Profiling immuno-metabolic mediators of vitamin B12 deficiency among metformin-treated type 2 diabetic patients in Ghana

Samuel Asamoah Sakyi; Edwin Ferguson Laing; Richard Mantey; Alexander Kwarteng; Eddie-Williams Owiredu; Richard Ephraim Dadzie; Benjamin Amoani; Stephen Opoku; Bright Oppong Afranie; Daniel Boakye

doi:10.1371/journal.pone.0249325

. 2021 Mar 30;16(3):e0249325. doi: 10.1371/journal.pone.0249325

Profiling immuno-metabolic mediators of vitamin B₁₂ deficiency among metformin-treated type 2 diabetic patients in Ghana

Samuel Asamoah Sakyi ^1,^*, Edwin Ferguson Laing ¹, Richard Mantey ¹, Alexander Kwarteng ², Eddie-Williams Owiredu ¹, Richard Ephraim Dadzie ³, Benjamin Amoani ⁴, Stephen Opoku ⁵, Bright Oppong Afranie ¹, Daniel Boakye ⁶

Editor: Benjamin Udoka Nwosu⁷

¹Department of Molecular Medicine, School of Medicine and Dentistry, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

²Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

³Department of Medical Laboratory Technology, Faculty of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana

⁴Department of Biomedical Science, School of Allied Health Sciences, University of Cape Coast, Cape Coast, Ghana

⁵Department of Medical Diagnostics, Faculty of Allied Health Sciences, College of Health Sciences, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana

⁶Department of Epidemiological Methods and Etiological Research, Leibniz Institute for Prevention Research and Epidemiology (BIP-S), Bremen, Germany

⁷University of Massachusetts Medical School, UNITED STATES

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: samasamoahsakyi@yahoo.co.uk

Roles

Samuel Asamoah Sakyi: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing – original draft, Writing – review & editing

Edwin Ferguson Laing: Conceptualization, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing

Richard Mantey: Conceptualization, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing

Alexander Kwarteng: Conceptualization, Formal analysis, Investigation, Methodology, Resources, Writing – original draft, Writing – review & editing

Eddie-Williams Owiredu: Conceptualization, Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing

Richard Ephraim Dadzie: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing

Benjamin Amoani: Conceptualization, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing

Stephen Opoku: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing

Bright Oppong Afranie: Data curation, Formal analysis, Methodology, Validation, Writing – original draft, Writing – review & editing

Daniel Boakye: Conceptualization, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing

Benjamin Udoka Nwosu: Editor

PMCID: PMC8009370 PMID: 33784336

Abstract

Background

The association between prolong metformin usage and B12 deficiency has been documented. However, the prevalence estimates of metformin-induced vitamin B12 deficiency showed substantial disparity among studies due to varied study definitions of vitamin B12 deficiency. Metformin blocks the calcium dependent absorption of the vitamin B12-Intrinsic Factor complex at the terminal ileum. Lack of intrinsic factor due to the presence of auto-antibodies to parietal cells (IFA) could lead to vitamin B12 deficiency and subsequently cause peripheral neuropathy. We investigated the prevalence of vitamin B12 deficiency using more sensitive, combined markers of vitamin B12 status (4cB12) and the immuno-biochemical mediators of vitamin B12 deficiency.

Methods

In this observational study, 200 consecutive consenting metformin-treated T2DM patients, aged 35 and above, attending the diabetic clinic at KATH were recruited. Vitamin B₁₂ deficiency was classified based on the Fedosov age-normalized wellness quotient. Anthropometric measurement was taken as well as blood samples for immunological and biochemical mediators. Peripheral neuropathy was assessed using the Michigan Neuropathy Screening Instrument (MNSI). Statistical analysis was performed using the R Language for Statistical Computing.

Results

Using the combined indicator (4cB₁₂), the prevalence of metformin induced vitamin B12 deficiency was 40.5% whilst the prevalence of MNSI-Q and MNSI-PE diabetic neuropathy was 32.5% and 6.5% respectively. Participants with vitamin B₁₂ deficiency had significantly higher levels of IFA, GPA, TNF-α, TC, LDL and albumin compared to those with normal vitamin B₁₂ levels (p < 0.05). Correlation analysis revealed a statistically significant negative association between 4cB₁₂ and the immunological markers [IFA (rs = -0.301, p<0.0001), GPA (rs = -0.244, p = 0.001), TNF-α (rs = -0.242, p = 0.001) and IL-6 (rs = -0.145, p = 0.041)]. Likewise, 4cB₁₂ was negatively associated with TC (rs = -0.203, p = 0.004) and LDL (rs = -0.222, p = 0.002) but positively correlated with HDL (rs = 0.196, p = 0.005).

Conclusion

Vitamin B12 deficiency and diabetic neuropathy are very high among metformin-treated T2DM patients and it is associated with increased GPA, IFA, TNF-α and cardiometabolic risk factors (higher LDL and TC and lower HDL). Upon verification of these findings in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12) in the management of patients with T2DM treated with metformin in Ghana.

Introduction

Prevalence of Diabetes Mellitus (DM) has risen to epidemic proportions in both developed and developing countries. Sub-Saharan Africa is the worse hit with an expected projection of 23.9 million DM cases in the next three decades [1]. Type 2 Diabetes Mellitus (T2DM) is the most abundant form of DM, and accounts for approximately 90% of all DM cases [2]. In Ghana, the disease has affected an estimated 6% of the urban populace [3].

Updated clinical treatment guidelines, including that of the American Diabetes Association, and the European Association for the Study of Diabetes propose that metformin should be initiated with concurrent lifestyle modifications [4–6]. Metformin enhances glucose tolerance in patients with T2DM by lowering basal and postprandial plasma levels of glucose [7]. Serum levels of vitamin B₁₂ have been reported to be inversely linked with the duration of T2DM and dose of metformin [1, 3, 8], with an average of 10 to 30% of patients exhibiting malabsorption of vitamin B₁₂ [9, 10].

The American Diabetes Association guidelines now recommend periodic evaluation for vitamin B12 deficiency in patients taking metformin [11]. However, measurement of vitamin B₁₂ is not done periodically among T2DM patients in Ghana even though a recent study has indicated a high prevalence of metformin-induced vitamin B₁₂ deficiency in Ghana [12]. T2DM patients with peripheral neuropathy, who have been treated with metformin for more than 6 months had lower serum vitamin B₁₂ and a more severe clinical peripheral neuropathy [13]. This implies that vitamin B₁₂ deficiency-induced peripheral neuropathy may be confused with Diabetic Peripheral Neuropathy (DPN) in the absence of the assessment of B₁₂ levels [14–16].

