Homocysteine and Nutritional Biomarkers In Cognitive Impairment

doi:10.21203/rs.3.rs-1725172/v1

Background: Cognitive impairment is a progressive disorder that affects the aging population. With the increase in the mean age of our population, it is becoming a public health problem. Homocysteinemia has been implicated in cognitive impairment. While it is modulated by vitamins B12 and folate, it acts through MMPs 2 and 9.

Objectives: To assesses the relationship of cognitive impairment with homocysteine, B12, folate, and MMPs 2 and 9, with the intention of detecting cases of mild cognitive impairment which are potentially reversible.

Materials and Methods: Blood samples were drawn from 73 enrolled subjects, with and without cognitive impairment on basis of Montreal cognitive assessment (MoCA) score <25 or >25, respectively. Homocysteine, B12, folate, and MMPs2 and 9 were estimated. Correlation between MoCA score and these parameters was elucidated.

Results: After adjusting for age and sex, homocysteine was the only significant independent predictor of MoCA score. Cut off of homocysteine for prediction of MoCA <25 was derived at 13.5 µmol/L (PPV=59.6%; NPV=79.2%; Sensitivity=84.8%; Specificity=50%). The equation derived for calculation of MoCA score from homocysteine is:

MoCA score = 32.893 + [(-0.223)(homocysteine in mmol/L)]

Conclusions: Homocysteine >13.5mmol/L predicts low MoCA (<25) with 84.8% sensitivity and 50% specificity. So patients with a Hcy >13.5mmol/L should be carefully evaluated for the presence/progression of dementia and administered vitamins of the B group as a measure towards amelioration of the modifiable risk factor of cognitive decline, i.e. homocysteinemia.

Homocysteine

vitamin B12

folate

nutrition

cognitive impairment

Over the last two centuries, life expectancy has doubled globally [1]. In fact the number of elderly subjects in the world is expected to increase from 93 million observed in 2011 to an expected 323 million in 2050, India being the second highest contributor after China [2]. We are, therefore, faced with a consequent increase in prevalence of cognitive impairment [3]. At the same time, there is a continued burden of nutritional deficiencies in India [4]. Many biomarkers have been implicated in the development of cognitive impairment, notable amongst them being homocysteine. This molecule has the distinction (as opposed to other markers) of being modifiable through the appropriate administration of vitamins of the B group [5] It has been demonstrated that subjects in the highest quartile of homocysteine levels were more than two times as likely to be in the lowest quartile of neurobehavioral test scores as compared to those in the lowest quartile of homocysteine levels [6]. Also, serum homocysteine levels have been shown to bear a significant negative correlation with serum levels of vitamin B₁₂ as well as folate [5]. In addition, it has been postulated that the neuromodulatory effects of raised serum homocysteine act through the altered expression of matrix metalloproteinases [MMPs] especially MMPs 2 and 9 [7,8]. This study evaluates the role of homocysteine and these nutrient biomarkers in cognitive impairment (CI) as defined by Montreal Cognitive Assessment (MoCA) score in North Indian urban population [9].

From amongst patients presenting to the neurology OPD with complaint of forgetfulness, a total of 73 subjects were enrolled over a period of 3 months. All subjects were subject to evaluation of higher mental functions, including lobar functions, by a detailed questionnaire. Also, MoCA score was calculated for all subjects. They were then segregated into those with and without CI (MoCA score<25 and 25-30, respectively). Thus, we included 40 subjects with CI and 33 subjects without CI. Blood samples were drawn and the following analytes were estimated:

Vitamin B₁₂ and folate by chemiluminescent immunoassay
Homocysteine by microparticle enzyme immunoassay
MMP 2 and MMP 9 by ELISA

The data generated was subject to preliminary statistical analysis by SPSS version 20. All numerical variables were expressed as mean±SEM or median and interquartile range as the case may be. Significance of difference between the two groups was derived. Since MoCA score represents the cognitive ability, the Spearman correlation of all variables with MoCA score was derived along with their significance by univariate analysis. Further, the data was subject to multivariate logistic regression and the emerging significant factor, homocysteine, was evaluated for its prediction of MoCA<25 through its ROC, AUC NPV, PPV, sensitivity and specificity. Significance was ascertained at a p<0.05.

The descriptive statistics derived are given in Table 1. It was observed that age and homocysteine were the only parameters found significantly different in the two groups of subjects – those with and those without cognitive impairment; vitamin B12, folate, MMP2 and MMP9 were not significantly different in these two groups [Table 1].

