The Relationship between Dietary Patterns and Depression is mediated by Serum Level of Folate and Vitamin B12: A Case-Control Study

Background: Major depressive disorder is among main worldwide causes of disability. The low medication compliance rates in depressed patients as well as the high recurrence rate of the disease can bring up the nutrition-related factors as a potential preventive or treatment agent for depression. The aim of this study was to investigate the association between dietary patterns and depression via the intermediary role of the serum folate and vitamin B12, total homocysteine, tryptophan, and tryptophan/competing amino acids ratio. Methods This was an individually matched case-control study in which 110 patients with depression and 220 healthy individuals, who completed a semi-quantitative food frequency questionnaire were recruited. In addition, to measure the serum biomarkers 43 patients with depression and 43 healthy people were randomly selected from the study population. To diagnose depression the criteria of Diagnostic and Statistical Manual of Mental Disorders, fourth edition, were utilized. Results The ndings suggest that the healthy dietary pattern was signicantly associated with a reduced odds of depression (OR: 0.75; 95% CI: 0.61-0.93) whereas the unhealthy dietary pattern increased it (OR: 1.382, CI: 1.116–1.71). The mediation analysis showed that the healthy dietary pattern was associated with a reduced risk of depression via increased serum levels of the folate and vitamin B12; however, the unhealthy dietary pattern was associated with increased risk of depression via decreased serum levels of folate and vitamin B12, based on tree adjusted logistic regression models. Conclusion Dietary patterns may be associated with depression by changing the serum levels of folate and vitamin B12. Further studies are required to conrm the mechanism.

utilized. Results The ndings suggest that the healthy dietary pattern was signi cantly associated with a reduced odds of depression (OR: 0.75; 95% CI: 0.61-0.93) whereas the unhealthy dietary pattern increased it (OR: 1.382, CI: 1.116-1.71). The mediation analysis showed that the healthy dietary pattern was associated with a reduced risk of depression via increased serum levels of the folate and vitamin B12; however, the unhealthy dietary pattern was associated with increased risk of depression via decreased serum levels of folate and vitamin B12, based on tree adjusted logistic regression models. Conclusion Dietary patterns may be associated with depression by changing the serum levels of folate and vitamin B12. Further studies are required to con rm the mechanism.

Background
Depression is considered as one of the most common psychological conditions. It is a major causative factor for the disability-adjusted life years lost in the world and currently 322 million people in the world are suffering from the disorder (1). Depression is highly prevalent in the developed and developing countries. Nearly 16 million American adults have experienced a major depression, in 2015 (2). In Iran, results of investigations show that 4.1 percent of adults are affected by major depressive disorders (MDD), while women are 1.95 times more probable to develop MDD than men (3).
It has been long con rmed that diet affects depression. Previous studies investigating the association between diet and depression have mostly considered on food groups (4,5), foods (6) or nutrients (7); whereas, few studies have focused on the dietary patterns. Considering the complex interactions between nutrients and foods, dietary pattern approach was suggested by nutritional epidemiologists to study the diet-disease relationships (8). Morever, this approach provides a more comprehensive insight into the dietbrain relation (9).
There is a body of documents indicating the association between dietary patterns and risk of depression.
A study on Australian women showed that lower odds of depression was associated with a "traditional" dietary pattern including high amounts of vegetables, fruit, meat, sh, and whole grains (9). Another study suggested adherence to "processed food" as a dietary pattern increased the risk of depression, while "whole food" consumption decreased it among British middle-aged women. The nding was con rmed by other researchers (10,11). There are some studies which have considered the potential role of biomarkers and food intake in depression simultaneously (12,13).
However, the mechanism(s) by which dietary factor(s) affect depression is not clearly understood.
Previous studies, on the association between food patterns and depression rarely considered biochemical measures along with dietary patterns.
Ergo, the mechanism through which dietary intake patterns and depression are co-related is not clearly understood. In the present study, the mediatory role of folate, vitamin B12, tHcy, Trp, and Trp/Caa ratio in modi cations of dietary patterns to decrease risk of depression is investigated.