Determination of B12 status and results interpretation is not very forthright since different B12 evaluation methods have different specificity and accuracy. Vitamin B12 deficiency is diagnosed by measurements of total serum B12 (sB12), methylmalonic acid (MMA), holo-transcobalamin (holoTC), and total homocysteine (Hcy). The use of individual markers has been demonstrated to be an inadequate reflection of true vitamin B₁₂ status. Against this, Fedosov proposed the utilization of combined indicator of vitamin B₁₂ status using two or more of the following markers: sB12, MMA, holoTC and Hcy [17]. However, most studies use only one or two of these biochemical tests in defining vitamin B12 status, leading to contradictory and inconsistent prevalence. It is thus imperative to use combined direct markers of serum vitamin B12 status (sB12 and hTC) and the metabolic markers (MMA and Hcy) (ω; combined indicator of vitamin B12 status using all four indicators (4cB12) in defining vitamin B12 deficiency) [17].

Gastric parietal cells secret intrinsic factor (IF) which plays a vital role in the absorption of vitamin B₁₂ in humans. It has been reported that metformin blocks the calcium dependent absorption of the vitamin B₁₂-Intrinsic Factor complex at the terminal ileum [18, 19]. Impaired IF production can also occur when there is autoimmune destruction of parietal cells in the context of type 1 diabetes. Lack of IF due to the presence of intrinsic factor antibodies (IFA) could lead to vitamin B₁₂ deficiency and subsequently cause peripheral neuropathy, megaloblastic or pernicious anemia [13, 20]. Furthermore, patients with positive anti-gastric parietal cell antibodies (GPA) may present with vitamin B₁₂ deficiency. However, in a study by Wang et al [20], only about 12.9% of GPA positive patients actually had pernicious anemia in accordance with the WHO definition. This suggests that a greater proportion of GPA positive cases may have been precipitated by other conditions. Yet, the role of these immunological mediators in vitamin B12 deficiency among metformin treated T2DM patients has not been evaluated. Additionally, B₁₂ deficiency has been shown to be linked with elevated levels of pro-inflammatory cytokines (such as tumor necrosis factor-alpha (TNF-α) as and biochemical markers related to cardiometabolic risk (glycemia, insulin resistance and lipid profile parameters) in T2DM [21, 22]. Moreover, metformin has been shown to inhibit IL-6 signaling via decreasing IL-6 receptor expression and primary adipocytes cultured in low vitamin B12 conditions showed increased gene expression of pro-inflammatory cytokines including interleukin-6 (IL-6) [23, 24]; however, the relationship is not fully explored in humans.

Thus, studies assessing the relationship between GPA, IFA, TNF-α, IL-6 as well as cardiometabolic risk factors and vitamin B12 deficiency among metformin-treated T2DM patients are warranted. Identifying these relationships might elicit specific interventions which may preclude further complications in these patients. This study thus investigated vitamin B12 deficiency using the more sensitive combined indicator of vitamin B₁₂ status (4cB12) and evaluated the immune-metabolic mediators associated with B₁₂ deficiency among clinically-diagnosed T2DM treated with metformin. The findings from this study will sensitize clinicians to assess metformin-treated T2DM patients for vitamin B₁₂ deficiency and related immuno-biochemical disorders to optimize care for these patients.

Materials and methods

Study design

This hospital-based observational study was conducted at the diabetic clinic of the Komfo Anokye Teaching Hospital (KATH) in the Ashanti Region of Ghana, from January 2018 to March 2019. KATH has approximately one thousand-bed facility and serves as a main referral point for parts of the northern and middle belts of Ghana. The hospital has a diabetic clinic which is frequented by more than 100 patients per week [25].

Ethical consideration

This study was approved by the Committee on Human Research Publication and Ethics (CHRPE) of the School of Medicine and Dentistry (SMD), Kwame Nkrumah University of Science and Technology (KNUST) (CHRPE/AP/013/18) and the ethical review board of KATH. Participation was voluntary and written informed consent was obtained from each participant after the aims and objectives of the study had been explained to them.

Study population

Two hundred (200) patients diagnosed with T2DM attending the diabetic clinic at KATH and who gave informed their consent were enrolled into the study. Since T2DM is predominantly a disease of the old, we included participants who were above 35 years of age.

Inclusion and exclusion criteria

Clinically diagnosed T2DM patients treated with metformin, aged 35 years and above receiving medical care at the out patients’ department of the diabetic unit of KATH were recruited onto the study. The diagnosis of type 2 diabetes mellitus was made based on American Diabetes Association (ADA) criteria (based on defective progressive insulin secretory on the background of insulin resistance using fasting plasma glucose ≥7.0 mmol/L; glycated hemoglobin ≥6.5%; oral glucose tolerance test 11.1 mmol/L) [26]. Patients with history of anemia, blood transfusion, thyroid illness, chronic alcoholism, renal insufficiency, gastric surgery as well as patients on chronic parenteral or enteral nutritional support, on B₁₂ supplementation (parenteral or oral), proton pump inhibitors, malabsorption syndrome, liver disease, vegetarians and pregnant women were excluded from this study. Patients with type 1 diabetes (diagnosed based on high plasma glucose in addition to autoimmune indicators such as autoantibodies to insulin, autoantibodies to glutamate decarboxylase (GAD65)) were excluded.

Questionnaire administration

Validated structured questionnaires were administered to gather information on demo-graphic characteristics, anthropometric variables, medications history including dosage, duration and symptoms of neuropathy. Patients’ medical records were retrieved from the hospital’s archive and reviewed to obtain additional relevant information such as medications used.

Assessment of peripheral neuropathy using the Michigan Neuropathy Screening Instrument (MNSI)

Neuropathy was assessed using the MNSI. The MNSI history questionnaire was self-administered and consists of responses that are collated to obtain a total score as described by Feldman et al. [27]. A score of ≥ 7 was considered abnormal.

Physical assessment

In all assessments, we ensured that the foot was warm (>30°C). During the MNSI assessment, a healthcare professional inspected both feet for malformations, calluses, dry skin, fissures and infections. Other deformities such as overlapping toes, joint subluxation, halux valgus, hammer toes, prominent metatarsal heads, flat feet, amputation and medial convexity (Charcot foot) were also assessed. Any abnormality observed on each foot (including ulcerated foot) received a score of 1.

Vibration sensation

Vibration sensation was assessed bilaterally using a 128 Hz tuning fork placed over the dorsum of the great toe on the boney prominence of the distal interphalangeal joints (DIP) joint. Patients (with eyes closed) were asked to indicate when they could no longer sense the vibration from the tuning fork. If the examiner felt vibration for 10 or more seconds on his or her finger, then vibration was considered decreased. Vibration was scored as 1) present if the examiner sensed the vibration on his or her finger for < 10 seconds, 2) reduced if sensed for ≥ 10 or 3) absent (no vibration detected.)

