Improvement in vitamin B₁₂ status of Wistar rats by supplementing the diet with Chlorella vulgaris biomass

Graphic abstract

The sources of bioavailable vitamin B₁₂ are limited, and most of them are animal-derived. Chlorella vulgaris, a freshwater microalga, is known for immune system boosting, nutraceutical properties and presence of a natural form of vitamin B₁₂. The present study focused on the in vivo evaluation of the Chlorella biomass as a source of bioavailable vitamin B₁₂ to alleviate the vitamin B₁₂ deficiency status of Wistar rats. Experimental animals were evaluated for the vitamin B₁₂ deficiency-related circulatory marker (serum vitamin B₁₂) and functional markers (plasma homocysteine and urinary methylmalonic acid), haematological and histological changes. The results showed that an increase of 2.4-fold in urinary methylmalonic acid (13.01 ± 0.89 µmoles moles of creatinine⁻¹), 2.6-fold in plasma homocysteine (17.18 ± 3.57 µmole L⁻¹), and 48% decrease in serum vitamin B₁₂ levels (252.69 ± 1.46 pg mL⁻¹) in vitamin B₁₂ deficient group compared to control animals. The Chlorella biomass supplementation in the diet led to the restoration of the functional and circulatory markers, hematological parameters, and vitamin B₁₂ content of kidney and liver to control levels. The Chlorella biomass supplementation increased the erythrocyte precursors and MAST cells in the bone marrow and also normalized the histological features of kidney, liver, and lung tissues. The results suggest that the vitamin B₁₂ from the Chlorella biomass was bioavailable and facilitated the improvement of vitamin B₁₂ status in deficient rats.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04901-9) contains supplementary material, which is available to authorized users.

Introduction

Vitamin B₁₂ plays a vital role in different types of body metabolic processes, such as DNA synthesis, one-carbon metabolism, and red blood cell formation (Sandeep et al. 2019). In general, the vitamin B₁₂ requirement of the human body is low (2.4 µg day⁻¹) (Stabler and Allen 2004), but its deficiency leads to low haemoglobin levels, megaloblastic anemia, neurological dysfunction, sperms atrophy, dysfunction of the reproductive system in both male and females (Moll and Davis 2017; Tetsunori et al. 1992). Vitamin B₁₂ synthesized in the intestine is not sufficient for humans, and hence need to obtain vitamin B₁₂ through food. Most of the available vitamin B₁₂ sources are animal-derived and therefore the vegans are more prone to a risk of vitamin B₁₂ deficiency (Watanabe 2007). Commercially, vitamin B₁₂ is produced through the fermentation process using genetically engineered microorganisms (Watanabe et al. 2002; Watanabe 2007).

Few microalgae and macroalgae have been widely used as food/feed supplements due to their nutritional values (Watanabe et al. 2014). Recent studies have shown the presence of natural forms of vitamin B₁₂ (i.e., methylcobalamin, hydroxy cobalamin) in macroalgae viz. Porphyra yezoensis (nori) (VandenBerg et al. 1991) and microalgae viz. Spirulina, Chlorella, Dunaliella sp. (Kumudha and Sarada, 2015; Kumudha et al. 2015). VandenBerg et al. (1991) reported the bioavailability of vitamin B₁₂ from Spirulina and nori. Further, Watanabe et al. (2002) validated vitamin B₁₂ bioavailability from Porphyra yezoensis (nori), and Madhubalaji et al. (2019) from Spirulina in rats. However, there are no reports on the bioavailability of vitamin B₁₂ from Chlorella. Recent studies have shown that the natural form of vitamin B₁₂ (Methylcobalamin) is more bioavailable than the commercially available cyanocobalamin (Paul and Brady 2017).

Chlorella is one of the GRAS (Generally Regarded as Safe) status microalgae and is used as a food supplement world over. Chlorella is also recognized as a nutraceutical ingredient by Food Safety and Standards Authority of India (FSSAI), Government of India (FSSAI 2016). Owing to the presence of growth factor, carotenoids, protein, lipids, chlorophyll, vitamins, and other bioactive molecules, Chlorella has been shown to have antiulcer, antioxidant, and anti-tumour (Noda et al. 1996) properties. The beneficial effects such as mice reproduction, laying hen performance (Halle et al. 2009), higher nutritional value (Janczyk et al. 2005), reduction in psychological stress, protection from stress-induced ulcer (Tanaka et al. 1997) were shown with Chlorella biomass supplementation.

In the present study, Chlorella vulgaris was cultivated in 1000 L raceway pond, and the biomass was quantified for its vitamin B₁₂ content by microbiological assay and HPLC methods. The bioavailability of vitamin B₁₂ from Chlorella vulgaris biomass was validated in the rat model. Circulatory (serum vitamin B₁₂ content) and functional markers (urinary methylmalonic acid and homocysteine), were used to assess the vitamin B₁₂ deficiency mediated haematolgical, biochemical, and histological changes in experimental animals. The present study signifies the potential of Chlorella vulgaris biomass as an alternative vegan source of vitamin B₁₂.