Sample size and study design
It was an individually matched case-control observational study. To extract dietary patterns, 110 patients with depression and 220 healthy individuals were recruited. The participants included 260 female (87 depressed and 173 healthy) and 70 male (22 depressed and 48 healthy).We selected the depressed patients from psychiatric clinic of Baharloo hospital in district 15, Imam Hossein hospital in district 7 and Tehran University student counseling center in district 6 through non-probable convenience sampling. After adjusting for age and sex, healthy individuals were selected from the area of residence of hospital patients and students of Tehran University. The samples selection and data collection were performed during October 2012 to June 2013.
To measure serum folate, vitamin B12, tHcy, Trp, and Trp/Caa ratio a total of 86 individuals (n=43 equally distributed in case and control groups) were randomly selected from the participants.

Inclusion criteria
People with an age range of 18 to 65 years, resided in Tehran, and who have been diagnosed with the disease for a maximum of 3 months were entered in case group. The participants included 260 female (87 depressed and 173 healthy) and 70 male (22 depressed and 48 healthy).

Exclusion criteria
People with cognitive impairment or other psychotic illnesses, severe depression or lacking the ability to cooperate and answer the questions, hormonal disorders such as Addison's, Cushing's disease, hyperthyroidism, hypothyroidism, hyperparathyroidism, cancer, heart disease, diabetes, stroke, bromyalgia, kidney, or liver failure, multiple sclerosis, Parkinson disease, a history of trauma, cuts, fractures, bleeding, burns, and other similar events in the past three months resulting in unconsciousness and hospitalization, chronic and infectious diseases such as HIV, mononucleosis, tuberculosis, viral hepatitis, and pneumonia in the past two weeks, those who consumed anti-depression drugs, those who had addiction to alcohol or drugs at the time of the study or in the past three months, body mass index (BMI) ≥40 kg/m2, pregnancy and lactation at the time of the study or in the past year, a special diet in the past two months, any type of special diet for more than two months in the past year, B12 injection more than once in the past six months, B-complex injection more than once per month for at least six months in the last year, and any dietary supplement in injection or oral form in the past three months were excluded from the study.

Recruitment and Matching
Our samples were recruited from two psychiatric clinics in Tehran, capital of Iran. The participants' major depressive disorder(s) was diagnosed by psychiatrists us ing the criteria of the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) (18). These patients had no history of depression in the past year. For the control group, lack of major depres sion disorder(s) was con rmed based on Beck Depression Inventory question naire (BDI-II) (19), standardized in Iran (20). The control group members had no history of depression in the past year.
To invite eligible individu als, the interviewers went to each patient's residential area and described the aim of the study. Patients were matched with controls based on gender, age, and residential area. Each patient with depression was matched with two members of the control group.
To identify the dietary patterns, the open Epi software was used (21,22).

Assessment of covariates
A demographic questionnaire was used to collect information about participants' general characteristics and some confounders. To calculate BMI weight and height were obtained from all participants. For quantitative measurement of anxiety, as a confounder, the Iranian standardized Beck Anxiety Inventory was utilized (23). Participants' dietary intakes in the last 12 months were assessed using a valid and reliable semi-quantitative food frequency questionnaire (FFQ) (24). A validated physical activity questionnaire consisting of nine levels of activity was applied (25,26).

Assessment of biochemical markers
In order to measure the biochemical markers, 5 ml blood samples were collected from participants after 12 hours of fasting from. The samples were transferred into tubes with no anticoagulant. After centrifuging for 20 min at 1,500 g in room temperature, the serum was separated and stored at -70°C. Serum folate and vitamin B12 were measured by gamma method (SimulTRAC-SNB Radioassay Kit; Becton Dickinson, Orangeburg, NY, USA). Total homocysteine (tHcy) concentration was also analyzed using the modi ed immuno-enzyme method (Axis-Shield, UK). Plasma levels of amino-acids were determined by a modern High-Performance Liquid Chromatography (HPLC) device with uorescent detector. Initial preparation involved the precipitation of serum protein with methanol. Simultaneous derivatization was performed with ortho-phthaldehyde. After derivatization, 10μl of each sample was injected into the HPLC device. The device model was YoungLin Acm 3000 HPLC (Young Lin Instrument Co. Ltd., Anyang, Korea). Later, HPLC was carried out on a 4.6 × 250 mm column of Inertsil ODS 5 μm, at 37 °C in the wavelength range of 340-420 nm, which took one hour for each sample. Within-and between-assays precisions were 4.8-6.8 and 6.5-8.5 for all amino acids, respectively.