Muscle stretch reflexes

The ankle reflexes were examined using an appropriate reflex hammer (e.g. Trommer or Queen square). The ankle reflexes were elicited in a sitting position with the foot when the patient is relaxed. For the reflex, the foot was passively positioned and the foot dorsiflexed slightly to obtain optimal stretch of the muscle. The Achilles tendon was tapped directly. If the reflex was obtained, it was graded as present. If the reflex was absent, the patient was asked to perform the Jendrassic maneuver (i.e., hooking the fingers together and pulling). Reflexes elicited with the Jendrassic maneuver alone were designated “present with reinforcement.” If the reflex was absent, even with the Jendrassic maneuver, the reflex was considered absent. The total possible score is 8 points and, by the scoring algorithm we used, a score greater than or equal to 2.5 was considered abnormal [27].

Blood pressure measurement

Blood pressure was measured using a calibrated sphygmomanometer with appropriate cuff sizes and stethoscope in accordance with the recommendation of the American Heart Association (American Heart Association 2012). The measurement was taken with participants in sitting position and after having rested for at least 10 min. Triplicate readings were taken 5 min apart and the mean value was recorded to the nearest 1.0 mm Hg.

Measurement of Body Mass Index (BMI)

The height of the participants was measured to the nearest meter using a wall-mounted ruler and weight was measured in light clothing without shoes, and in an upright position (to the nearest 0.1 kg) using a bathroom scale (BR9012, Zhongshan Camry Electronic Co. Ltd, Guangdong, China). BMI was calculated as weight (kg) divided by height squared (m²).

Blood sample collection and processing

Five milliliters (5ml) of venous blood sample was collected from each participant into both serum gel separator tube (3ml) and EDTA tubes (2ml). EDTA samples were processed for analysis immediately for hemogram and samples in serum gel separator tube were centrifuged at 1500 rpm for 3 min. The serum was stored in cryovials at −80°C until vitamin B12, and methyl malonic acid (MMA) assays were performed.

Measurement of hematological parameters

Both thick and thin blood films were performed by standard protocols as described by [28]. Full blood count (FBC) was estimated using an automatic hematological analyzer (SYSMEX XP-2000i, Japan).

Biochemical assays

Serum samples collected from the participants were analyzed for the concentrations of vitamin B₁₂, GPA and IFA by solid phase sandwich ELISA using the DuoSet ELISA kit (R&D Systems, Inc., USA) according to the manufacturer’s instructions. To improve the diagnosis of vitamin B₁₂ deficiency, methylmalonic acid and homocysteine blood levels were also measured, as elevated levels are sensitive indicators of tissue vitamin B₁₂ deficiency [29]. These markers of vitamin B₁₂ deficiency have been shown to improve the sensitivity and specificity for detecting vitamin B₁₂ deficiency especially among type 2 diabetic patients with borderline serum vitamin B₁₂ concentrations of 200–400 pg/ml and subtle haematological manifestations [30]. Positive GPA was defined as values >20 pmol/l whereas IFA positivity was also defined by >1.53 AU/mL.

Classification for vitamin B₁₂ deficiency

Vitamin B₁₂ deficiency was classified based on the Fedosov age-normalized wellness quotient [ω; combined indicator of vitamin B₁₂ status using all four indicators (4cB₁₂)] [17]. The wellness score was calculated according the formula: ω (4cB₁₂) = log₁₀[(sB₁₂*hTC)/(MMA*Hcy)]_test−[lr_norm/(1- (Age/230)^2.6)], where the direct markers of vitamin B₁₂ status (sB₁₂ and hTC) are reported in pmol/L and the metabolic markers (MMA and Hcy) are reported in μmol/L. The test portion of the equation refers to the tests of the participant. The second component of the equation describes the “normal” logarithmic ratio (lr_norm) predicted based on the age of the participant. The absolute values of the correction factor lr_norm decrease with increasing age: for age ≤ 40 years, the lr_norm = 3.750; 41–60 years (3.678); 61–80 years (3.561) and >80 years (3.436). Participants with ω< -0.5 were considered vitamin B₁₂ deficient in accordance with previous studies [17, 31, 32].

Statistical analysis

Statistical analysis was performed using the R Language for Statistical Computing version 3.6.0 [33]. Categorical data were presented as frequencies (percentages). For continuous data, normality was checked using Shapiro-Wilk’s test, as well as visual inspection with Q-Q plots. Parametric and nonparametric data were presented as mean ±SD and median (interquartile ranges), respectively. Distribution of the indicators of vitamin B₁₂ (4cB₁₂, sB₁₂, hTC, MMA, Hcy) were presented with density plots. Comparison of immuno-metabolic parameters based on vitamin B₁₂ status was performed using one-way analysis of covariance (ANCOVA) with adjustment for age, sex, BMI, duration of diabetes, and dosage and duration metformin therapy. Hierarchical clustering by Spearman’s correlation was used to assess relationship between the indicators of vitamin B₁₂ and immuno-metabolic parameters. Logistic regression analysis was used to evaluate the association between vitamin B12 status and diabetic neuropathy. All tests were two-sided and p-value < 0.05 was considered statistically significant.

Results

A total of 200 diabetic patients with mean age of 59.00±9.11 years were included in this study. Most of the participants were females (71.0%), have had diabetes for 13.92±6.19 years and had been on metformin therapy for 12.88±6.15 years. Furthermore, 32.5% and 6.5% presented with neuropathy based on MNSI-Q and MNSI-PE, respectively. Other clinical, immunological, metabolic and hematological parameters are shown in Table 1.

Table 1. Demographic, clinical and immune-metabolic profile of the entire study participants.

Variables	Mean ±SD
Age (years)	59.00±9.11
BMI (kg/m²)	26.10±3.70
SBP (mmHg)	132.21±17.60
DBP (mmHg)	77.35±9.93
DM duration (years)	13.92±6.19
Metformin duration (years)	12.88±6.15
Sex	Frequency (%)
Female	142 (71.0)
Male	58 (29.0)
Metformin dosage (mg/day)
1000	32 (16.0)
2000	130 (65.0)
3000	38 (19.0)
MNSI-Q
Neuropathy absent	135 (67.5)
Neuropathy present	65 (32.5)
MNSI PE
Neuropathy absent	187 (93.5)
Neuropathy present	13 (6.5)
Circulation impairment
Absent	112 (56.0)
Present	88 (44.0)
General asthenia
Absent	110 (55.0)
Present	90 (45.0)
Immunological parameters
IFA (AU/mL)	1.10 (1.0–1.21)
GPA (pmol/l)	5.20 (3.71–6.80)
TNF-α (pg/ml)	42.67 (30.00–50.92)
IL-6 (pg/ml)	66.67 (58.17–85.00)
Metabolic parameters	Median (IQR)
FPG (mmol/L)	6.90 (6.00–9.05)
HbA1c (%)	6.90 (6.00–8.20)
TC (mmol/L)	5.10 (4.20–6.30)
TG (mmol/L)	1.60 (1.20–1.90)
HDL (mmol/L)	1.30 (0.90–1.90)
LDL (mmol/L)	3.02 (2.23–4.32)
Serum albumin (g/dl)	4.10 (4.05–4.20)
Corrected calcium (mmol/L)	9.04 (8.82–9.44)
Hematological parameters
TWBC (10³/μL)	5.12±1.71
RBC (10⁶/μL)	4.02±0.60
Hemoglobin (g/dL)	12.05±1.54
HCT (%)	34.12±4.79
MCV (fL)	83.39±7.94
MCH (pg)	30.82±2.44
MCHC (g/dL)	36.56±1.87
PLT (10³/μL)	187.0 (155.0–210.0)

