Chronic kidney disease (CKD) is a common noncommunicable condition globally, and a powerful independent risk factors for multiple adverse outcomes, including development of end stage renal disease, acute kidney injury, cardiovascular events and premature death.1,2 CKD is defined as the persistent urine abnormalities, structural abnormalities or impaired excretory renal function suggestive of a loss of functional nephrons or an estimated glomerular filtration rate (eGFR) less than 60 ml/min/1.73 m2, persisting for 3 months or more, irrespective of the cause.3,4 CKD affects 8% to 16% of the population worldwide,5 however its prevalence in Pakistan has been reported as 100 per million population a year.6 Patients with CKD are at higher risk for nutritional deficiencies due to medication interactions, dietary restrictions and malnutrition that sometimes may lead to deficiency of essential vitamins especially vitamin B-12 (Cobalamin).7,8

Vitamin B12 is essential for optimal cardiovascular, hematopoietic and macrocognitive function in the body.9 Vitamin B-12 deficiency has central role in homocysteine metabolism, a non-protein amino acid synthesized by methionine that can be recycled into methionine by B group Vitamins.10 Elevated homocysteine levels in CKD patients and the role of hyperhomocysteinemia as a cardiovascular risk and important risk factor for early death and progression of CKD in this population is still to be fully elucidated and deserves further investigation. However, it has been established that low B12 levels have been associated with higher serum homocysteine.11 So, it is important to identify the B-12 levels in patient with CKD. In a hospital based cross sectional study, Patil RG and colleagues determined the prevalence of Vitamin B12 deficiency in CKD patients and they reported that Vitamin B12 deficiency was observed in 56% of CKD patients. Significant association was observed between presence of neurological symptoms and presence of vitamin B12 deficiency (13.6% in normal B-12 levels and 42.9% in deficient patients; p-value 0.03). Similarly mean duration of CKD was also significantly associated with presence of Vitamin B12 deficiency (Normal 5.32±3.73 and Deficient 8.42±3.51 years; p-value < 0.01).12

Some past international studies have been conducted on B-12 deficiency in CKD patients but there is scarcity of local literature on frequency of B-12 deficiency. Taking into consideration the high impact of vitamin B-12 deficiency on the health of CKD patients, this study is being conducted to detect the burden of vitamin B-12 deficiency in CKD patients of our local population. If high prevalence is noted in these patients, early detection of this common deficiency would be ensured in future in all the CKD patients. Moreover, B-12 supplements will be prescribed in all the deficient patients by the clinicians. Early and prompt detection as well as treatment of vitamin B-12 deficiency in CKD patients would ultimately help to reduce the overall associated morbidity and mortality in these patients.

STUDY DESIGN: Cross-sectional study

SETTING SETTING: Department of Nephrology, Shifa International Hospital, Islamabad

DURATION: Six months (26-03-2020 to 26-09-2020)

SAMPLE SIZE: The overall sample size in our study was 95 patients.

SAMPLE TECHNIQUE: Consecutive (non- probability) sampling technique

SAMPLE SELECTION

INCLUSION CRITERIA

• All the patients who are diagnosed with CKD as per operational definition
• Both male and female patients
• Aged 18 to 75 years

EXCLUSION CRITERIA

• Vegetarians or patients on current parenteral or enteral nutritional support
• Patients on use of proton pump inhibitors or vitamin B-12 supplements
• Patients with malabsorptive syndrome as confirmed through clinical history
• Patients with history of gastric or intestinal surgery

DATA COLLECTION PROCEDURE

Permission and approval was sought from the hospital ethical and research committee for conduction of study. All patients visiting CKD and fulfilling the inclusion criteria of the study were enrolled from Nephrology Department of Shifa International Hospital, Islamabad. A well-informed written consent was signed from all the patients. Detailed history of present illness and medication history was taken by the trainee researcher followed by thorough physical examination. 5ml of venous blood sample was drawn by the senior phlebotomist from all the enrolled patients using aseptic technique in a BD vacutainer tube, and were sent to the hospital laboratory for biochemistry analysis. Vitamin B-12 deficiency was diagnosed as per our operational definition. Demographic details of all patients and study results were entered in the predesigned proforma

DATA ANALYSIS PROCEDURE

Data was entered and analyzed using computer software SPSS version 17. Mean and standard deviation was calculated for quantitative variables like age, BMI, duration of CKD, and serum vitamin B-12 levels. Frequency and percentages were calculated for qualitative variables like gender, stage of CKD and vitamin B-12 deficiency. Effect modifiers like age, gender, stage of CKD, BMI and duration of CKD were controlled by the stratification. Post stratification Chi square test was applied and p-value ≤0.05 considered significant.