Table 1

Baseline demographics and mean/median values for analytes estimated
Parameter	Subjects without CI (n = 33)	Subjects with CI (n = 40)	p value
Age in years, Mean (Range)	38.66±1.89 (25–65)	52.13±2.87 (22–78)	< 0.0001
Gender F/M	21/12	16/24	-
MOCA Score, Mean (Range)	27.59±0.29 (25–30)	19.33±0.74 (5–24)	< 0.0001
Vitamin B12 (pg/ml) Median (IQR)	415 (240.5-517.5)	220 (144–620)	ns (0.136)
Homocysteine µmol/L Mean (Range)	11.39±0.93 (7–36)	16.87±1.61 (5–49)	0.009
Folic Acid (ng/ml) Mean (Range)	9.45±1 (4–23)	10.67±1.12 (5–24)	ns (0.474)
MMP 2, Mean (Range) µg/ml	96.28±5.62 (4-139)	88.65±4.43 (4-137)	ns (0.092)
MMP 9, Mean (Range) µg/ml	89.69±16.40 (4-426)	161.74±25.83 (4-600)	ns (0.065)

Amongst the parameters assessed, age and homocysteine were the only ones that were significantly different in the two groups (with and without cognitive impairment)

Vitamin B₁₂ and folate were found to bear a significant negative correlation to homocysteine as shown in Table 2.

Table 2

Spearman’s correlation of homocysteine with vitamin B₁₂ and folate
Homocysteine vs	Correlation r	p value
Folic Acid	-0.351	0.003
Vitamin B₁₂	-0.438	< 0.0001
Homocysteine had a negative correlation with both vitamin B12 and folate

Table 3 gives the correlation of MoCA score to all the parameters estimated – homocysteine, folate, vitamin B12, MMP2 and MMP9. Of these, MoCA correlation only to homocysteine and vitamin B₁₂ were significant. Univariate analysis revealed that only homocysteine and vitamin B12 had a significant correlation with MoCA score.

In addition to estimating the circulating levels of the MMPs 2 and 9, the samples were processed for MMP 2 RNA expression as well. MMP2 gene is located on chromosome 16 at position 12.2.

Table 3

Spearman’s correlation of MoCA score with Homocysteine, vitamin B₁₂, folate, MMP2 and MMP9
MoCA score vs	Correlation r	p value
Homocysteine	-0.386	0.001**
Folic Acid	0.092	0.454
Vitamin B₁₂	0.224	0.005*
MMP 2	0.075	0.542
MMP 9	-0.129	0.290

Ct (constant threshold) was measured for expression of the SOI (sequence of interest; in this case, gene of interest) and RS (reference sequence or house-keeping gene) which was GAPDH in this case.

ΔCt was calculated

∴ ΔCt = CtSOI - CtRS

[Difference between Ct of SOI and Ct of RS, mostly house-keeping gene]

∴ ΔCt denotes the expression of MMP 2 gene.

Expression of MMP2 was calculated after normalization with GAPDH expression.

Pearson’s correlation was performed for the serum homocysteine levels and the MMP 2 gene expression. This is shown in Table 5.

Table 5

Homocysteine and Δ constant threshold of MMP 2 and their correlation
Analyte	Homocysteine In µmol/L	ΔConstant threshold of MMP 2 gene [no.of repeats]	Pearson’s correlation
Mean	12.84 ± 7.7	10.64 ± 2.48	p = 0.002

Univariate analysis of the data revealed that MoCA scores significantly correlated with homocysteine and vitamin B12. The correlation graphs are given in Fig. 1.

This data was further subject to multivariate logistic regression for evaluation before and after adjusting for sex and age. It emerged that homocysteine was the only significant independent variable for MOCA scores after adjusting for age and sex, as given in Table 6.

Table 6

Multivariate logistic regression for prediction of MoCA score
	Not Adjusted					Adjusted for Age, sex
	R Squared	Unstandardized Coefficients		t	p value	R Squared	Unstandardized Coefficients		t	p value
	R Squared	Beta	Std. Error	t	p value	R Squared	Beta	Std. Error	t	p value
(Constant)	0.224	29.717	1.623	18.309	< 0.001**	0.469	32.893	2.569	12.804	< 0.001
Homocysteine in µmol/L		-0.314	0.074	-4.223	< 0.001**		-0.223	0.066	-3.393	0.001**
Vitamin B12 In pg/mL		-0.005	0.002	-2.929	0.005**		-0.002	0.001	-1.373	0.174
signifies p value highly significant Durbin-Watson statistic 1.75**

Further, it was elucidated that the Durbin-Watson statistic was 1.75, which indicates a good positive autocorrelation. The statistical output was used to derive an equation for the prediction of MoCA based on plasma homocysteine levels.