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MNSI-Q: Michigan Neuropathy Screening Instrument Questionnaire; Michigan Neuropathy Screening Instrument Physical Examination (MNSI-PE); IFA: Intrinsic factor antibody; GPA: Gastric parietal cell antibody; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin 6; FPG: Fasting plasma glucose; HbA1c: Glycated hemoglobin; TC: Total cholesterol; TG: Triglyceride; HDL: High density lipoprotein; LDL: Low density lipoprotein; TWBC: Total White Blood Cells; RBC: Red Blood Cells; HCT: Haematocrit; MCV: Mean Cell Volume; MCH: Mean Cell Haemoglobin; MCHC: Mean Cell Haemoglobin Concentration; PLT: Platelet count.

The average sB₁₂, hTC, MMA and Hcy were 142.10 (59.00–178.90) pmol/L, 27.02 (15.03–45.66) pmol/L, 0.17 (0.14–0.20) μmol/L and 6.50 (4.35–13.80) μmol/L, respectively. The median Fedosov’s wellness score (ω) was -0.18 (-0.97–0.32) (Fig 1A). Using the combined indicator (4cB₁₂), the prevalence of vitamin B₁₂ deficiency was 40.5% (Fig 1B).

Fig 1 — (A) Density plots of vitamin B12 indicators; (B) Bar graph displaying the prevalence of vitamin B12 deficiency using 4cB12. 4cB12 (ω): combined vitamin B12 indicator, Hcy: Homocysteine, hTC: holotranscobalamin, MMA-methylmelanoic acid, SB12-Serum vitamin B12.

IFA (1.18 (1.00–1.30) vs 1.00 (0.98–1.20), AU/ML, p = 0.003), GPA (5.80 (4.10–7.90) vs 4.80 (3.60–6.50) pmol/l, p = 0.005) and TNF-α (45.00 (33.47–56.67) vs 41.67 (28.33–48.33), pg/ml p<0.0001) levels were significantly higher among the participants with vitamin B₁₂ deficiency compared to those with normal vitamin B₁₂ levels. The levels of IL-6 did not differ significantly between the deficient and non-deficient group (Fig 2).

Participants with vitamin B₁₂ deficiency presented with significantly higher TC (5.60 (4.60–6.80) vs 5.10 (4.10–6.00) mmol/L, p = 0.01), LDL (3.08 (2.46–4.84) vs 2.86 (2.15–3.85) mmol/L, p = 0.005) and albumin (4.20 (4.10–4.30) vs 4.10 (4.00–4.20) g/l, p = 0.021) levels compared to those with normal vitamin B₁₂ levels (Fig 3).

Although vitamin B12 deficient participants had marginally higher mean cell volume (macrocytosis) and platelet count, there was no statistically significant association between hematological parameters and vitamin B12 status (S1 Table in S1 File).

Correlation analysis revealed a statistically significant negative association between 4cB₁₂ and the immunological markers [IFA (rs = -0.301, AU/ml, p<0.0001), GPA (rs = -0.244, pmol/l, p = 0.001), TNF-α (rs = -0.242, pg/ml, p = 0.001) and IL-6 (rs = -0.145, pg/ml, p = 0.041)]. Likewise, 4cB₁₂ was negatively associated with TC (rs = -0.203 mmol/l, p = 0.004) and LDL (rs = -0.222, mmol/l, p = 0.002) but positively correlated with HDL (rs = 0.196, mmol/l, p = 0.005). A negative correlation was also observed between sB₁₂ and IFA (rs = -0.341, p<0.0001), GPA (rs = -0.234, p = 0.001) and TNF-α (rs = -0.169, p = 0.016) whereas hTC correlated negatively with only TNF-α (rs = -0.22, p = 0.002). Conversely, MMA correlated positively with AIF (rs = 0.189, p = 0.007), GPA (rs = 0.262, p<0.0001), IL-6 (rs = 0.185, p = 0.009) and calcium (rs = 0.145, p = 0.041) but negatively with HDL (rs = -0.19, p = 0.007). Correspondingly, Hcy showed a positive correlation with the immunological markers [AIF (rs = 0.278, p<0.0001), GPA (rs = 0.342, p<0.0001), TNF-α (rs = 0.174, p = 0.014), IL-6 (rs = 0.186, p = 0.008) as well as TC (rs = 0.211, p = 0.003) and LDL (rs = 0.217, p = 0.002) but a negative correlation with HDL (rs = -0.18, p = 0.011) (Fig 4).

Fig 4 — Hierarchical clustering by Spearman’s correlation was used to assess relationship between adipokines and their ratios with obesity indices. Red-yellow-white coloration represents negative (-) to positive (+) correlation coefficient. *; p<0.05, **; p<0.01, ***; p<0.0001.

Among the 32.5% and 6.5% presented with neuropathy based on MNSI-Q and MNSI-PE, respectively, a higher proportion also had vitamin B12 deficiency (20.0% and 4.0% for MNSI-Q and MNIS-PE, respectively). Furthermore, vitamin B12 deficiency was significantly associated with over 3-fold increase in the odds of neuropathy compared to the non-deficient counterparts by MNSI-Q (COR = 3.67, 95% CI (1.97–6.82), p<0.0001). Although vitamin B12 deficiency was also associated with higher odds of neuropathy by MNSI-PE, the association was not statistically significant (Table 2). Of note, a higher dose of metformin (3000mg/day) but not duration of treatment was associated with increased odds of vitamin B12 deficiency and neuropathy (S2–S4 Tables in S1 File).

Table 2. Association between vitamin B12 status and diabetic neuropathy.

Vitamin B12 status	MNSI-Q			p-value
	Neuropathy absent	Neuropathy present	COR (95% CI)
Non-deficient	94 (47.0)	25 (12.5)	1
Deficient	41 (20.5)	40 (20.0)	3.67 (1.97–6.82)	<0.0001
	MNSI-PE
Non-deficient	114 (57.0)	5 (2.5)	1
Deficient	73 (36.5)	8 (4.0)	2.50 (0.79–7.93)	0.120

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Discussion

The current study investigated the prevalence of vitamin B12 deficiency among T2DM patients treated with metformin using the more sensitive combined indicator of vitamin B12 status (4cB12) and immuno-biochemical mediators of vitamin B12 deficiency.