Materials and methods

Chemicals, assay kits, columns

Cyanocobalamin (CN-B₁₂), methylcobalamin (CH₃-B₁₂) and amberlite XAD-2 column were procured from Supelco, Sigma-Aldrich (Bangalore, India). C18 Atlantis HPLC column (T 5.0 µm, 4.5 × 250 mm) and Sep-Pak cartridges were procured from Waters Corporation, Milford, MASS USA. MRS Broth and vitamin B₁₂ assay medium were obtained from Hi-Media, Bangalore, India. HPLC grade methanol and analytical grade chemicals were used. Easi-extract vitamin B₁₂ immunoaffinity column was procured from R-Biopharm Rhone Ltd, (Glasgow, UK). Rat homocysteine ELISA kit (Crystal Chem. Inc. USA) was used.

Organisms and culture

Lactobacillus delbrueckii MTCC 911 strain was procured from Microbial Type Culture Collection, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Chlorella vulgaris strain collected from the NEERI campus, Nagpur, India (Madhubalaji et al. 2020) was used in the present study (Hereafter referred to as Chlorella).

Open raceway pond cultivation and biomass characterization

Chlorella was cultivated in an outdoor open raceway pond with an operating volume of 1000 L using modified BBM media. The Chlorella biomass was harvested through a continuous centrifuge (Westfalia separator, Germany) and dried using a spray dryer (Model BE1216, Bowen, Somerville, NJ). The spray-dried biomass was analyzed for its proximate composition, and the same biomass was used for the in vivo bioavailability studies of vitamin B₁₂.

Proximate composition analysis

Total protein content (%) of the Chlorella vulgaris biomass was analyzed by using a protein analyzer (Thermo flash 2000 N/protein analyzer), moisture content of the biomass was analyzed by a moisture analyzer (Model: MA35, Sartorius, Germany). The carbohydrate content of the biomass was estimated by the phenol–sulphuric acid method (Madhubalaji et al. 2020). Total chlorophyll and carotenoids from the biomass were extracted using acetone, the absorbance of the extracts was measured at 645, 662, 470 nm, and their contents were calculated using Litchtenthaler equations (Niroula et al. 2019). Lipid was extracted with chloroform and methanol (2:1)and quantified gravimetrically as detailed earlier ( Vidyashankar et al. 2013). Ash content of the biomass was analyzed using the standard AOAC method (AOAC 2000).

Vitamin B₁₂ analysis

Extraction and purification of vitamin B₁₂ from Chlorella vulgaris biomass

Vitamin B₁₂ from spray-dried microalgal biomass was extracted by aqueous method and purified with XAD-2 and Sep-Pak columns as detailed earlier (Kumudha et al. 2015).

Detection, quantification of vitamin B₁₂ using HPLC and microbiological assay methods

The purified B₁₂ sample from Chlorella biomass was injected into the reverse phase HPLC column pre-equilibrated with methanol. The vitamin B₁₂ was eluted with a linear gradient of methanol (50% methanol containing 0.1% (v/v) acetic acid) for 53 min, with a flow rate of 1 ml min⁻¹ by using C-18 Atlantis HPLC column (T 5.0 µm, 4.5 × 250 mm) (Kumudha and Sarada 2015). The standard curve plotted with various concentrations of methylcobalamin, and B₁₂ content of the biomass sample was estimated.

Vitamin B₁₂ was quantified by using Lactobacillus delbrueckii MTCC 911 culture. The standard vitamin B₁₂ (range of 0.01–0.2 µg mL⁻¹) was prepared in distilled water and inoculated with Lactobacillus delbrueckii, and a standard curve was drawn. A purified vitamin B₁₂ extract from Chlorella vulgaris biomass, as explained earlier, was used for the quantification of vitamin B₁₂. After overnight incubation, Lactobacillus delbrueckii culture growth was measured at 600 nm using a Shimadzu spectrophotometer (UV-160A) (Kumudha et al. 2015; Watanabe et al. 2002). Based on the vitamin B₁₂ standard curve, vitamin B₁₂ content in Chlorella sample was estimated.