MoCA score = 32.893 + [(-0.223)(homocysteine in µmol/L)]

The ROC curve was then drawn and area under the curve was quantified as shown in Fig. 3.

The characteristics of homocysteine > 13.5 µmol/L (the cut-off derived from the ROC curve) as a predictor for low MOCA score is given in Table 7.

Table 7

Predictive characteristics of homocysteine derived from the ROC curve
Homocysteine Cut-off	PPV	NPV	Sensitivity	Specificity
13.5 µmol/L	59.6%	79.2%	84.8%	50%

Summary of results

Amongst vitamin B12, folate, homocysteine and the MMPs 2 and 9, cognitive score (MoCA) indirectly correlated with homocysteine and vitamin B12. After adjusting for age and sex, homocysteine was the only significant independent predictor of MoCA score. Cut off of homocysteine for prediction of MoCA <25 was derived at 13.5 µmol/L (PPV=59.6%; NPV=79.2%; Sensitivity=84.8%; Specificity=50%). The equation derived for calculation of MoCA score from homocysteine is:

MoCA score = 32.893 + [(-0.223)(homocysteine in μmol/L)]

The term cognition encompasses all the brain functions related to memory, learning, spatial orientation, reasoning, judgement, planning, and problem solving. The Diagnostic and Statistical Manual of Mental Disorders 5 (DSM 5) defines key domains of cognitive function: executive function, learning and memory, perceptual motor function, language complex attention, and social cognition. Cognitive impairment is defined as deficits in cognitive ability that are acquired (as opposed to developmental), typically represent decline and may have an underlying brain pathology. The causes may vary between different types of disorders but most include damage to the portions of brain involved in cognitive abilities, i.e. frontal and temporal cortex, basal ganglia, limbic system (thalamus, hypothalamus, hippocampus, amygdala, cingulate gyrus) and even the cerebellum. The common causes are degenerative dementias, stroke with vascular/mixed dementia, genetic causes, traumatic brain injuries, metabolic factors of which homocysteinemia is well-known. Neuropathologically, in such cases, there may be depositions of β amyloid as plaques, tau protein tangles, presence of Lewy bodies, occurences of ischemic demyelination, infarcts with angiopathic changes in the brain. There has been supporting evidence suggesting association of homocysteinemia in cognitive functions from previous studies [10–13].

In 2002, approximately 5.4 million (22.2%) people of the USA who were elderly (> 70 years old) had cognitive impairment [14]. Of these, 10–15% of those with homocysteinemia progressed to Alzheimers Disease, whereas only 1-2.5% of those without homocysteinemia progressed to AD [15]. Qadri et al measured vitamins B12 and folate and homocysteine in 283 subjects. They observed that subjects in the lowest quartile of folate had an increased odds ratio for mild cognitive impairment (3.1) and dementia (3.8). they also observed that those who had homocysteinemia had an increased odds ratio (4.3) for dementia [16]. In a population based study by Schafer et al, linear regression models revealed that homocysteine was consistently and strongly associated with worse neurobehavioral test performance. The same study elucidated that subjects in the highest quartile of homocysteine levels were more than two times as likely to be in the lowest quartile of neurobehavioral test scores as those in the lowest quartile [17].

Through a case series, McCaddon demonstrated the beneficial effects of vitamin B12 and folate supplements on homocysteine as well as the cognitive score [18]. Malouf and Grimley conducted a randomized clinical trial in elderly subjects with raised circulating homocysteine levels with and without cognitive impairment. In those without cognitive impairment, they administered a daily dose of 800 mcg of folate for 3 years and demonstrated better global functioning (p = 0.033), better memory storage (p = 0.006). and better information processing speed (p = 0.016). Those who had cognitive impairment were daily administered choline esterase inhibitors and 1mg of folate for the same period resulting in better overall response (OR = 4.06; p = 0.02) and a better Nurse’s Observation scale for Geriatric Patients (p = 0.002) [19].