Prevalence of vitamin B12 deficiency

Vitamin B12 deficiency is diagnosed by measurements of total B12, methylmalonic acid (MMA) holo-transcobalamin (holoTC), and total homocysteine (Hcy) in blood. However, most studies use only one or two of these biochemical tests in determining vitamin B12 deficiency and this has often led to inconsistent and contradictory results. The current study however classified Vitamin B12 deficiency using combined direct markers of vitamin B12 status (sB12 and hTC) and the metabolic markers (MMA and Hcy) based on the Fedosov age-normalized wellness quotient [ω; combined indicator of vitamin B12 status using all four indicators (4cB12)] [34]

Using this combined indicator (4cB₁₂), the prevalence of vitamin B₁₂ deficiency among T2DM patients was 40.5%. Sparre Hermann et al. reported a 26.7% prevalence of vitamin B12 deficiency in metformin-treated T2DM patients based on holoTC in Sweden [35]. Studies by Ahmed et al., in South Africa and Qureshi et al., reported deficiencies of 28.1%, and 33% respectively [10, 36]. A related study in Ghana, by Yakubu et al reported vitamin B12 deficiency of 32.1% using serum vitamin B12, methylmalonic acid as markers [12]. The varied definitions of vitamin B12 deficiency by the use only one or two biochemical indicators of vitamin B12 deficiency these studies could explain these wide variations in the reported prevalence. Moreover, differences in cultural and religious beliefs in different geographic regions of the world, could also affect the observed variations. Of note, the high rate of vitamin B12 deficiency in this study compared to previous studies could be suggestive that the use of individual markers (serum B12, MMA, hTC or Hcy) underestimates the vitamin B12 status among diabetic populations.

Influence of immunological mediators

The current study observed that intrinsic factor antibody (IFA), gastric parietal cell antibodies (GPA) and tumor necrotic factor alpha (TNF-α) levels were significantly higher among vitamin B₁₂ deficient T2DM patients compared to those with normal vitamin B12 levels. Antonio Cabrera de León et al in a similar study in the Canary Island estimated a higher prevalence of GPA (7.8%) [30]. Patients with increased positive GPA are susceptible to gastric parietal cell destruction that could lead to a deficiency in IF and subsequent blockage in the absorption of vitamin B12 in the terminal ileum [19, 24] Additionally, vitamin B12 absorption requires IF and auto-immune destruction of IF results in decreased vitamin B12 levels as consistent with this study [13, 20].

Furthermore, low vitamin B₁₂ levels has been reported to be associated with increased pro-inflammatory cytokines levels and could account for the high TNF-α levels among vitamin B₁₂ deficient participants compared to the non-deficient counterpart [21, 22]. Our study also observed that an increment in GPA, IF, and TNF-a led to the increase in methylmalonic acid (MMA), holo-transcobalamin (holoTC), and total homocysteine (Hcy) (4cB12) [Fig 4]. Thus, GPA, IF and TNF-α are independent risk factors associated with vitamin B₁₂ deficiency among metformin-treated T2DM.

Relationship between vitamin B₁₂ and biochemical parameters

One major function of Vitamin B₁₂ is to act as a coenzyme in the synthesis of succinyl-CoA from methylmalonyl-CoA (MM-CoA) [37]. In the event of vitamin B₁₂ deficiency, this reaction is blocked which results in subsequent accumulation of MM-CoA and thus causing increased levels of serum methylmalonic acid (MMA) [38] as observed in this study. Our study observed that vitamin B₁₂ deficiency was associated with hyperhomocysteinemia. This findings was corroborated by Masoud et al, who observed a high prevalence of vitamin B₁₂ deficiency and hyperhomocysteinemia in adults with T2DM in Oman [39]. Similarly, Weikert et al., in their population based prospective study reported the association between reduced serum B₁₂ levels and increased serum homocysteine levels [40].

Additionally, participants with vitamin B12 deficiency had higher cardiometabolic risk factors such as high TC and LDL compared to those with normal vitamin B12 levels. This finding is consistent with a study by Al-Daghri et al. who found vitamin B12 levels to be a negative predictor of HDL levels [21]. These highlight the importance of maintaining adequate vitamin B₁₂ concentrations in reducing the inflammatory and cardiometabolic risks associated with T2DM.

Prevalence of peripheral neuropathy

The current study employed one of the best-known methods in determining diabetic neuropathy; the Michigan Screening instrument [27, 41], the MNSI-Q and MNSI-PE and observed diabetic neuropathy of 32.5% and 6.5%, respectively, among our study population. The reported prevalence of diabetic neuropathy is 16.6% in Ghana [42], 29.5% in Ethiopia [43], 56.2% in Yemen [44]. The variations in the prevalence may be due to differences in the types of diabetes, the different methods of patient selection, the sample size and diagnostic criteria employed. The higher prevalence of neuropathy in this study compared to previous prior study in Ghana was not unexpected since a more sensitive diagnostic instrument was employed in this current study. Furthermore, the duration and dosage of metformin have been identified as independent risk factors for diabetic neuropathy and most of our study population has had diabetes and been treated with metformin for more than 12 years. The study also found vitamin B12 deficiency to be associated with increased risk of neuropathy. This finding is in harmony with a study by Alvarez et al. who found an inverse relationship between plasma level of vitamin B12 and diabetic neuropathy [45]. Other studies have reported similar findings among T2DM patients treated with metformin [46, 47] although there are also conflicting reports [48]. One practical limitation of this study was our inability to perform nerve conduction studies to ascertain whether there was nerve damage among our participants as sometimes, problems with the electrical activity in nerves can cause pain, tingling, or weakness in muscles. We could not also perform nerve biopsy, which has been suggested as gold standard for determination of the presence and types of neuropathy. Therefore, electrophysiological findings could not be confirmed. Another limitation of this study is the relatively small sample size and the cross-sectional nature of the design. The current study design lacked comparator and a control group of healthy subjects, or participants who are not on metformin, this makes it difficult to make causal inference. They are however, useful for establishing preliminary evidence in planning a future advanced study, subsequently, further case-control and prospective studies are warranted.

Conclusion

Vitamin B12 deficiency and diabetic neuropathy are very high among metformin-treated T2DM patients and it is associated with increased GPA, IFA and TNF-α. Moreover, vitamin B12 deficiency is associated with cardiometabolic risk factors (higher LDL and TC and lower HDL). Upon verification of these findings in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12) in the management of patients with T2DM treated with metformin in Ghana.

Supporting information

S1 File

(DOCX)

Click here for additional data file.^{(19.6KB, docx)}

Acknowledgments

The authors are grateful to all T2DM participants, the staff of Komfo Anokye Teaching Hospital, Diabetic Clinic for their participation and immense support in making this research a success.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

KNUST/Kref/2018

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PLoS One. doi: 10.1371/journal.pone.0249325.r001

Decision Letter 0

Benjamin Udoka Nwosu

15 Jan 2021

PONE-D-20-39903

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PLOS ONE

Dear Dr. Sakyi

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

As you can see from the Reviewers' comments below, the principal areas of concern are in the study design and the applicability of the Fedosov methodology

The Reviewers' would want you to include a comparator group consisting of either patients without a diagnosis of type 2 diabetes, or patients with type 2 diabetes but were not treated with metformin.