Animals

Twenty-four healthy albino Wistar male rats weighing 50–60 g were used. The “Institutional Animal Ethics Committee (IAEC), CSIR-CFTRI, Mysore” approved the experimental study, which followed the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Registration No: 49/Go/ReBi/s/1999/CPCSEA), Government of India, New Delhi, India. Before starting the experiment, the rats were acclimatized for seven days by giving water and standard diet ad libitum. During the acclimatization period, rats were housed in separate polypropylene cages with 2 animals per cage, maintained at 25 ± 2 °C with 12:12 h light: dark cycle, and 40–60% relative humidity. After the acclimatization period, the rats were divided into 4 groups, with six rats in each group (3 animals per cage), i.e., control group, B₁₂ deficient group, B₁₂ deficient group supplemented with Chlorella biomass at the level-1(referred to as Chlorella-1X), and B₁₂ deficient group supplemented with Chlorella biomass at the level-2 (referred to as Chlorella 2X). (refer Supplementary material Table 1 for more details).

Diet

AIN-93G composition was used for diet preparation (Reeves 1997). The control diet was prepared with a vitamin mix, including vitamin B₁₂ (Standard AIN-93 diet). Vitamin B₁₂ deficient diet was prepared with the vitamin mix devoid of Vitamin B₁₂. For Chlorella biomass supplemented diet, the vitamin B₁₂ deficient diet was mixed with a known quantity of Chlorella biomass. The amount of Chlorella supplemented was based on the vitamin B₁₂ content in the Chlorella biomass. The diet was supplemented with Chlorella biomass at two levels (1) 41.5 g Chlorella biomass per Kg diet (Chlorella 1X) to match half of the vitamin B₁₂ content of the control diet. (2) 83 g of Chlorella biomass per Kg diet (Chlorella 2X), providing an equal amount of B₁₂ as in control diet. The inclusion of the Chlorella biomass at 8.3% in diet had a negligible effect on the overall proximate composition of the diet, except for a 1–2% increase in protein and a 2–3% decrease in carbohydrates. The vitamin mix used in the preparation of diet was purchased from Hi-Media, Mumbai, India. The mineral mix was purchased from SRL, India. The diet was prepared in powdered form and stored at 4 °C. Further, cakes were made freshly from the powdered diet and fed to the rats. Each rat had free access to food and water. Weight of leftover diet and water was recorded daily. Rat body weight was recorded at regular intervals. Diet composition and vitamin mix ingredients are mentioned in the supplementary material Tables 2 and 3.

Blood collection, haematological and biochemical parameters

Animals were kept for overnight starvation before collecting the blood samples. At the end of the ten weeks treatment, the blood was drawn from CO₂ anesthetized animals by retro-orbital method for the estimation of vitamin B₁₂. At the end of the 13th week, the animals were anesthetized with CO_2, and blood samples were collected in heparinized tubes from the sacrificed animals by heart puncture and placed immediately on ice. Heparin was used as an anticoagulant. Immediately after collection, blood was subjected to haematolyzer (Model: Sysmex XP-100, Kobe, Japan) for analyzing hematological parameters viz. haemoglobin (HGB) concentration, red blood cells (RBCs), white blood cells (WBCs), Packed cell volume (PCV), Mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), Mean corpuscular haemoglobin concentration (MCHC), platelet count (PC) and leukocyte count. Plasma samples were obtained from the blood collected in heparinized tubes by centrifugation at 1000 × g for 10 min and samples were stored at − 80 °C. Serum was obtained by centrifugation of blood (at 1000 × g for 10 min) after coagulation and was used for further biochemical analysis. Selected organs were collected from the sacrificed rats and immediately washed with saline, weighed and kept at − 80 °C until further processing. The femur bone of Wistar rats was collected, cleaned, and contents were flushed into a tube containing 1 ml of fetal bovine serum and were centrifuged at 500 × g for 10 min. Further, the pellet was suspended with few drops of fresh serum; slides were prepared and air-dried. The dried slides were stained with Giemsa stain (Pugalendhi et al. 2009).

Vitamin B₁₂ content in serum, liver and kidney

Liver and kidney tissues were thawed on ice, each organ was cut into small pieces of about 1 g and homogenized using a tissue homogenizer (TH, Omni International, GA, United States) in 10 ml acetate buffer (10 mM, pH 4.8). The homogenate was incubated at 98 °C in a water bath with 20 mg of KCN for 30 min in the dark. Further, the homogenate was centrifuged at 8000 × g  for 10 min. The supernatant fraction was used for vitamin B₁₂ estimation by microbiological assay, as mentioned under vitamin B₁₂ analysis. Vitamin B₁₂ in serum was analyzed by Rat vitamin B₁₂ ELISA assay kit (KINESIS Dx, Los Angeles, USA) (Madhubalaji et al. 2019) as per the manufacturer instructions.

Functional markers and biochemical parameters

Plasma levels of homocysteine in experimental rats were determined by Rat Homocysteine (HCY) ELISA kit (KINESISDx, Los Angeles, USA), urinary Methylmalonic acid (uMMA) was analyzed by using MMA ELISA kit (E2091Ge EIAab, Wuhan, China) (Madhubalaji et al. 2019) according to the manufacturer's instructions. The biochemical parameters of liver and kidney homogenates and serum were measured by using commercially available standard biochemical assay kits (Agappe, India) that follows the modified International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) method (Madhubalaji et al. 2019). The kits were calibrated as per the manufacturer's instructions, and assays were performed.