A total of ninety-five (n=95) adult patients irrespective of gender with age between 18-75 years who were diagnosed CKD as per our operational definition were included in the study. Patients who were vegetarians or patients on current parenteral or enteral nutritional support were excluded. Patients taking proton pump inhibitors or vitamin B-12 supplements and patients with malabsorptive syndrome or history of gastric or intestinal surgery were also excluded from the study. Vitamin B-12 deficiency was diagnosed as per our operational definition. Data analysis showed that 63.2% (n=60) of patients were male with mean age of 50.55±14.97 years, while 36.8% (n=35) of patients were female with mean age of 52.77±13.49 years (table 1).

Demographic analysis showed that mean BMI was 26.91±2.88 kg/m², mean duration of CKD was 4.12±2.27 years and mean vitamin B-12 level was 208.06±161.12 pg/mL (table 2). Most of the patients 49.5% (n=47) were belonged to stage V CKD. Detailed results are described in table 3. Patients were further classified into different groups on the basis of age, BMI and duration of CKD which are graphically illuminated in figure 1, 2, and 3.

In our study, vitamin B-12 deficiency was found in 57.9% (n=55) of patients (table 4).

Table 1: Demographic Profile of the total study sample (age and gender distribution)

Table 2: Demographic Profile of the study Population (BMI, Duration of CKD, Vitamin B-12 Levels)

Table 3: Distribution of total study population on the basis of stage of CKD

Figure 1: Distribution of total study population in different BMI groups

Figure 2: Distribution of total study population in different age groups

Figure 3: Distribution of total study population in different groups on basis of duration of CKD

