Vitamins E is the collective name of a group of eight different compounds α-, β-, γ-, and δ-tocopherols and the corresponding four tocotrienols (30). One of these, α-tocopherol, is the most abundant and active form in humans and is accepted as a major free radical that eliminates antioxidants and protects biological molecules from detrimental oxidative modifications (31) and lipid peroxyl radicals. The tocopherols can trap propagating radical intermediates that are produced during lipid peroxidation and arrest chain reactions (32). In contrast with vitamin B, vitamin E is insoluble in water, but it is a fat-soluble vitamin and can solely be synthesized in plastids. All tocopherols are found in large quantities in leaves, buds, seeds in the germination state, but also in plant and vegetable oils, therefore only photosynthetic organisms are able to synthesize vitamin E (30). Moreover, vitamin E has multiple applications, as it is widely used as dietary supplement alongside other micronutrients, such as vitamin C and contributes to reducing the risk or preventing diseases that occur due to oxidative changes in biological molecules. Many studies confirm that vitamin E has been found to have beneficial effects for animals (murine), humans and non-murine animals. Studies were developed on animal models, like mice, or cell lines to study various diseases. The latest study on obesity by Kato et al. (33) was conducted on mice fed with a high-fat diet and concluded that tocotrienols (T3s) exerted an anti-obesity effect, normalizing body mass, serum cholesterol and white adipose tissue (Figure 1). It suppressed liver damage, but no changes in cognitive dysfunction were detected. Furthermore, vitamin E mitigated the adverse effect on reproductive performance in mice treated with TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (34) and it can inhibit the oxidative stress induced by some heavy metals such as arsenic, cadmium, and alcohol. In this way, it considerably reduces the harmful effects on the functions of the reproductive system (spermatogenesis) (35). When the rats were treated with different doses of vitamin E over a period of 30 days, their kidneys and livers were affected, but proved it has no adverse effect on the testes. As such, El-Hak et al. (36) recommended exercising caution when taking high doses of vitamin E. Biological and histological modifications that appear due to exposure of mice to CPF (chlorpyrifos) during gestation and lactation were reversed by an oral supplementation of vitamin E, either to the dams or to their pups (37).

Regarding human studies, in a group of employees working on extremely low frequency electromagnetic fields (ELF-EMFs) and treated with vitamin E and C, the results show that these antioxidants can increase the activity of the non-enzymatic antioxidant defense system and protect DNA from damage (Figure 1). To evaluate cell death induced by DNA damage Hosseinabadi et al. (38) used Annexin V FITC/P.I. flow cytometry assay to assess early/late apoptosis and necrosis induced by external factors. Apparently, receiving these vitamins affect the viability of the investigated cells.

Also, vitamin E is being studied in the prevention and treatment of different types of cancer. The use of flow cytometry (FACscanto II system) and FITC Annexin V kit (39) has demonstrated the antiproliferative effect of α-tocopherol on oral cancer cells ORL-48 at a low dose and vitamin E has also been reported to induce apoptosis in erythroid leukemia and in prostate and breast cancer cells (40) (Figure 1).

The same technique was used for non-murine animal studies with FACSca, BD Biosciences (San Jose, CA, USA) (41) to determine the lymphocyte proliferation and peripheral blood mononuclear cell (PBMC) in 15 healthy dogs fed with different types of diet supplemented with either sunflower oil, menhaden fish oil and α-tocopherol acetate for 12 weeks. The results show that the high plasma levels of vitamin E appear to inhibit this suppression of the lymphoproliferative response. Also, the result of flow cytometric measurements (PAS III, Partec GmbH, D-Munster) indicate that vitamin E exerts significant protecting effects on metabolic processes reflected by intracellular glutathione, pH and cellular viability after UVB irradiation (42). On the other hand, even though it is not yet clear how vitamin E reaches the mitochondrial membranes in cells, it is evident that its administration can reduce mitochondrial oxidative dysfunction (32).

Hu and his team (43) proved that GPX4 and vitamin E cooperatively maintain lipid redox balance and prevent ferroptosis in HSPCs, while O’Brien et al. (44), used flow cytometry (FACSCalibur and CellQuest software–Becton Dickinson Instruments, Cambridge, MA, USA) to investigate the effect of dietary supplementation with Vit E and/or Se on immunological parameters in cats. The results show that vitamin E supplementation significantly increased the phagocytic activity and lymphocyte proliferation at a moderate dose (225 mg/kg DM) and did not alter the percentage expression of cell surface markers. This dose appears adequate to enhance some parameters of cat immune function.