Organ weight and histological findings

Experimental animals were sacrificed at the end of the 13 weeks and the major tissue organs viz. liver, lungs, heart, kidneys, brain, small intestine, large intestine, stomach, cecum, thyroid, testis and spleen were dissected out, washed with 0.9% chilled saline, weights were recorded and observed for lesions. A part of the kidney and liver were stored immediately at − 80 °C for the analysis of vitamin B₁₂. Primary tissues were preserved in 10% neutral buffered formalin. These tissues were fixed, processed, trimmed and embedded in paraffin sections at a thickness of 4 μ using a microtome, stained with haematoxylin and eosin, microscopically observed for histological variations (Madhubalaji et al. 2019).

Statistical analysis

Data values were presented as mean ± standard deviation (SD). Differences with probability level (p-value) < 0.05 were considered significant. All the data between groups for multiple comparisons of hematological parameters, methylmalonic acid, vitamin B₁₂ content, homocysteine and other biochemical parameters measured in serum and tissue homogenates were analyzed by one-way ANOVA and post hoc analysis (Tukey’s test) (Madhubalaji et al. 2019) using Minitab 18 software.

Results and discussion

Vitamin B₁₂ deficiency is one of the major problems faced by vegans, as B₁₂ sources are animal-derived (Watanabe 2007). Vitamin B₁₂ acts as a cofactor for 20 different types of enzymes in eukaryotes and also involved in various metabolisms viz. nucleic acid, one-carbon metabolism (Adaikalakoteswari et al. 2017), fatty acid metabolism (Pawlak et al. 2014). Chlorella is known for its growth factor and nutraceutical properties. The proximate composition of Chlorella biomass indicates protein 34% (w/w), carbohydrates 25% (w/w), total lipid 16.79% (w/w) and ash content of 7.26% (w/w) (Table 1). Vitamin B₁₂ (methylcobalamin) content in biomass was found to be the same by both HPLC and microbiological methods, which was in the range of 30–32 µg per 100 g of Chlorella biomass. In the present study, the Chlorella biomass was used as a natural source of vitamin B₁₂ (Kumudha et al. 2015) and supplemented, along with B₁₂ deficient diet to rats. In in-vivo studies, achieving complete vitamin B₁₂ deficiency is not possible due to gut microbes. Still, continuous supplementation of vitamin B₁₂ deficient diet made the partial vitamin B₁₂ deficiency onset, which was further investigated and confirmed by considering vitamin B₁₂ content, B₁₂ biomarkers, haematological, and biochemical parameters.

Table 1.

Proximate analysis of Chlorella powder

Parameters	Content
Protein (% w/w)	34.74 ± 2.41
Carbohydrates (% w/w)	25.37 ± 10.80
Total lipids (% w/w)	16.79 ± 0.92
Chlorophyll-A (mg/100 g)	766.74 ± 19.13
Chlorophyll-B (mg/100 g)	220.06 ± 23.96
Total Chlorophyll (mg/100 g)	986.81 ± 49.95
Carotenoids (mg/100 g)	338.55 ± 14.90
Moisture content (% w/w)	9.70 ± 0.61
Ash content (% w/w)	7.26 ± 3.33

Values are represented as mean ± standard deviation (significant at p < 0.05)

Rat body weight, food, and water intake

Wistar rats were supplemented with Chlorella biomass in the AIN-93 diet at 4.15 and 8.3% levels for 13 weeks. No significant difference in the mean body weight of rats was noticed among experimental groups except Chlorella 2X biomass fed group, in which a marginal decrease in weight of rats was noticed (Supplementary material Fig. 1a). Water intake and mean food intake of rats data are provided in Supplementary material Figs. 1b, c, respectively. It can be noted that deficient group rats' water and feed intake were higher compared to the control group. The feed consumption and water intake by animals of Chlorella supplemented group was less than the control group, and a marginal decrease in weight gain was observed in the Chlorella 2X fed group (Supplementary material Fig. 1a). Previous reports have shown that Chlorella has a slimming effect, and its supplementation causes a decrease in weight (Janczyk et al. 2005).

Haematological profile

The haematological profile of the experimental rats is presented in Table 2. No significant differences were observed in haematological parameters of animals in the experimental groups except for RBC and HGB. In deficient rats, a marginal decrease in HGB (14%) was observed compared to control. A decrease in haemoglobin content is considered as one of the indicators of vitamin B₁₂ deficiency (Aktas et al. 2014). However, in the Chlorella biomass fed group, haemoglobin content was found to be higher than control indicating improvement in hemoglobin with Chlorella biomass supplementation. A slight improvement in RBC count was also observed in animals of the Chlorella biomass supplemented group (Table 2). Similar results have been reported earlier (Janczyk et al. 2005).

