Vitamin B12

The B-PROOF study is a randomized, placebo-controlled, double-blind, parallel intervention study. B-PROOF is an acronym for 'B-vitamins for the PRevention Of Osteoporotic Fractures'. This large multi-centre project is carried out in The Netherlands by a consortium from Erasmus MC (EMC, Rotterdam), VU University Medical Center (VUmc, Amsterdam) and Wageningen University (WU, Wageningen), the latter acting as coordinator. The study aimed to include 3000 subjects, aged 65 years and older, with elevated plasma homocysteine concentrations (≥ 12 μmol/L). The intervention period is 2 years. Participants were randomly allocated in a 1:1 ratio to the intervention group or to the control group. We stratified for study centre, sex, age (65-80 years, ≥ 80 years), and homocysteine concentration (12-18 μmol/L, ≥ 18 μmol/L). The intervention group receives a daily tablet with 500 μg vitamin B₁₂ and 400 μg folic acid and the control group receives a daily placebo tablet. Both tablets contain 15 μg (600 IU) of vitamin D₃ to ensure a normal vitamin D status [35 (link)]. The intervention and placebo tablets, produced by Orthica, Almere, the Netherlands, are indistinguishable in taste, smell and appearance. The random allocation sequence and randomization were generated and performed using SAS 9.2 by an independent research dietician.
Recruitment took place from August 2008 until March 2011. The B-PROOF study has been registered with the Netherlands Trial Register http://www.trialregister.nl under identifier NTR 1333 since June 1, 2008 and with ClinicalTrials.gov under identifier NCT00696514 since June 9, 2008. The WU Medical Ethics Committee approved the study protocol, and the Medical Ethics committees of EMC and VUmc gave approval for local feasibility.

A total of 2470 participants were recruited, including 1151 cognitively normal controls (NC), 898 patients with amnestic mild cognitive impairment (aMCI), and 421 patients with mild Alzheimer disease (AD).
We recruited the controls using cluster sampling in Jingansi Community Shanghai, China. The inclusion criteria for NC were: age between 50 and 90; no memory complaints verified by an informant; cognitively normal, based on the absence of significant impairment in cognitive functions or activities of daily living (ADL); Clinical Dementia Rating (CDR)  =  0; and Hamilton depression rating scale (HAMD) scored ≤ 12 on the 17-item scale in past 2 weeks. They had adequate visual and auditory acuity to allow cognitive testing. Participants with any significant neurologic disease and psychiatric disorders/psychotic features were excluded.
All the patients with aMCI and AD were recruited from the Memory Clinic, Huashan Hospital, from Jun 2004 to Oct 2011.They finished the laboratory tests and cranial CT/MRI scan, and had no clinically significant abnormalities in vitamin B12, folic acid, thyroid function (free triiodothyronine-FT3, free tetraiodothyronine-FT4, thyroid stimulating hormone-TSH), rapid plasma regain (RPR), or treponema pallidum particle agglutination (TPPA).
The aMCI patients were diagnosed according to the following criteria19]: (1) cognitive complaints verified by an informant; (2) cognitive impairment lasting more than 3 months; (3) Mini-mental state examination-Chinese version (C-MMSE)[25] (link) ≥ cut-off score for adjusted education; (4) Abnormal objective memory impairment documented by scoring below the age and education adjusted cutoff on an episodic memory test (Auditory Verbal Learning Test); (5) preserved basic ADL/minimal impairment in complex instrumental functions; (6) etiology unknown; (7) normal sense of hearing and sight; (8) has not met diagnostic criteria of dementia based on the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA).
The AD patients (n = 421) met the following criteria: (1) diagnosed as probable AD according to the NINCDS-ADRDA; (2) no obvious medical, neurological or psychiatric diseases or psychological dysfunction including anxiety and depression within the previous one month; (3) no visual or auditory deficit.

In this study, Rhodopseudomonas palustris (NCIMB 1774) was used as model organism. Cells were precultured in Van Niels medium—a fast-growing medium containing (per 1 L): 1 g K₂HPO₄, 0.5 g MgSO₄, 10 g yeast extract and the balance deionised water (du Toit and Pott 2021 (link)). After autoclaving (121 °C, 20 min), 10 mL of sterile glycerol (4 M) was added to the medium aseptically (du Toit and Pott 2021 (link)). Bacterial cells were suspended in the medium and grown anaerobically in 500 mL Schott bottles under argon atmosphere. Temperature was maintained at 35 °C (± 0.2 °C) and light intensity was calibrated to 200 W m⁻² (± 20 W m⁻²) in the wavelength range of 500–1100 nm (tungsten filament incandescent light, Eurolux^©, South Africa) (Bosman et al. 2022a (link)) using a handheld spectrophotometer (RGB Photonics, Qmini VIS–NIR). Culturing time was approximately five days to allow cells to reach the mid-logarithmic phase.
All growth and hydrogen production experiments were conducted using a Rhodospirillaceae medium containing (per 1 L): 0.6 g K₂HPO₄, 1.7 g KH₂PO₄, 0.02 g MgSO₄·7H₂O, 0.005 g CaCl₂·2H₂O, 0.4 g NaCl, 0.3 g Na₂S₂O₃, 0.0005 g ferric citrate, 0.0002 g para-aminobenzoic acid, and 1 mL of trace element solution containing (per 1 L): 70 mg ZnCl₂, 100 mg MnCl₂·4H₂O, 60 mg H₃BO₃, 200 mg CoCl₂·6H₂O, 20 mg CuCl₂·2H₂O, 20 mg NiCl₂·6H₂O, and 40 mg NaMoO₄·2H₂O (Pott et al. 2014 (link)). The medium was autoclaved (121 °C, 20 min) and the pH measured at 7.2. Also aseptically added to the medium after autoclaving was a vitamin solution containing (per 1 L): 1.2 g thiamine HCl and 0.01 g cyanocobalamin (filter-sterilized), and lastly 10 mL of 5 M sterile glycerol (final concentration of 50 mM) and 5 mL of sterile glutamic acid (final concentration of 10 mM) were added to the medium aseptically (Pott et al. 2014 (link)).