There is also the need to exclude the diagnosis of type 1 diabetes in the subjects because of high prevalence of other autoimmune conditions in your cohort.

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Reviewer #2: Yes

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Reviewer #1: The study addresses an important problem of B12 deficiency associated with metformin use. However, there are many, well conducted prior studies (close to 200) across the globe which have conclusively demonstrated this association. This study is from Ghana, and the authors acknowledge another study from Ghana that demonstrated similar conclusions (This reviewer could not retrieve the citation for this study on Pubmed as referenced by Yakubu at al which showed a lower prevalence of B12 deficiency).

There are several limitations to the study:

1. It is a relatively small study (n=200) and there is no control group.

2. There is a very high prevalence of Vitamin B12 deficiency (40.5%) reported, but in the absence of any control (for example, age/gender matched individuals not on Metformin) it is difficult to attribute this high prevalence only to metformin use. There are numerous other prior studies that are larger, with appropriate controls, which show lower prevalence.

3. In the absence of a control group, a before treatment B12 compared to B12 after treatment with Metformin would provide useful data. This was not done as this is an observational study.

4. The study uses a relatively little used and poorly validated computational model (Fedosov combined marker) to diagnose Vitamin B12 deficiency. Low B12 levels alone are a sensitive indicator of deficiency ( up to 95% sensitive depending on cutoff) (NEJM, 2013. 368:149-60) . In those with borderline low Vitamin B12 (200-300 pg/ml), an elevation of MMA and Homocysteine add to the sensitivity, but both need to be elevated as homocysteine alone can be high in folate deficiency. MMA elevations may not be specific as other conditions including renal disease can cause elevations. Therefore these should be used in situations where Vitamin B12 is borderline low. Holotranscobalmin is neither specific or sensitive but can be useful in pregnancy. There is little data on validity of Fedosov model (Reference 18 in current article) which has been cited only 15 times since its publication in 2013. Reading the original paper reveals this is a theoretical construct which is validated against cognitive impairment in the elderly and hematological impairments.

5. The data correlating B12 deficiency with neurological impairments (two clinical measures of peripheral neuropathy) and immunological parameters does not provide any new information

6. This study shows rates of Vitamin B12 deficiency (40.5%) which is far higher than almost all prior studies, but the non-standard measure (Fedosov wellness quotient) may be the reason for this discrepant finding.

7. Since the average B12 in the group, on page 14, line 287 is reported as 142 pmol/L (which is below the threshold for severe Vit B12 deficiency 148 pmol/L or 200 pg/ml), it would be informative to know what was the percentage of B12 deficiency using just the threshold of 148 pmol/L which is very specific cutoff

8. The study needs to emphasize what new knowledge it adds to the topic of B12 deficiency in Metformin treated patients.

Minor Problems:

Labelling/legends in Figure 1 need to be clearer

Reviewer #2: In the manuscript “Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana” Sakyi et al investigated the immuno-biochemical mediators of vitamin B12 deficiency using Fedosov age normalized wellness quotient. Fedosov proposed the utilization of combined indicator of vitamin B12 status using two or more of the following markers: total serum B12, methylmalonic acid, holo-transcobalamin, and total homocysteine. Comparison of immuno-metabolic parameters based on vitamin B12 status was performed using one-way analysis of covariance (ANCOVA) with adjustment for age, sex, BMI, duration of diabetes, and dosage and duration metformin therapy. The authors report that the prevalence of metformin induced vitamin B12 deficiency was 40.5% whilst the prevalence of diabetic neuropathy was > 67%. Also, Vitamin B12 deficient patients had higher levels of intrinsic factor, gastric parietal cell antibody, TNF-α, total cholesterol and LDL-cholesterol. They conclude that prevalence of Vitamin B12 deficiency and diabetic neuropathy are high among metformin-treated patients with type 2 diabetes and suggest that routine measurement of Vitamin B12 should be included in the management of patients with type 2 diabetes treated with metformin.

Comments:

Strength: a reasonably elaborate study on treatment with metformin and Vitamin B12 deficiency

Major

1. It would be appropriate to clarify how the diagnosis of type 2 diabetes was made. The presence of gastric parietal cell antibody makes it imperative to exclude type 1 diabetes

2. A comparator group such as patients without diabetes or patients with type 2 diabetes but who were not treated with metformin would be required to justify the conclusion that the prevalence of Vitamin B12 deficiency and peripheral neuropathy are high in patients treated with metformin in Ghana. Could there be the possibility that B12 deficiency may be high in the general population.

3. Is there any correlation between the dose/duration of treatment with metformin and Vitamin B12 deficiency/peripheral neuropathy?

4. 2017 ADA guidelines recommended periodic monitoring of Vitamin B12 in patients treated with metformin over a long period of time, suggesting routine measurement in all patients treated with metformin may not be cost effective.

Minor

1. Type II diabetes should read Type 2 diabetes

2. “on metformin” is better rendered as “treated with metformin” throughout the manuscript

3. Figure 4 appears confusing, a table might be easier to understand

**********

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Reviewer #1: Yes: Samir Malkani

Reviewer #2: No

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PLoS One. 2021 Mar 30;16(3):e0249325. doi: 10.1371/journal.pone.0249325.r002

Author response to Decision Letter 0

8 Feb 2021

The Editor,

PLOS ONE

Dear Sir/Madam,

RESPONSE TO REVIEW COMMENTS

The authors very much appreciate the timely and scrupulous review of our manuscript (PONE-D-20-39903). Kindly find below the response to the reviewers’ comments. Tracked changes have been employed to highlight texts revised per the reviewers’ recommendations. Revised statements indicated in this response are in “quotation marks”.

Reviewer #1

Comment 1: It is a relatively small study (n=200) and there is no control group.

Response: Thank you for your comment. This observational study is a baseline study to evaluate to use of Fedosov combined marker in Ghanaian population which has not been explored. However, a case-control study with large sample size will add a layer of knowledge. We acknowledged this as a limitation worth exploring by future studies (line 433-4).

“Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.”

Comment 2: There is a very high prevalence of Vitamin B12 deficiency (40.5%) reported, but in the absence of any control (for example, age/gender matched individuals not on Metformin) it is difficult to attribute this high prevalence only to metformin use. There are numerous other prior studies that are larger, with appropriate controls, which show lower prevalence.

Response: Thank you for your comment. Although this observational study reported a very high prevalence of vitamin B12 deficiency (40.5%) with the absence of a control group compared to other studies conducted elsewhere, this was not unexpected for reasons such as disparities in environmental and genetic factors that may impact their levels. Additionally, differences in the parameters (MMA, serum B12, etc) considered in determining B12 deficiency is another factor. In an effort to accurately capture patients with true vitamin b12 deficiency, we relied on the combined 4cB12.