This fat-soluble vitamin is a component of the vitamin K family. There are two types of natural vitamin K: vitamin K1 and vitamin K2 (45). One of these is more specific for hepatic activating blood clotting factors, while the other one, vitamin K2, is used to activate various extra-hepatic proteins which are more dependent on vitamin K2 than K1. The most common form of vitamin K2 in the human diet is menaquinone-4 and menaquinone-7 (MK-4 and MK-7) (45–48). The effects of vitamin K on the immune response have been shown to be beneficial in various diseases, including inflammatory diseases and cancer (49). During the fermentation process, microorganisms such as Gram-positive bacteria produce a type of compound known as menaquinone, which is actually vitamin K2. This compound is referred to as menaquinone-n due to its 5-carbon-unsaturated carbon chain (50). Another K-family compound is K3, which is synthetically produced without a side chain (51).

In the nervous system, vitamin K plays a vital role in regulating the activity of certain enzymes involved in sphingolipid metabolism and synthesis. This substance is known to play a role in various cellular events, such as the formation of new cells and the maintenance of cellular communication (52). Studies show that to gain a more substantial anti-ferroptosis effect, fully reduced forms of vitamin K, which include phylloquinone and menaquinone, confer this benefit (Figure 2). The conventional function of this group of naphthoquinones, which is linked to blood clotting, is that it acts as a factor for γ-glutamyl carboxylase (Figure 2). One of the most important factors contributing to the efficient reduction of vitamin K to its hydroquinone form is the presence of ferroptosis suppressor protein 1 (FSP1). This is a NAD(P)H-ubiquinone reductase that is a component of the anti-ferroptosis process (53–55) (Table 1). In this respect, the analysis was conducted through flow cytometry, using BODIPY 581/591 C11 staining. Pfa1 cells were analyzed using a flow cytometer (CytoFLEX and CytExpert 2.4, Beckman Coulter) with a 488-nm laser paired with a 530/30 nm bandpass filter (56). Moreover, while the effects of 6-hydroxydopamine (6-OHDA) on the maintenance of cell viability were not significant, it was observed that the administration of vitamin K2 can protect the growth and maintenance of pheochromocytoma Cell Line 12 (PC12) cells in the Parkinson’s disease. The effects of vitamin K2 on the development and maintenance of PC12 cells have been evaluated using various methods. For instance, the number of apoptotic cells was evaluated using PI staining and flow cytometry (22). The levels of glutathione (GSH), cell viability, and apoptosis were increased in the presence of vitamin K2. However, the levels of reactive oxygen species (ROS) and bcl-2 like protein 4 (Bax) were significantly decreased in the presence of 6-OHDA (Figure 2). In one study, Sada et al. investigated the effects of vitamin K2 on the maintenance of cell viability. They found that it inhibited the activation of the apoptosis-inducing factor in the cell (24). The GATA-binding factor 1 (erythroid transcription factor) mediates cell apoptosis by activating the protein anti-B-cell lymphoma-extra-large (Bcl-XL), a factor that contributes to hematopoietic development.

Certain analyses were performed through flow cytometry, such as cell differentiation (24). In another study, Gehrmann et al. (57) found that vitamin K2 can reduce the production of reactive nitrogen species (RNS) through the NFkB-iNOS pathway. Due to its low toxicity, vitamin K has been considered a potential cancer-fighting agent (Figure 2). However, there are only a few studies that have investigated the effects of this substance on the growth and maintenance of cancer cells (21). The results of flow cytometry analysis revealed that the presence of vitamin K1 and vitamin K2 did not induce cell cycle arrest or apoptosis in PCL/PRF/5 cells (21). Also, the presence of vitamin K2 in patients with different types of leukemia can have an apoptosis-inducing effect on their newly isolated cells. Multi-color flow cytometry was the technique used (25). Moreover, it was also reported that vitamin K2 can suppress the proliferation of T-cells in osteoporotic patients. This study was conducted using flow cytometry based on carboxyfluorescein succinimidyl ester (CSFE) dye dilution. The T-cell proliferation was then determined by CFSE dye dilution of CD3 positive using an AccuriC6 flow cytometer (58) (Table 1). As such, poor bone development and an increased risk of cardiovascular disease are some of the effects of vitamin K deficiency. The rare occurrence of vitamin K toxicity is attributed to the presence of a component called menadione, which is not used in humans. When this happens, it can cause various symptoms such as jaundice and hemolytic anemia. The toxic effects of menadione can be triggered by the increased levels of oxygen that the liver takes in. This increases lipid peroxidation, which then leads to cell damage and death (59).

