamin, 250–1000 μg /day, given orally for 1 month, may be an effective treatment for cobalamin deficiencies not related to pernicious anemia.     

Causal Effects of Homocysteine,Folate, and Cobalamin on Kidney Function: A Mendelian Randomization Study

Blood homocysteine level and related vitamin levels are associated with various health outcomes. We aimed to assess causal effects of blood homocysteine, folate, and cobalamin on kidney function in the general population by performing Mendelian randomization (MR) analysis. Genetic instruments for blood homocysteine, folate, and cobalamin levels were introduced from a previous genome-wide association (GWAS) meta-analysis of European individuals. Summary-level MR analysis was performed for the estimated glomerular filtration rate (eGFR) from the CKDGen consortium GWAS that included 567,460 European ancestry individuals. For replication, allele-score-based MR was performed with an independent U.K. Biobank cohort of 337,138 individuals of white British ancestry. In summary-level MR for the CKDGen data, high genetically predicted homocysteine levels were significantly associated with low eGFR (per 1 standard deviation, beta for eGFR change −0.95 (−1.21, −0.69) %), supported by pleiotropy-robust MR sensitivity analysis. Genetically predicted high folate levels were significantly associated with high eGFR change (0.86 (0.30, 1.42) %); however, causal estimates from cobalamin were nonsignificant (−0.11 (−0.33, 0.11) %). In the U.K. Biobank data, the results were consistently identified. Therefore, a high blood homocysteine level causally decreases eGFR. Future trials with appropriate homocysteine-lowering interventions may be helpful for the primary prevention of kidney function impairment.     

Transient plasma cobalamin elevation in patients with pneumonia – two case reports

We report two cases of transient significantly elevated plasma cobalamin (B12) in geriatric patients acutely admitted with fever, increased C-reactive protein and X-ray verified pneumonia. Extensive diagnostic workup did not reveal kidney or liver disease, neither any signs of cancer. Furthermore, none of the patients had received therapeutic B12 supplementation prior to admission. In both cases, plasma B12 normalized at an out-patient control few months later. We were not able to identify the reason for the initial B12 elevation in any of the patients, since none of the usually recognized causes were evident. Since both patients had an infection and temporarily elevated B12, we suggest a possible inflammatory response or a vitamin B12 production by the infectious agents as the cause. Both suggestions, however, need further exploration.     

In vitro studies of gastric juice in patients with food-cobalamin malabsorption

Food-cobalamin absorption depends on the initial release of cobalamin from its binders in food. Therefore, the characterization of patients' gastric juices and their behavior in this process was undertaken. Pentagastrin-stimulated gastric juice specimens from three patients with severe food-cobalamin malabsorption, six patients with mild malabsorption, and five patients with normal absorption were tested for pH, pepsin, intrinsic factor content, and anin vitro method that quantitates transfer of cobalamin from egg yolk to gastric R binder. Transfer of cobalamin correlated best within vivo egg yolk-cobalamin absorption test results in the 14 patients (r=0.731,P<0.005). Transfer also correlated inversely with gastric juice pH (r=–0.619,P<0.02). Basal gastric juice specimens, with their higher pH, from the same subjects failed to promote cobalamin transfer until their pH was lowered to 1.0–1.3. Pepsin levels did not correlate within vitro transfer or with absorptionin vivo; nevertheless, raising the low pepsin concentration of one stimulated gastric juice improved transfer, while inhibiting pepsin activity with pepstatin A inhibited transfer. Mixing experiments with selected stimulated gastric juices demonstrated that poorin vitro transfer, which in a few cases seemed unrelated to pH or pepsin levels, was not due to any inhibitory activity of such gastric juices. These studies confirm that gastric acid and pepsin play a central role in releasing food-bound cobalamin and transferring it to R binder, but suggest that other, still unidentified gastric defects occasionally contribute to impaired transfer; the latter defects are not inhibitory in nature but seem to involve the absence of a permissive activity. The finding that the ability of a gastric juice to promote the transfer of cobalaminin vitro was the best overall indicator of a patient's ability to absorb food cobalaminin vivo suggests that gastric juice defects are responsible for most cases of food-cobalamin malabsorption. The phenomenon may also provide a practicalin vitro estimate of a patient's ability to absorb food cobalamin.This study was supported by grant DK 32640 from the National Institutes of Health.     

