The design project entitled “Design of a treatment protocol to improve the health of B12 deficient patients” is carried out as an individual design project of PDEng program in Chemical Product Design. This project is commenced for the B12 Institute, Rotterdam, which aims to improve the diagnosis and treatment of patients with vitamin B12 and folate deficiency. The experience of the B12 Institute showed that the current diagnostic and treatment protocol is not sufficient to improve the health of some severe patients. This design project aims to provide knowledge beyond the classical theories of B12 deficiency, such that an extended protocol can be recommended to enhance the recovery and health of B12 deficient patients. The recommended protocol consists of diagnostic means and subsequent corrective actions. The approach of the project is divided into 4 major steps: 1. study of the biochemistry of vitamin B12 cellular metabolism; 2. generation of hypotheses on disorders related to B12 deficiency according to the biochemistry study; 3. recommendation of biomarkers/diagnostic tools; 4. recommendation of corrective actions. This report discusses the hypotheses of the vitamin B12 cellular inactivity, which extends the theory of the classical B12 deficiency. The hypotheses revolve around the failure of the B12 cellular activation due to enzymes defects and the activation cofactors deficiencies, as well as the failure of B12 co-enzymatic function due to oxidative stress. A series of vicious cycles between the overlooked causes and impacts in B12 inactivity is also described. The highlights of the hypotheses are summarized as follows: • B12 deficiency causes folate cycle block, which leads to glycine deficiency, • Glycine deficiency leads to glutathione deficiency and collagen deficiency, • Glutathione deficiency causes an elevated oxidative stress, and vice versa, • Collagen deficiency leads to intestinal bacterial dysbiosis, • Intestinal bacterial dysbiosis causes the production of bacterial toxins, including formaldehyde, • An excess of formaldehyde exacerbates oxidative stress and damages to the body, and may as well inactivate cellular B12. However, we found that the current knowledge on the mechanisms explaining a lower B12 enzyme activity may still be insufficient to explain the whole condition and issues related to B12 cellular inactivity. Finally, we designed a mini study to obtain evidence on a number of the hypotheses, especially those related to oxidative stress. New biomarkers for the extension of the diagnostic tools are explored. The new biomarkers are expected to provide better tools to explain the condition and symptoms of the patients. In addition, several supplements are recommended as corrective actions of the anticipated issues disclosed by the new biomarkers. The concepts and the results of the project are expected to provide new insights for the medical research and practice of B12 deficiency treatment, as well as to provide major improvement to the health of the patients. @en