Table 2.

Hematological profile of Wistar rats

Parameters	Control	B12 deficient	Chlorella 1X	Chlorella 2X
WBC (103 µL−1)	22.43 ± 6.70a	19.83 ± 0.40a	22.12 ± 2.82a	21.34 ± 0.37a
RBC (106 µL−1)	7.57 ± 0.35a	7.54 ± 0.56ab	8.69 ± 0.64ab	9.49 ± 0.48b
HGB (g dL−1)	14.66 ± 0.2b	12.65 ± 0.49a	17.3 ± 0.68c	17.12 ± 0.44c
HCT (% v/v)	43.33 ± 1.00a	41.05 ± 1.69a	52.23 ± 1.68a	51.22 ± 0.99a
MCV (fL)	53.2 ± 1.24a	54.96 ± 1.20a	53.23 ± 0.98a	54.03 ± 1.45a
MCH (pg)	18.2 ± 0.26a	18.23 ± 0.15a	18.2 ± 0.43a	17.9 ± 0.45a
MCHC (g dL−1)	33.16 ± 0.05a	34.33 ± 0.20a	33.14 ± 0.29a	33.1 ± 0.17a
PLT (103 µL−1)	442 ± 10.51a	406 ± 8.76a	452 ± 24.22a	389 ± 17.34a
LYM (% v/v)	82.43 ± 1.16a	75.56 ± 2.51a	79.63 ± 0.45a	79.5 ± 5.03a
LYM COUNT (103 µL−1)	18.46 ± 5.35a	15.8 ± 0.43a	16.9 ± 2.10a	16.96 ± 2.13a

Values are average of minimum 3 animals and represented as mean ± standard deviation, 1X indicates 41.3 g of Chlorella per kg diet, and 2X indicates 83 g of Chlorella per kg diet

HCT Hematocrit, HGB Hemoglobin, MCH Mean corpuscular haemoglobin, MCHC Mean corpuscular haemoglobin concentration, MCV Mean corpuscular volume, PC Platelet count, PCV Packed cell volume, RBC Red blood cells, WBC White blood cells

^abcMean values with different superscript were significantly different (p < 0.05)

Microscopic observation of bone marrow cells

Bone marrow cells collected from femur bone were fixed on the slides, stained by Giemsa stain, and observed under a microscope (Pugalendhi et al. 2009). Bone marrow cells imaging showed an increase in the erythrocyte precursors and MAST cells in the Chlorella biomass fed group compared to the control group (Fig. 1), suggesting Chlorella biomass facilitated the production of more erythrocyte precursors.

Fig. 1.

a Increased MAST cells in the bone marrow of Chlorella biomass supplemented group. b Increased erythrocyte precursors in the bone marrow of Chlorella biomass supplemented group. c Bone marrow cells of the control group

Biochemical parameters

Serum biochemical parameters viz. creatinine, glucose, iron, total protein, alkaline phosphatase, SGOT, SGPT, cholesterol, triglycerides, ferritin, transferrin, and urea were analyzed, and results are presented in Table 3. No significant difference in creatinine, total protein, alkaline phosphatase, SGOT, SGPT, transferrin levels were observed among the experimental groups. However, an increase in triglyceride (37%), cholesterol (21%) levels in vitamin B₁₂ deficient group was observed (Table 3). Vitamin B₁₂ deficiency might have increased the expression levels of adipo/lipogenic genes and altered miR, which causes the accumulation of triglycerides (Adaikalakoteswari et al. 2017). Vitamin B₁₂ regulates the SERBF1 and LDLR genes (Adaikalakoteswari et al. 2015), and deficiency of vitamin B₁₂ may caused an increase in the level of cholesterol. Whereas in Chlorella biomass supplemented vitamin B₁₂ deficient groups, the cholesterol levels were comparable to control. A 23% increase in glucose levels was observed in the vitamin B₁₂ deficient group. Ling and Chow (1954) reported that vitamin B₁₂ regulates carbohydrate metabolism, and vitamin B₁₂ deficiency causes an increase in blood glucose levels. Chlorella biomass supplementation to the diet of vitamin B₁₂ deficient group led to lowering of the glucose levels to even lower than the control animals (Table 3). Iron and vitamin B₁₂ are interdependent (Moll and Davis 2017), and in B₁₂ deficient rats, a significant decrease of iron (25%) was observed. Chlorella is rich in iron content, and an increase in iron content (11%) was observed with Chlorella biomass supplementation compared to control, indicating that iron from Chlorella biomass is bioavailable in experimental rats. Similarly, vitamin B₁₂ affects the alkaline phosphatase metabolism (Kim et al. 1996). Vitamin B₁₂ deficiency resulted in a slight decrease in alkaline phosphatase (17%) compared to the control group. Overall, Chlorella biomass supplementation resulted in lower levels of glucose, and creatinine, while higher levels of iron and urea compared to the control group. Similar results have been reported previously with Chlorella biomass supplementation (Janczyk et al. 2005).