Vitamin B12-dependent biosynthesis ties amplified 2-methylhopanoid production during oceanic anoxic events to nitrification

Bacterial hopanoid lipids are ubiquitous in the geologic record and serve as biomarkers for reconstructing Earth’s climatic and biogeochemical evolution. Specifically, the abundance of 2-methylhopanoids deposited during Mesozoic ocean anoxic events (OAEs) and other intervals has been interpreted to reflect proliferation of nitrogen-fixing marine cyanobacteria. However, there currently is no conclusive evidence for 2-methylhopanoid production by extant marine cyanobacteria. As an alternative explanation, here we report 2-methylhopanoid production by bacteria of the genus Nitrobacter, cosmopolitan nitrite oxidizers that inhabit nutrient-rich freshwater, brackish, and marine environments. The model organism Nitrobacter vulgaris produced only trace amounts of 2-methylhopanoids when grown in minimal medium or with added methionine, the presumed biosynthetic methyl donor. Supplementation of cultures with cobalamin (vitamin B₁₂) increased nitrite oxidation rates and stimulated a 33-fold increase of 2-methylhopanoid abundance, indicating that the biosynthetic reaction mechanism is cobalamin dependent. Because Nitrobacter spp. cannot synthesize cobalamin, we postulate that they acquire it from organisms inhabiting a shared ecological niche—for example, ammonia-oxidizing archaea. We propose that during nutrient-rich conditions, cobalamin-based mutualism intensifies upper water column nitrification, thus promoting 2-methylhopanoid deposition. In contrast, anoxia underlying oligotrophic surface ocean conditions in restricted basins would prompt shoaling of anaerobic ammonium oxidation, leading to low observed 2-methylhopanoid abundances. The first scenario is consistent with hypotheses of enhanced nutrient loading during OAEs, while the second is consistent with the sedimentary record of Pliocene–Pleistocene Mediterranean sapropel events. We thus hypothesize that nitrogen cycling in the Pliocene–Pleistocene Mediterranean resembled modern, highly stratified basins, whereas no modern analog exists for OAEs.

Hopanoids are a structurally diverse class of isoprenoid lipids that are involved in bacterial membrane homeostasis by mediating membrane organization and stress response (1–4). As chemical fossils, hopanoids and their diagenetic products are ubiquitous in the geologic record where they serve as important biomarkers for our planet’s biogeochemical and microbial evolution from the Proterozoic onward (5–8). Specifically, a subgroup of hopanoids methylated at the C-2 position (2-methylhopanoids) has been used as biomarkers for cyanobacteria (9) and invoked as evidence for the proliferation of nitrogen-fixing cyanobacteria during intervals such as Mesozoic ocean anoxic events (OAEs) (10, 11) and the Paleocene–Eocene Thermal Maximum (12).The importance of hopanoids as biomarkers has sustained interest in understanding their sources and their role in bacterial physiology, leading to multiple recent studies that challenge prior assumptions (2, 3, 13–15). Specifically, the occurrence of 2-methylhopanoids in diverse alphaproteobacteria, including the anoxygenic phototroph Rhodopseudomonas palustris (16) and other freshwater and soil bacteria (17–21), illustrates that 2-methylhopanoids are not exclusive to cyanobacteria. Although 2-methylhopanoids predominantly originate from cyanobacteria in environments such as freshwater and lagoonal microbial mats (22, 23), it is plausible that other bacteria could have contributed to the geologic record of 2-methylhopanoids. However, it is unlikely that freshwater cyanobacteria were primary contributors to the accumulation of 2-methylhopanoids in offshore marine environments during Mesozoic OAEs. Thus, numerous uncertainties surround the origin of 2-methylhopanoids in the geological record and the reasons behind their prevalence during episodes of ocean anoxia.Screening of genomes and metagenomes for the hpnP gene encoding a hopanoid C-2 methyltransferase (24) has led to the identification of a small subset of freshwater and soil bacteria as putative 2-methylhopanoid producers but has not revealed instances in marine cyanobacteria (24, 25). A more recent gene homology analysis suggested the presence of the hpnP gene in diverse marine cyanobacteria (26). However, 2-methylhopanoids have not been detected in any of these cyanobacteria (15), suggesting this recent study (26) may have detected related genes of different function.Identification of common source organisms and elucidation of the underlying biochemistry of 2-methylhopanoid biosynthesis could help constrain the factors controlling their geologic record. Based on previous detection of the hpnP gene in a species of the alphaproteobacterial genus Nitrobacter (24), we hypothesized that Nitrobacter spp. could be an important but previously overlooked source of 2-methylhopanoids. Nitrobacter spp. are nitrite-oxidizing bacteria (NOB) that are abundant in soil, fresh water, and the oceans where they share an ecological niche with other NOB (Nitrospina, Nitrospira, Nitrococcus spp.), ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (27–29).Here, we use a combination of genomic analyses and culture experiments with the model organism Nitrobacter vulgaris AB1 to elucidate the factors driving 2-methylhopanoid biosynthesis in this taxon. Our results suggest that the reaction mechanism is not only dependent on a radical S-adenosylmethionine (SAM) enzyme (23) but also on the enzymatic cofactor cobalamin (vitamin B₁₂). Because Nitrobacter spp. are cobalamin auxotrophs, we hypothesize that synergistic interaction between Nitrobacter and cobalamin-producing nitrifying archaea could be an important control on 2-methylhopanoid production. This hypothesis is consistent with enhanced production of 2-methylhopanoids under conditions of intensified oxidative nitrogen cycling in past environments.     