Table 4: Frequency of deficiency of vitamin B-12 in total study sample

Vitamin B12 (B12) is a water-soluble vitamin that plays a key role in the normal function of the nervous system and blood formation along with serving as a cofactor for the formation of methionine from homocysteine. Normally, B12 is released from dietary protein in the stomach and binds to intrinsic factor (IF). The B12-IF complex is absorbed in the ileum via the cubilin receptor.13 Defects in cubilin, a proximal tubular membrane protein, have been associated with both megaloblastic anemia and tubular proteinuria.14-16 Cubilin also acts to reabsorb the majority of filtered albumin from the urine and recently, genome-wide association studies have identified SNPs in CUBN in association with albuminuria and B12 levels.17- 19 B12 deficiency is associated with anemia and neurological disorders. Through its central role in homocysteine metabolism, low B12 levels have been associated with higher serum homocysteine, a risk factor for cardiovascular disease. However, in CKD homocysteine levels increase as GFR declines, independent of B12 levels.20 Patients affected by chronic kidney disease (CKD) or end-stage renal disease (ESRD) have a shorter life expectancy than those with normal renal function, primarily due to the dramatic increase in mortality.21 Chronic haemodialysis treatment is associated with a 10 to 50-fold higher risk of premature death than in the general population, and cardiovascular disease (CVD) represents the leading cause of death in ESRD patients.22,23 Although patients with CKD and ESRD display elevated homocysteine levels, the role of hyperhomocysteinemia as a cardiovascular and mortality risk factor in this population is still to be fully elucidated and deserves further investigation.24, 25 Furthermore, the high prevalence of hyperhomocysteinemia in patients with CKD has increased interest in speculating the role for hyperhomocysteinemia as a risk factor for the progression of CKD. The role of folic acid and vitamin B12 role is well recognized, as they are not only essential cofactors for homocysteine metabolism, but their homeostasis disruption may be related directly to cardiovascular risk and CKD progression.26, 27 The impact of cobalamin deficiency on renal function and the benefits of vitamin B12 supplementation have been investigated in several studies. An analysis on a cohort of 2965 subjects from the Framingham Heart Study, replicated in 4445 participants from NHANES 2003–2004, did not highlight a relationship between B12 deficiency, albuminuria, and reduced kidney function.28 Surprisingly, another study by Soho et al. showed an association between high vitamin B12 levels and all-cause mortality in dialysis patients. The authors explain this unexpected finding in view of the chronic inflammatory status characterizing uremic patients and postulate that this mechanism is an evolutionary adaptation to prevent B12 uptake from infectious organisms in peripheral tissues. Therefore, higher circulating B12 levels may actually reflect functional B12 deficiency in the peripheral tissues, leading to hyperhomocysteinemia and increased cardiovascular risk.29 Vitamin B-12 deficiency associated anemia is an important contributing factor in the clinical manifestations associated with deteriorating renal function.30 There is increased incidence of cardiovascular dysfunction, cognitive impairment and sleep disturbances in anaemic patients with CKD. It is also associated with progression of renal disease and increased mortality. Increased healthcare costs and worsened quality of life are other issues of concern in CKD patients with cobalamin deficiency and anemia. 31 Therefore, evaluation of prevalence and etiological profile of vitamin B-12 deficiency in CKD patients at the regional level is important from the point of view of optimum allocation of health care resources, especially in resource limited settings in developing countries like Pakistan. To overlook the status of B-12 deficiency in our local population, we conducted this cross-sectional study and for this purpose, we enrolled ninety-five (n=95) adult patients irrespective of gender with age between 18-75 years who were diagnosed CKD. Patients who were vegetarians or patients on current parenteral or enteral nutritional support were excluded. Patients taking proton pump inhibitors or vitamin B-12 supplements and patients with malabsorptive syndrome or history of gastric or intestinal surgery were also excluded from the study. Vitamin B-12 deficiency was diagnosed as per our operational definition. Our study results showed that 63.2% (n=60) of patients were male with mean age of 50.55±14.97 years, while 36.8% (n=35) of patients were female with mean age of 52.77±13.49 years. We further analyzed that mean BMI was 26.91±2.88 kg/m2, mean duration of CKD was 4.12±2.27 years and mean vitamin B-12 level was 208.06±161.12 pg/mL. Most of our patients (49.5%) were belonged to stage V CKD. In our study, vitamin B-12 deficiency was found in 57.9% (n=55) of patients. Stratification was done for vitamin B-12 deficiency on the basis of gender, age, stage of CKD, BMI and duration of CKD. Chi-square test was employed and it was evaluated that statistically significant difference (p-value ≤ 0.05) was noted for duration of CKD and stage of CKD. However, we found no significant difference for age, gender and BMI. Our study results are in concordance with previously published studies. In a cross-sectional study, GD Anil et al, studied the hematological profile and vitamin B12 levels in CKD patients in a tertiary care hospital. Among 50 patients 68% had Hb between 5-10gm% and 18% had Hb less than 5-10gm% and 18% had Hb less than 5gm%, 14% had more than 10%. 54% of patients had serum B12 levels less than 187, which considered deficient and the rest 46% had level between 187-883. These results are similar to our study results. Authors concluded that B-12 deficiency anemia is prevalent among CKD patients of which 86% had moderate degree of anemia which was the most frequent finding in both sexes and the degree of anemia was more severe in females as compared to males, most of the patients being elderly. Majority of the patients in this study revealed serum B12 deficiency which also caused elevated serum homocysteine levels in the patients.32 In another hospital based cross sectional study, Patil RG and colleagues, determined the prevalence of Vitamin B12 deficiency in CKD patients and the contributory role of dietary factors to Vitamin B12deficiency and this study also endorsed our study results. For this purpose, they included 50 patients suffering from CKD. Laboratory investigations included complete blood count, urine examination, blood sugar profile, lipid profile and renal function test. Serum vitamin B12 & homocysteine levels were done by CMIA. Out of total 50 subjects, 31 were males and 19 were females with a mean age of 45.26 ± 9.63 years. Vitamin B12 deficiency was observed in 56% of CKD patients. Significant association was observed between presence of neurological symptoms and presence of vitamin B12 deficiency. Similarly mean duration of CKD was also significantly associated with presence of Vitamin B12deficiency. They concluded that CKD patients showed high prevalence of Vitamin B12 deficiency. Most of the patients had associated neurological, haematological and gastro-intestinal symptoms. Hence all the treating nephrologists should anticipate the deficiency of Vitamin B12in CKD patients and take appropriate measures for its control.12 Recently, similar to our study, Dandge VA and Variya D ruled out the vitamin B12 deficiency in CKD patients. Sample size of 80 cases of CKD patients aged between 18-80 year were included in study. Serum vitamin B12 Level was checked, correlation B12 deficiency with duration of CKD was observed. It was observed in this study that, 47 (58.75%) cases of CKD had vitamin B12 deficiency that was nearly similar to our study results. The mean duration of CKD is more in B12deficient group as compared to normal Group and also finds the higher proportion of vitamin B12 deficiency in CKD patients. They concluded from the study that there is high prevalence of vitamin B12 deficiency in CKD patients besides other causes of anemia. Hence, serum vitamin B12 level testing should be recommended routinely in patients with chronic kidney disease and all the treating nephrologists/physicians should anticipate the deficiency of vitamin B12 in CKD patients and take appropriate measures to prevent associated morbidity and mortality of CKD.33 Current review literature and our study results reflected a strong association of vitamin B-12 deficiency and CKD. No past study has evaluated the cobalamin levels in these patients of our local population, so present study would be a reference study for future researches. However, our study has few limitations too. We haven’t studied and corelated the associated co-morbid and clinical profile of the patient with the study results. Like diabetes and use of diabetic drugs especially metformin has established role in B-12 deficiency. Secondly, we haven’t determined the nutritional status of the patients that is the biggest factor for the development of B-12 deficiency. Furthermore, we haven’t studied the outcome of B-12 deficiency such as development of neurological disorder and anemia in these patients. It could be very helpful in reducing the morbidity and mortality in these patients. In our study, we also missed the diagnosis of homocysteine levels in these patients that is influenced by the vitamin B-12 deficiency and play a vital role in development of CVD and other fatal diseases in CKD patients. Most of the limitations are due to the less availability of financial resource and shorter study time. These limitations can be minimized by conducting a larger scale study funded by the public organization.

High frequency of vitamin B-12 deficiency has been observed in our local patients with CKD. A statistically significant (p-value ≤0.05) association for cobalamin deficiency was noted for duration of CKD and stage of CKD.