Table 3.

Effect of Chlorella biomass feeding on serum biochemical parameters of Wistar rats

Parameters	Control	Vitamin B12 deficient	Chlorella 1X	Chlorella 2X
Creatinine (mg dL−1)	1.87 ± 0.004a	1.87 ± 0.03a	1.89 ± 0.01a	1.76 ± 0.08b
Glucose (mg dL−1)	118.56 ± 28.92b	146.01 ± 5.46a	107.61 ± 27.39c	84.28 ± 9.35c
Iron (µg dL−1)	204.19 ± 12.06b	153.70 ± 12.34a	228.41 ± 8.92b	222.22 ± 18.39b
Total protein (g dL−1)	8.41 ± 0.17a	9.21 ± 0.68a	7.32 ± 0.36a	8.09 ± 0.88a
Alkaline Phosphatase Activity (U L−1)	648.57 ± 32.47a	541.01 ± 78.27a	596.47 ± 88.83a	637.26 ± 10.46a
SGOT Activity (U L−1)	52.54 ± 6.97a	55.63 ± 14.66a	52.83 ± 34.06a	53.53 ± 17.67a
SGPT Activity (U L−1)	24.72 ± 0.96a	24.02 ± 7.82a	24.02 ± 4.19a	28.23 ± 7.42a
Cholesterol (mg dL−1)	128.33 ± 8.03b	156.23 ± 3.26c	92.38 ± 6.77a	123.56 ± 14.74b
Triglycerides (mg dL−1)	137.23 ± 7.73a	188.48 ± 7.90c	145.10 ± 8.93a	163.94 ± 5.39b
Ferritin (ng mL−1)	50.33 ± 3.78bc	52.49 ± 1.8c	45.33 ± 6.63ab	40.33 ± 4.48a
Transferrin (mg dL−1)	14.88 ± 0.75a	15.61 ± 1.64a	14.14 ± 1.7a	14.5 ± 1.36a
Urea (mg dL−1)	20.41 ± 0.38a	20.46 ± 0.32a	30.25 ± 1.43c	24.64 ± 0.05ab

Values are average of minimum 3 animals and represented as mean ± standard deviation, 1X indicates 41.3 g of Chlorella per kg diet, and 2X indicates 83 g of Chlorella per kg diet

^abcMean values with different superscript were significantly different (p < 0.05)

Vitamin B₁₂ content in liver and kidney

Vitamin B₁₂ is primarily stored in the liver and kidney organs. Kidneys have a vital role in vitamin B₁₂ regulation. Therefore, vitamin B₁₂ content accumulated in the liver and kidney could serve as one of the vitamin B₁₂ bioavailability indicators. The vitamin B₁₂ content in the liver and kidney tissue of animals of all the groups are presented in Fig. 2b. The control group showed the vitamin B₁₂ content of 9.86 ± 0.55 ng g⁻¹ and 15.98 ± 2.30 ng g⁻¹ in liver and kidney tissues, respectively. The vitamin B₁₂ deficient group showed 1.76 fold (5.60 ± 0.13 ng g⁻¹), and 1.85 fold (8.622 ± 0.48 ng g⁻¹) lowered vitamin B₁₂ contents, respectively, in their liver and kidney tissues. The vitamin B₁₂ levels in the liver and kidney tissues of the group supplemented with Chlorella biomass were similar to the control group, i.e., 10.02 ± 0.32 and 15.25 ± 0.52 ng g⁻¹ respectively at the 1X level of supplementation and 10.03 ± 0.054 and 15.66 ± 0.16 ng g⁻¹ respectively at 2X level of supplementation. The results suggest that the vitamin B₁₂ from Chlorella biomass was bioavailable to rats. The lower vitamin B₁₂ content in liver and kidney tissues of B₁₂ deficient rats was reported earlier (Madhubalaji et al. 2019). Earlier reports showed that the accumulation of vitamin B₁₂ in the liver and kidney tissues might indicate true and pseudo forms (VandenBerg et al. 1991). However, Yetley et al. (2011) reported that circulatory (serum vitamin B₁₂) and functional markers (uMMA, Hcy) are true indicators of vitamin B₁₂ deficiency. Therefore to acquire more accuracy and confirmation on the bioavailability of vitamin B₁₂ from Chlorella biomass, circulatory as well as functional markers of vitamin B₁₂ were determined in the present study.