Functional Characterization and Categorization of Missense Mutations that Cause Methylmalonyl‐CoA Mutase (MUT) Deficiency

Methylmalonyl‐CoA mutase (MUT) is an essential enzyme in propionate catabolism that requires adenosylcobalamin as a cofactor. Almost 250 inherited mutations in the MUT gene are known to cause the devastating disorder methylmalonic aciduria; however, the mechanism of dysfunction of these mutations, more than half of which are missense changes, has not been thoroughly investigated. Here, we examined 23 patient missense mutations covering a spectrum of exonic/structural regions, clinical phenotypes, and ethnic populations in order to determine their influence on protein stability, using two recombinant expression systems and a thermostability assay, and enzymatic function by measuring MUT activity and affinity for its cofactor and substrate. Our data stratify MUT missense mutations into categories of biochemical defects, including (1) reduced protein level due to misfolding, (2) increased thermolability, (3) impaired enzyme activity, and (4) reduced cofactor response in substrate turnover. We further demonstrate the stabilization of wild‐type and thermolabile mutants by chemical chaperones in vitro and in bacterial cells. This in‐depth mutation study illustrates the tools available for MUT enzyme characterization, guides future categorization of further missense mutations, and supports the development of alternative, chaperone‐based therapy for patients not responding to current treatment.     

应用数码显微镜评价重组牛碱性成纤维细胞生长因子辅助治疗萎缩性舌炎的临床效果

目的：应用数码显微镜观察重组牛碱性成纤维细胞生长因子（recombinantbovinebasicfibroblastgrowthfactor,rb-bFGF）辅助治疗萎缩性舌炎的疗效。方法：39例维生素B12缺乏的萎缩性舌炎患者,随机进入治疗组（21例）和对照组（18例）。采用数码显微镜,检测患者初次就诊及第7、14d复诊时舌乳头形态,同时记录舌痛、味觉改变的程度。比较两组患者在初诊及两次复诊时上述3项指标的改善情况。结果：第7d复诊,治疗组患者总体舌黏膜萎缩的改善程度高于对照组（P〈0．05）,其舌痛存在时间短于对照组护（P〈0．05）;第14d复诊,两组治疗效果趋于一致。结论：rbFGF能够减轻炎症反应、促进舌乳头生长并缩短疼痛时间。     

Long-term effects of nitrous oxide anaesthesia on laboratory and clinical parameters in elderly Omani patients: a randomized double-blind study

AIMS: This study examined the long-term effects of nitrous oxide anaesthesia on serum levels of cobalamin and folate, red cell folate levels and haematological parameters, and neurological status in elderly Omani patients. METHODS: Sixty-nine consecutive patients undergoing ophthalmic surgery were randomly and double-blind assigned to nitrous oxide or propofol anaesthesia. They met the following entry criteria: age 55 years or above, no major organ failure, no clinical signs or symptoms of cobalamin or folate deficiency, mean cell volume (MCV) cobalamin and/or folate substitution therapy during the preceding months. Serum levels of cobalamin and folate, red cell folate levels, and haematological parameters were measured prior to anaesthesia and 3-5 weeks later. At that time, the patients also underwent thorough neurological examination. RESULTS: Data of 51 patients were complete and considered for analysis. In both nitrous oxide and propofol group, the range of exposure time was comparable (+/-1 h). In the nitrous oxide group, a slight but significant decrease in haemoglobin, Hct, and red blood cell count (RBC) (P < 0.001) was observed, whereas there was a mild increase in mean cell haemoglobin (MCH) and mean cell volume (P < 0.05). In addition, there was a significant decrease in serum folate levels (P < 0.05). Hct and RBC decreased slightly in the propofol group (P < 0. 05), whereas there was a small increase in MCH. There was no difference between the two anaesthetics with regard to serum cobalamin and red cell folate levels, but there was a significant decrease in serum folate levels in the nitrous oxide group compared to those in the propofol group. Three patients with pre-existing low red cell folate levels, who were randomized to nitrous oxide anaesthesia, developed clinical symptoms of folate deficiency. CONCLUSION: This study showed that short-term (40-80 min) nitrous oxide anaesthesia did not affect cobalamin levels but reduced serum folate levels in this elderly population. Although this reduction was clinically irrelevant, some patients with pre-existing asymptomatic folate deficiency developed nitrous oxide-induced folate deficiency.