Vitamin B5 is water soluble and can be synthesized from the diet and intestinal microflora (63). It is found in most foods and is considered the precursor of CoA, as it participates in many metabolic reactions, especially in energy-producing reactions such as fat and carbohydrate metabolism, and less in protein metabolism (64). As a precursor of CoA and acyl carrier proteins, vitamin B5 increases mitochondrial activity by restoring CoA levels (65) (Figure 3). Therefore, coenzymes containing pantothenic acid are involved in energy reactions and are also specific to lipid metabolism (66). According to a recent study, the differentiation of some cytotoxic cells into cells that produce interleukin, e.g., IL-22, is stimulated by vitamin B5 and CoA. They are, in fact, CD8 + Tc22 cells. By exogenous administration of CoA to reprogrammed T-cells, they can adopt the CD8 + Tc22 phenotype. Tc22 cells that produce IL-22 are considered to be the cells with the strongest antitumor activity from CD8 + T-cell subsets. In mice, they were observed to have a treatment-enhancing action through programmed death-ligand 1 (PD-L1) antibodies against cancer (67).

According to current literature, the increased circulating levels of B5 should be correlated with a good state of health that is reflected in effective immune responses (63, 68). Moreover, by increasing the level of SOD in the lungs and inhibiting the accumulation of neutrophils, B5 derivatives significantly attenuate pulmonary edema (69) (Figure 3). In addition, in patients with endometriosis, B5 derivatives decrease tumor necrosis factor alpha (TNF-α) levels and reduce oxidative stress (70) (Figure 3). Also, vitamin B5, alongside other vitamins, is capable of improving mental health (71). In a study by Pourcel et al. (72) it was demonstrated by flow cytometry that cultured cells have an improved metabolic capacity in the presence of vitamin B5 compared to unexposed cells (Table 1). The percentage of fluorescent cells was determined using the CyAn ADP flow cytometer (Beckman Coulter), as well as the fluorescence intensity of GFP-positive cells. In short, the protein production and cell homeostasis state may be stimulated by the lack of vitamin B5 (72). In the case of tuberculosis, it has been demonstrated that vitamin B5 has antibacterial effects. In vivo, the growth of Mycobacterium tuberculosis was suppressed by the administration of vitamin B5. The mechanisms involved belong to innate, as well as adaptive immunity. It was discovered that the stimulation of TNF-α and IL-6 expression simultaneously with the intensification of signal molecules such as ERK and AKT can be achieved by vitamin B5. Initially, neutrophils are harvested and washed. After they are stained on ice for 30 min with mapping cell population (mAp) mixtures that were previously fluorescently conjugated or isotopes that matched the control (73). The technique used was also flow cytometry in the assessment of the phagocyte capacity of cells and could be performed after pretreatment with vitamin B5 and then with the help of fluorescent Texas-Red-labeled MTB H37Rv (74). Another area where vitamin B5 is quite used as treatment is the management of dyslipidemia. It is responsible for lowering total cholesterol and LDL (75) (Figure 3). Interestingly, there are studies that show that in pregnant or breastfeeding women, the administration of B5 in large doses may cause episodes of diarrhea or even intestinal pain (76). When there is a deficit of estrogens, a diet rich in pantothenic acid has an improving effect on the loss of bone mass. This effect was evaluated with the help of flow cytometry, which mainly analyzed cell apoptosis with several concentrations of pantothenic acid used in this study. The effects of different concentrations of pantothenic acid on cell viability were followed as Annexin V/PI Apoptosis Detection Kit (KeyGEN, Biotech) was used to obtain these results (77) (Table 1).

The vitamin includes a group of 6 chemical compounds that have the pyridine ring in common. VB6 is considered an essential micronutrient due to the incapacity of the human body and animals to synthetize it (78, 79). In contrast, VB6 can be produced by bacteria, fungi and plants. In humans, it can be assimilated from food (80). A lack of dietary variety is usually the main cause of B6 deficiency, both for animals (81) and for humans (82). Cereals, poultry, beef, and potatoes are the main sources of VB6 (82, 83). Given the importance of this vitamin for the health of all living organisms, research nowadays is testing genetic engineering techniques to create commercial crops with a greater ratio of VB6 (82, 84, 85).