This study demonstrated that in our population, vitamin B12 bears a significant correlation with the cognitive score, whereas folate does not. Should we presume then that folate may not impact cognitive scores in the North Indian urban population? Maybe. In favour of this assumption is our retrospective data of approximately 48,000 subjects where we observed that the prevalence of deficiency of vitamin B12 and folate are 44.1% and 2.2%, respectively, which could explain the lack of correlation of the cognitive scores with folate [20]. In the current study, multivariate analysis, after adjusting for age and sex, elucidated that, amongst the analytes studied, homocysteine alone impacted the cognitive score. Further calculations indicated the cut-off of homocysteine as 13.5 µmol/L and an equation has been derived for calculating the MoCA score from homocysteine. In one of our previous studies, we demonstrated that the beneficial effects of B12 as well as folate in reducing homocysteinemia was irrespective of the initial levels of these vitamins, which means that with or without prevalent deficiency, homocysteine was lowered.[5] The role of homocysteine in causation of cognitive decline may be segregated into short-term and long-term events as given in the Fig. 4 below.

Short-term and long-term events in the progression of vitamin (B12, B6 and folate) deficiency and homocysteinemia-induced cognitive decline and its prevention: Early preclinical events could include successively – vitamin deficiencies, homocysteinemia, inhibition of post-synaptic GABA receptors and elevated MMP9, and phosphorylation of tau proteins. The long-term events could include accumulation of the phosphorylated tau proteins and amyloid-β-peptides into neurofibrillary tangles and amyloid plaques which would ultimately culminate in cognitive decline. Modified from Bhargava, 2018.[13]

Hence, it may be postulated that if homocysteine be reduced, through administration of the vitamins B12 and folate, the cascade of events may be prevented. This could reduce the burden of cognitive impairment and thence of dementia/Alzheimer’s disease.

It is, therefore, suggested that all subjects > 50 years of age (the lower age limit of our subjects demonstrating cognitive impairment) should be tested for circulating levels of homocysteine and those with a level > 13.5 µmol/L should be evaluated and followed up for cognitive decline. At the same time, they may be administered vitamins of the B group to reduce homocysteine and prevent or decrease the possibility of cognitive decline.

Further studies may be conducted to assess the molecular mechanism of cognitive impairment due to homocysteinemia.

Homocysteine > 13.5µmol/L predicts low MoCA (< 25) with 84.8% sensitivity and 50% specificity. So patients with a Hcy > 13.5µmol/L should be carefully evaluated for the presence/progression of dementia and administered vitamins of the B group as a measure towards amelioration of the modifiable risk factor of cognitive decline, i.e. homocysteinemia.

Funding:
This study was funded by the Research Development Program (RDP) of Sir Ganga

Ram Hospital, New Delhi, India, under project no: 4.9.35-010.

Competing interests:
The authors have no relevant financial or non-financial interests to disclose.

Author Contributions:
Seema Bhargava conceptualized the study, reviewed literature, formulated the design of the study, performed biochemical analyses for estimation of homocysteine, vitamin B12 and folate, compiled the data, participated in analysis of the data, participated in writing and editing the manuscript and formulated the hypothesis.

Prahlad K Sethi helped in formulation of the design of the study, identified subjects,

assessed their cognitive abilities, reviewed and edited the manuscript.

Anuradha Batra helped in formulation of the design of the study, identified subjects,

assessed their cognitive abilities, reviewed and edited the manuscript.

Sangeeta Choudhury helped in formulation of the design of the study, performed analyses of samples for MMPs 2 and 9, helped in statistical analysis, helped in editing the manuscript.

Parul Takkar helped in compilation of data and performed statistical analyses

Anjali Manocha, Mamta Kankra and Parul Singla helped in review of literature, formulation of the design of the study, performing biochemical analyses for estimation of homocysteine, vitamin B12 and folate, compilation of data, editing of manuscript.

Ethics approval:
This study was approved by the institutional ethics committee under no: EC/01/16/925.

Consent to participate:
Informed consent was obtained from all individual subjects included for

participating in the study and publishing of the data as per the consent form approved

by the ethics committee.

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Table 4 is not available with this version

No competing interests reported.

Homocysteine and Nutritional Biomarkers In Cognitive Impairment

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Journal Publication

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Abstract

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Introduction

Materials And Methods

Results

Discussion

Conclusions

Declarations

References

Table

Additional Declarations

Status:

Journal Publication

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