Fig. 2.

a Vitamin B₁₂ content in serum, b Vitamin B₁₂ content in kidney and liver tissue homogenates, c Homocysteine levels in serum and plasma, d urinary Methylmalonic acid (uMMA) levels of experimental rats, Values are expressed as mean ± SD (p ≤ 0.05), 1X indicates 41.3 g of Chlorella per kg diet and 2X indicates 83 g of Chlorella per kg diet

Circulatory marker (Serum vitamin B₁₂ content)

Vitamin B₁₂ contents in the serum of experimental rats are presented in Fig. 2a. The serum vitamin B₁₂ level was found to be 466.88 ± 28.36 pg mL⁻¹ in the control group, while it was 50% less than control in the B₁₂ deficient group (202 ± 32 pg mL⁻¹). The serum vitamin B₁₂ levels in the groups supplemented with Chlorella biomass at 1X, and 2X levels were found to be comparable to the control group with values of 448.58 ± 13.55 and 449.08 ± 15.18 pg mL⁻¹, respectively. Herrmann and Obeid (2008) reported that serum vitamin B₁₂ is an insensitive and unspecific indicator of vitamin B₁₂ deficiency , and it determines the vitamin B₁₂ status outside of the cells. So, functional markers (viz. methylmalonic acid and homocysteine), which indicate the true intracellular form of vitamin B₁₂ were treated as key indicators of vitamin B₁₂ deficiency.

Functional markers of vitamin B₁₂

Plasma, serum homocysteine levels

The onset of vitamin B₁₂ deficiency is indicated by elevated levels of functional markers viz. methylmalonic acid (Adaikalakoteswari et al. 2017) and homocysteine (Madhubalaji et al. 2019). Plasma and serum homocysteine levels are presented in Fig. 2c. The plasma homocysteine levels were elevated in the vitamin B₁₂ deficient group (17.18 ± 3.57 µmol L⁻¹) compared to the control group (6.55 ± 0.0515 µmol L⁻¹). Similar results of increased homocysteine levels in vitamin B₁₂ deficient conditions were reported earlier (Madhubalaji et al. 2019). In Chlorella biomass supplemented deficient group of animals, plasma homocysteine levels were similar to the control rats (6.53 ± 0.18 µmol L⁻¹). A similar trend was observed in the serum homocysteine levels, as presented in Fig. 2c. The restoration of normal levels of homocysteine indicates the recovery of vitamin B₁₂ deficiency in animals whose vitamin B₁₂ deficient diet was supplemented with Chlorella biomass. Piyathilake et al. (2000) have reported that along with vitamin B₁₂, folate and pyridoxine are also involved in the conversion of homocysteine to methionine. The Chlorella biomass has been reported to have folate (Edelmann et al. 2019). Therefore the restoration of homocysteine levels in the deficient group on supplementation with Chlorella biomass could be due to the mutual effect of vitamin B₁₂ and folate available in the Chlorella biomass. Hence, to confirm that the vitamin B₁₂ from Chlorella biomass was indeed bioavailable to animals, one more functional marker of vitamin B_12, i.e., uMMA was studied.

Urinary MMA levels

In methyl malonyl Co-A Mutase to succinyl Co-A reaction, vitamin B₁₂ acts as a cofactor (Kamath 2017). In the absence of vitamin B₁₂, the conversion of methyl malonyl Co-A Mutase to succinyl Co-A becomes less, which results in elevated levels of methylmalonic acid (Adaikalakoteswari et al. 2015, 2017). Hence, methylmalonic acid is considered as a key functional marker of vitamin B₁₂ deficiency. Urinary methylmalonic acid levels are presented in Fig. 2d. The urinary MMA levels were elevated in the vitamin B₁₂ deficient group (13.01 ± 0.89 µmol /mole of creatinine) compared to the control group (5.33 ± 0.04 µmol mole of creatinine⁻¹). In the case of Chlorella biomass supplemented groups at 1X and 2X level, the uMMA levels of 5.27 ± 0.05 µmol mole of creatinine⁻¹ and 5.34 ± 0.12 µmol mole of creatinine⁻¹ respectively were observed, which were similar to control.

In vitamin B₁₂ deficient group, higher homocysteine levels of plasma, serum and urinary methylmalonic acid were correlated with lower vitamin B₁₂ content in serum, plasma, liver and kidney, indicating the vitamin B₁₂ deficient status of the animals. Whereas in the Chlorella biomass supplemented deficient groups, both the plasma and serum homocysteine and uMMA levels were similar to control. The vitamin B₁₂ content in serum, plasma, kidney, and liver of Chlorella biomass supplemented rats were comparable to control. Results confirm the availability of vitamin B₁₂ to Wistar rats from Chlorella biomass. In summary, biochemical parameters, circulatory and functional markers levels of deficient groups supplemented with Chlorella biomass at the two concentrations were comparable to control, suggesting that Chlorella supplementation at 1X level itself was sufficient to alleviate the vitamin B₁₂ deficiency. Therefore, Chlorella biomass has potential towards application as a food supplement to improve the vitamin B₁₂ status.