Comment 3: In the absence of a control group, a before treatment B12 compared to B12 after treatment with Metformin would provide useful data. This was not done as this is an observational study.

Response: Thank you very much for your comment. The current study was a cross -sectional study where one-point sample was taken. We have highlighted this as a limitation (line 433-4).

Comment 4: The study uses a relatively little used and poorly validated computational model (Fedosov combined marker) to diagnose Vitamin B12 deficiency. Low B12 levels alone are a sensitive indicator of deficiency (up to 95% sensitive depending on cutoff) (NEJM, 2013. 368:149-60). In those with borderline low Vitamin B12 (200-300 pg/ml), an elevation of MMA and Homocysteine add to the sensitivity, but both need to be elevated as homocysteine alone can be high in folate deficiency. MMA elevations may not be specific as other conditions including renal disease can cause elevations. Therefore, these should be used in situations where Vitamin B12 is borderline low. Holotranscobalmin is neither specific nor sensitive but can be useful in pregnancy. There is little data on validity of Fedosov model (Reference 18 in current article) which has been cited only 15 times since its publication in 2013. Reading the original paper reveals this is a theoretical construct which is validated against cognitive impairment in the elderly and hematological impairments.

Response: Thank you for your comment. Although serum B12 presents a good marker for B12 deficiency, several studies have highlighted inconsistencies in using serum B12 to define B12 deficiency. High serum B12 levels can be accompanied by signs of deficiency, and functional deficiency from tissue uptake defects and action of vitamin B12 at the cellular level have been implicated in this association [PMID: 26807790; PMID: 21733877]. There is also evidence that functional vitamin B12 deficiency can occur regardless elevated serum B12 levels and using MMA alone is limited. [PMID: 21733877]. Moreover, elevation of MMA is non-specific in the presence of renal impairment (a somehow common sequela in diabetes) but the use of MMA and homocysteine enhance the sensitivity to identify individuals with borderline vitamin B12 deficiency. Thus, the most accurate marker to true B12 deficiency remains to be identified. However, we believe that a model such as Fedosov 4cB12 which accommodates other markers in addition to serum B12 provides an opportunity.

Comment 5: The data correlating B12 deficiency with neurological impairments (two clinical measures of peripheral neuropathy) and immunological parameters does not provide any new information.

Response: Thank you for your comment. Studies correlating vitamin B12 status to diabetic neuropathy has produced conflicting results [PMID: 27730072; PMID: 28882470; PMID: 27716423]. The two clinical measure of peripheral neuropathy in this study will contribute to the understanding of the relationship between vitamin B12 deficiency is associated and risk of neuropathy.

Comment 6: This study shows rates of Vitamin B12 deficiency (40.5%) which is far higher than almost all prior studies, but the non-standard measure (Fedosov wellness quotient) may be the reason for this discrepant finding.

Response: Thank you very much for your comment. The high prevalence of vitamin B12 deficiency reported in our study (40.5%) is not extremely different from a related study in Ghana which reported high prevalence of B12 deficiency (32.1%) among T2DM patients on metformin [DOI: 10.1080/20905068.2019.1662647]. Additionally, similar to vitamin D deficiency which is very common in the study region despite adequate sunshine, our pilot study found that the use of only serum B12, which is commonly used in other populations overestimates B12 deficiency as evidenced in the higher prevalence of B12 deficiency (>50% of the population) based on serum B12 (Supplementary file: Table S4). In an effort to normalize and accommodate all relevant markers, we used the Fedosov wellness quotient.

Comment 7: Since the average B12 in the group, on page 14, line 287 is reported as 142 pmol/L (which is below the threshold for severe Vit B12 deficiency 148 pmol/L or 200 pg/ml), it would be informative to know what was the percentage of B12 deficiency using just the threshold of 148 pmol/L which is very specific cutoff.

Response: Thank you for your comment. As indicated in the response to comment 6, the use of serum B12 only in the study setting overestimates the prevalence of B12 deficiency, corroborating previous reports on the disparity between serum B12 levels and functional B12 deficiency (Kindly refer to response to comment 6 and Supplementary file: Table S4).

Comment 8: The study needs to emphasize what new knowledge it adds to the topic of B12 deficiency in Metformin treated patients.

Response: Thank you for your comment. Specifically regarding B12 deficiency in diabetes, as highlighted in line 120-3, the use of individual markers (serum B12, MMA, hTC, Hcy) has rather yielded inadequate reflection of true vitamin B12 status. The study thus highlights the likelihood of missing some cases of B12 deficiency when a single indicator is used compared to using multiple markers (line 120-3 & 378-80).

“Of note, the high rate of vitamin B12 deficiency in this study compared to previous studies could be suggestive that the use of individual markers (serum B12, MMA, hTC or Hcy) underestimates the vitamin B12 status among diabetic populations.”

Minor comment:

Comment 1: Labelling/legends in figure 1 need to be clearer

Response: Thank you for the observation. Labelling/legend in figure 1 has been made clearer.

----------------------------------------------------------------------------------

Reviewer #2

Major comments:

Comment 1: It would be appropriate to clarify how the diagnosis of type 2 diabetes was made. The presence of gastric parietal cell antibody makes it imperative to exclude type 1 diabetes

Response: Thank you for your observation. The inclusion and exclusion criteria have been clarified (line 177-86)

“The diagnosis of type 2 diabetes mellitus was made based on American Diabetes Association (ADA) criteria (based on defective progressive insulin secretory on the background of insulin resistance using fasting plasma glucose ≥7.0 mmol/L; glycated hemoglobin ≥6.5%; oral glucose tolerance test 11.1 mmol/L)”

“Patients with type 1 diabetes (diagnosed based on high plasma glucose in addition to autoimmune indicators such as autoantibodies to insulin, autoantibodies to glutamate decarboxylase (GAD65)) were excluded.”

Comment 2: A comparator group such as patients without diabetes or patients with type 2 diabetes but who were not treated with metformin would be required to justify the conclusion that the prevalence of Vitamin B12 deficiency and peripheral neuropathy are high in patients treated with metformin in Ghana. Could there be the possibility that B12 deficiency may be high in the general population.

Response: Thank you for your comment. It is possible that there is generally high prevalence of vitamin B12 deficiency in the study population given that a previous study reported over 30% prevalence using serum B12. Additionally, using only serum B12 levels, over 50% of our study population was deficient. Given that this is a baseline study, we have acknowledged the limitation of the cross-sectional design and suggested further studies in this regard (line 433-4).

“Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.”

Comment 3: Is there any correlation between the dose/duration of treatment with metformin and Vitamin B12 deficiency/peripheral neuropathy?

Response: Thank you for your comment. Higher dose of metformin (3000mg/day) was associated with increased odds of vitamin B12 deficiency and neuropathy. However, the duration of treatment with metformin was not associated with vitamin B12 deficiency or neuropathy (Supplementary file: Table S2 & S3).