As a critical co-factor, it is involved in more than 150 metabolic reactions and in cellular signaling. In addition, VB6 has the ability to decrease the final products of glycation (78, 86, 87). It was first discovered as a treatment of dermatitis in rats (88), then, with the increasingly active research of this micronutrient, it was demonstrated that several diseases like, diabetes, pneumonia, atherosclerosis, heart disease or even the resistance to COVID-19 can be connected with B6 deficiency (87, 89). Also, Field et al. revealed that supplemental VB6 increased surround suppression of visual contrast detection, decreased self-reported anxiety, and showed a trend toward depression reduction (90). Moreover, it seems that VB6 may be regarded as possible anti-tumorigenic food resource (91). Much of the research on the possible anticancer effects of VB6 is carried out by flow cytometry of utmost importance in the study of cancer (Figure 4). Using this technique, Pandey et al. (92) determined the effectiveness of transfection and poly (ester amine) gene transporter-coupled VB6 membrane transport in cancer cells. The technique was performed to see the efficiency expressed in percentages of the transfection with regard to the phosphoprotein enriched in astrocytes/t green fluorescent protein (PEA/tGFP) polyplexes and polyethyleneimine/t green fluorescent protein (PEI25k/tGFP) polyplexes with the VBPEA/GFP polyplexes (1 μg) in A549 cells. Harvested transfected cells were washed with 1× PBS. Subsequently, in order to determine the transfection percentage, the cells expressing GFP were again labeled by FACS (1 × PBS) (92). Also, flow cytometry was used to highlight that T-cell anti-tumor responses are determined by VB6 metabolism (Figure 4). Therefore, fluorochrome-conjugated monoclonal antibodies were used to stain the surface, cytoplasm, and/or nuclear components of single cell suspensions after the use of Near-IR Dead Cell Stain Kit (93) (Table 1).

Similar to B5, this vitamin is water-soluble. As it was previously mentioned, vitamin B9 is necessary to maintain regulatory T-cells (18). This vitamin may be uptaken from intestinal bacteria as well as from the diet (94). It was demonstrated that folate has two different places of absorption. The dietary form can be absorbed in the jejunum, while in the colon, there are bacteria that can synthesize folates. Thus, the bacteria from the colon represent a potential endogenous source of folates (95). Its role in the regulation of Treg cells was also evaluated by flow cytometry. Both the sorting and the actual method of flow cytometry were carried out by the following methods of Gohda et al. (96, 97) (Table 1). The first technique followed preincubation with a 10 g/ml anti-CD16/32 Ab mixture (BD Biosciences). However, in the CD3 T-cell fraction, this treatment increased the CD8 population and it is also responsible for stimulating the proliferation of nerve stem cells in the brain, which has a beneficial impact on neurodegenerative diseases (98). Moreover, vitamin B9 is capable to maintain and even proliferate the Schwan myelin-producing cells (99). Also, abnormalities of folic acid metabolism in the brain are correlated with increased apoptosis of astrocytes (Figure 5). In this respect, an annexin solution was used to stain the harvested cells. The procedure took 10–15 min, after which the cells were resuspended in the binding buffer. The detection of apoptotic cells was performed using a FACS-Calibur cytometer (BD Bioscience) (100). Unfortunately, it has been shown that this phenomenon can become favorable for the apparition of neurodegenerative disorders such as Alzheimer’s disease (101, 102). The decrease of apoptosis capacity is possible due to the folic acid ability to prevent oxidative stress. To this end, flow cytometry and Hoechst 33342 fluorescence staining were used to measure astrocyte apoptosis. The apoptotic character was determined by the condensation and fragmentation of the chromatin in the nucleus (103) (Table 1). Saxena et al. (104) observed a link between maternal folic acid levels and Autism Spectrum Disorders (ASD), due to its role as a precursor of S-adenosyl-methionine (SAM). SAM is considered the folate donor in the DNA methylation process. It has a role in maintaining amino acid homeostasis, and in the fight against reactive oxygen species, all due to its role as a cofactor in a multitude of one-carbon transfer reactions (105). In contrast, there are also some data that found a correlation between the over expression of folic acid receptors and several types of cancer, such as kidney, ovarian, and breast cancer (106–108). There is a trend nowadays to fortify certain foods with vitamin B. The negative effects of excess are not well known (109). Excess folic acid is converted in the liver under the action of dihydrofolate reductase into tetrahydrofolate. When folic acid is in excess, the enzyme is saturated, and folic acid can no longer be reduced. Thus, an excess of unreduced folic acid results, which can be considered a possible mechanism for carcinogenesis (109, 110). Moreover, excess folate can have repercussions on replication and ultimately on survival of cancer cells. In addition, when enzymes are saturated, their efficiency is reduced. Due to it, the metabolism of nutrients can be changed. Finally, the risk of some diseases can be increased (111). In contrast, an insufficient quantity of folic acid can cause DNA hypomethylation (112). Cytosine methylation in the genome is linked to the expression of certain genes (113). If the hypomethylation is specific to the promoter regions of some genes, then the control of transcription and distinctly the suppression of some genes can be modified. Thus, folic acid deficiency, which results in hypomethylation, may result in the induction of some proto-oncogenes. These are known for their role in tumorigenesis (114). Inactivation of promoter regions of suppressor genes can be caused by hypermethylation.