Relative organ weight

No significant differences were observed in the weight of the kidney, heart, adrenal gland, brain, lungs, small intestine, and large intestine among the experimental groups compared to the control group (Supplementary material Table 4). However, in deficient rats, 13% increase in weight of the liver was observed compared to control. A similar increase of liver weight in vitamin B₁₂ deficient animals has been previously reported by various researchers (Dryden and Hartman 1966; Tanaka et al. 1997; Tetsunori et al. 1992). Further, in vitamin B₁₂ deficient group, a slight (1.15-fold) increase in the thyroid gland compared to control was observed. Similar observations have been reported previously (Dryden and Hartman 1966). In case of the cecum, 41% and 97% increase in the weight was observed with Chlorella biomass supplementation at 1X and 2X levels, respectively, compared to the control group, which was in line with earlier reports (Janczyk et al. 2005).

Histological observations

Histo-micrographs of different organs indicated no significant changes or abnormalities in their histology except the liver, kidney, and lungs (Fig. 3). In vitamin B₁₂ deficient group, moderate to severe cytoplasmic degeneration hypertrophy and also necrosis were observed in liver tissue, as shown in Fig. 3a ii, whereas mild hypertrophy and cytoplasmic vacuolation were observed in Chlorella biomass fed group. In kidney histology, mild glomerular distention and moderate epithelial cell degeneration were seen in the deficient group (Fig. 3b ii). In the Chlorella biomass fed group, these features of kidney histology were similar to the control group. Severe peribronchial cellular infiltration and congestion were observed in the lungs of the B₁₂ deficient group, as shown in Fig. 3c ii. Mild cellular infiltration was found in the lungs of Chlorella biomass supplemented rats. In the case of other organs, the histology of the Chlorella biomass fed group was almost similar to the control group. No pathology was found in the control group.

Fig. 3.

a Liver tissue histo-micrographs (20X) (i) control (ii) Deficient—Moderate to severe cytoplasmic degeneration, hypertrophy and also necrosis (iii) Chlorella 1x—Mild to moderate cytoplasmic vacuolation (iv) Chlorella 2X-No Pathology/abnormality detected. b Kidney tissue histology photographs (20X) (i) Control (ii) Deficient—Mild glomerular distention and moderate epithelial cell degeneration (iii) Chlorella 1X—Mild glomerular distention and moderate epithelial cell degeneration (iv) Chlorella 2X—Moderate glomerular distention and tubular dilation. c Lungs tissue histology photographs (Magnification: 4X) (i) Control (ii) Deficient—severe peribronchial cellular infiltration and congestion (iii) Chlorella 1X—Moderate peribronchial cellular infiltration (iv) Chlorella 2X—Mild peribronchial cellular infiltration

Conclusion

Vitamin B₁₂ deficiency caused changes in circulatory marker (serum vitamin B₁₂), functional markers (uMMA, homocysteine), haematological and biochemical parameters. Vitamin B₁₂ deficiency mediated changes in Wistar rats were normalized by feeding the diet supplemented with Chlorella biomass. Besides, supplementation of Chlorella biomass improved blood glucose levels, erythrocyte precursors, MAST cells, and increased the cecum size. The results substantiate the bioavailability of vitamin B₁₂ from Chlorella biomass, which can form an alternate source of vitamin B₁₂ for vegans. Based on the present study, the clinical trials with human subjects can be undertaken to establish the bioavailability of vitamin B₁₂ from Chlorella, which is having GRAS status and is recognized as nutraceutical by FSSAI, Government of India.

Electronic supplementary material

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Abbreviations

AIN

American Institute of Nutrition

CPCSEA

Committee for the Purpose of Control and Supervision of Experiments on Animals

GRAS

Generally Regarded As Safe

IAEC

Institutional Animal Ethics Committee

IFCC

International Federation of Clinical Chemistry and Laboratory Medicine

HCT

Hematocrit

Hcy

Homocysteine

HGB

Hemoglobin

KCN

Potassium cyanide

MCH

Mean corpuscular haemoglobin

MCHC

Mean corpuscular haemoglobin concentration

MCV

Mean corpuscular volume

MMA

Methyl malonic acid

PC

Platelet count

PCV

Packed cell volume

RBC

Red blood cells

SGOT

Serum glutamic oxaloacetic transaminase

SGPT

Serum glutamic pyruvic transaminase

WBC

White blood cells

Funding

The authors thank the DBT, Govt. of India, New Delhi, India, for providing a research Grant (BT/PR10658/PFN/20/806/2013) to carry out this study.