Comment 4: 2017 ADA guidelines recommended periodic monitoring of Vitamin B12 in patients treated with metformin over a long period of time, suggesting routine measurement in all patients treated with metformin may not be cost effective.

Response: Thank you very much for your comment. The sentence has been rephrased.

Minor comment:

Comment 1: Type II diabetes should be read Type 2 diabetes.

Response: Thank you for the comment. ‘Type II diabetes’ has been replaced with ‘Type 2 diabetes’.

Comment 2: “on metformin” is better rendered as “treated with metformin” throughout the manuscript.

Response: Thank you for the suggestion. “on metformin” has been replaced with “treated with metformin” throughout the manuscript.

Comment 3: Figure 4 appears confusing; a table might be easier to understand.

Response: Thank you very much for your comment. Due to the large number of biochemical and immunological parameters together with the five indicators of vitamin B12, using a table will make it very clumsy. However, further description has been added to make the figure clearer.

Thank you once again for the timely review and scrupulous of our manuscript. Looking forward to hear favorably from you.

Sincerely,

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PLoS One. doi: 10.1371/journal.pone.0249325.r003

Decision Letter 1

Benjamin Udoka Nwosu

5 Mar 2021

PONE-D-20-39903R1

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PLOS ONE

Dear Dr. Sakyi,

Please provide a detailed discussion of the limitation of your study with respect to the lack of a comparator group. This is critical. Your current two-sentence response is not sufficient: 'Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.'

You have to discuss the concerns about his lack of comparator group, and what your group should have done in terms of including a control group of healthy subjects, or a group of patients with diabetes but were not receiving metformin.

Please submit your revised manuscript by March 15th, 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Benjamin Udoka Nwosu, MD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

Please provide a detailed discussion of the limitation of your study with respect to the lack of a comparator group. This is critical. Your current one sentence response, is not sufficient: 'Another limitation of this study is the relatively low sample size and the cross-sectional nature of the design. Further case-control and prospective studies are warranted.'

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

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Reviewer #2: Yes

**********

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Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: "It is imperative Ghana include periodic measurement of Vitamin B12 deficiency using the more sensitive combined indicators (4cB 12 ), in the management of T2DM patients treated with metformin" in concluding sentence of the abstract should read "if the findings of this study are verified in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12 ) in the management of patients with T2DM treated with metformin in Ghana.

**********

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PLoS One. 2021 Mar 30;16(3):e0249325. doi: 10.1371/journal.pone.0249325.r004

Author response to Decision Letter 1

8 Mar 2021

The Editor,

PLOS ONE

Dear Sir/Madam,

RESPONSE TO REVIEW COMMENTS R2

The authors very much appreciate review of our manuscript (PONE-D-20-39903). Kindly find below the response to the reviewers’ and editor’s comments. Revised statements indicated in this response are in “highlighted in red”.

Editor’s comment

Response: The limitations has been discussed further to read “Another limitation of this study is the relatively small sample size and the cross-sectional nature of the design. The current study design lacked comparator and a control group of healthy subjects, or diabetes who are not on metformin, this makes it difficult to make causal inference. They are however, useful for establishing preliminary evidence in planning a future advanced study, subsequently, further case-control and prospective studies are warranted”. Page 20, line 430-435.

Reviewer #2

Response: The concluding sentence of the abstract and main manuscript has been revised to read " Upon verification of these findings in a prospective case-control study, it may be beneficial to include periodic measurement of Vitamin B12 using the more sensitive combined indicators (4cB 12) in the management of patients with T2DM treated with metformin in Ghana. Page 3, line 78-81

Thank you once again for the timely review of our manuscript. Looking forward to hear favorably from you.

Sincerely,

Samuel Asamoah Sakyi

Corresponding author

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PLoS One. doi: 10.1371/journal.pone.0249325.r005

Decision Letter 2

Benjamin Udoka Nwosu

16 Mar 2021

Profiling Immuno-Metabolic Mediators of Vitamin B12 Deficiency Among Metformin-Treated type II Diabetes Patients in Ghana

PONE-D-20-39903R2

Dear Dr. Sakyi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Benjamin Udoka Nwosu, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

My comments have been addressed.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0249325.r006

Acceptance letter

Benjamin Udoka Nwosu

22 Mar 2021

PONE-D-20-39903R2

Profiling Immuno-Metabolic Mediators of Vitamin B₁₂ Deficiency Among Metformin-Treated Type 2 Diabetic Patients in Ghana

Dear Dr. Sakyi:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

Dr. Benjamin Udoka Nwosu

Academic Editor

PLOS ONE

Associated Data

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Profiling immuno-metabolic mediators of vitamin B12 deficiency among metformin-treated type 2 diabetic patients in Ghana

Samuel Asamoah Sakyi

Edwin Ferguson Laing

Richard Mantey

Alexander Kwarteng

Eddie-Williams Owiredu

Richard Ephraim Dadzie

Benjamin Amoani

Stephen Opoku

Bright Oppong Afranie

Daniel Boakye

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Study design

Ethical consideration

Study population

Inclusion and exclusion criteria

Questionnaire administration

Assessment of peripheral neuropathy using the Michigan Neuropathy Screening Instrument (MNSI)

Physical assessment

Vibration sensation

Muscle stretch reflexes

Blood pressure measurement

Measurement of Body Mass Index (BMI)

Blood sample collection and processing

Measurement of hematological parameters

Biochemical assays

Classification for vitamin B12 deficiency

Statistical analysis

Results

Table 1. Demographic, clinical and immune-metabolic profile of the entire study participants.

Fig 1. Distribution of vitamin B12 indicators and the prevalence of B12 deficiency.

Fig 2. Comparison of select immunological parameters based on vitamin B12 status.

Fig 3. Comparison of select biochemical parameters based on vitamin B12 status.

Fig 4. Correlation between indicators of vitamin B12 and the immunological parameters.

Table 2. Association between vitamin B12 status and diabetic neuropathy.

Discussion

Prevalence of vitamin B12 deficiency

Influence of immunological mediators

Relationship between vitamin B12 and biochemical parameters

Prevalence of peripheral neuropathy

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Benjamin Udoka Nwosu

Roles

Author response to Decision Letter 0

Decision Letter 1

Benjamin Udoka Nwosu

Roles

Author response to Decision Letter 1

Decision Letter 2

Benjamin Udoka Nwosu

Roles

Acceptance letter

Benjamin Udoka Nwosu

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Profiling immuno-metabolic mediators of vitamin B₁₂ deficiency among metformin-treated type 2 diabetic patients in Ghana

Classification for vitamin B₁₂ deficiency

Fig 2. Comparison of select immunological parameters based on vitamin B₁₂ status.

Fig 3. Comparison of select biochemical parameters based on vitamin B₁₂ status.

Fig 4. Correlation between indicators of vitamin B₁₂ and the immunological parameters.

Relationship between vitamin B₁₂ and biochemical parameters