Statistical analysis
The normality of covariates was tested by Kolmogorov-smirnov. To compare variables in two groups were used t-test or Mann-Whitney. The association between dietary patterns and depression was investigated by logistic regression models after adjusting for the confounders in multiple logistic regression models. In addition, the goodness of t for these logistic regression models was examined by logical con dence intervals and Hosmer and Lemeshow test. Furthermore, we used multiple logistic regression to test the mediatory variables (27).
The mediatory analysis was designed to determine the mediator variable, among the biochemical markers, in the relationship between dietary patterns and depression. Logistic regression was employed due to the dichotomous nature of the dependent variable. We designed tree models to determine whether a variable was a mediator or not.
After these analyses, we selected the mediator variables in the case that: 1. A signi cant association was con rmed between dietary patterns and depression 2. By adding the mediator variables into the rst model, the relationship between dietary patterns and depression did not remain signi cant. Since dietary pattern is related to depression via mediator variables, by adding mediators into the model, the relationship between dietary patterns and depression is transferred into the relationship between mediator variables and depression. Therefore, a third model should be designed to ensure the signi cant relationship between the mediator variables and depression.
3. In the third model, the relationship between the mediator variables and depression should be signi cant (27).
The healthy dietary pattern in this study was de ned as high in fruits as well as cruciferous, yellow, green, leafy, and other vegetables, low fat dairy, whole grains, nuts, and olives. Unhealthy dietary pattern was high in re ned grains and breads, high fat dairy, solid oils, liquid oils and mayonnaise, pickle, snacks, soft drinks, industrial fruits and juice, red meats, poultry, processed meats, and sweets.
In the current study, the mediatory analysis was performed after adjusting for the confounding variables.
In other words, the confounding variables were present in all three aforementioned models. All statistical analyses were carried out using SPSS (version 20; Chicago, IL).

Results
Some comparisons between groups are shown in Table 1 and 2. Physical activity as a confounder was signi cantly higher in control group than the patients (p=0.004) and daily energy intake was not signi cantly different between groups (p=0.06).
According to the ndings, we recognized two dietary patterns of healthy and unhealthy foods. The healthy dietary pattern led to signi cantly lower odds ratio for depression (OR: 0.39, CI: 0.17-0.92), whereas, the unhealthy dietary pattern resulted in signi cantly higher odds ratio (OR: 2.6, CI: 1.04-6.08). Table 3 illustrates the relationship between biochemical markers and depression before and after adjustment for the confounding factors. Based on the information tabulated in Table 3, just folate and vitamin B12 had signi cant relationship with depression.
The results showed that dietary patterns and depression were co-related via the intermediary role of folate and vitamin B12, which was initially due to the signi cant association of dietary patterns with depression as well as the signi cant association of folate and vitamin B12 with depression. Table 4 illustrates the association between dietary patterns and depression after adjusting for the confounding factors (Model 1). To test the hypothesis, folate and vitamin B12 were added into the regression model 2. By adding these covariates, the signi cance association between dietary patterns and depression was eliminated.
Therefore, these vitamins can be assumed as intermediate variables.
To con rm the results, the relation of folate and vitamin B12 with depression was examined (Table 4 -model 3). This mediatory role was con rmed with the signi cant results of model 3.
Based on Table 4, the healthy dietary pattern is related to decreased depression by increasing the serum level of folate and vitamin B12. However, the unhealthy dietary pattern is related to increased depression via reduction of the serum level of folate and vitamin B12. All three models were adjusted for participants' job, education, marital status, children number, smoking and hookah use, depression history, unemployment history in the past ve years, tragic events in the past six months and through the whole lifetime, daily energy intake, physical activity, and dietary patterns.

Discussion
We compared a number of variables among the two groups and there was not a signi cant difference. It shows that we've matched enough cases and controls su ciently.
Of course physical activity as a confounder was signi cantly higher in controls than the patients. Low levels of physical activity are expected in patients with depression, owing to the reduction of energy levels is one of the hallmarks of the disease. Additionally, daily energy intake was not signi cantly different between the patients and control group. Lack of signi cant difference in energy consumption between the two groups was probably due to the fact that patients were new cases. In other words, the duration of their disease was short and the disease-related disorders had not affected their food choices yet.
In the current study, as the most important result, the intermediary role of serum folate and vitamin B12 was investigated in the association between dietary patterns and depression. According to our results, the increase of serum folate and vitamin B12 in healthy dietary pattern and the decrease of these two vitamins in unhealthy dietary pattern were related to depression.
Generally, healthy dietary patterns contain more healthy foods such as folate and vitamin B12, which have a preventive effect against depression (28). In the healthy dietary pattern, fruits and vegetables are considered as healthy foods, which contain more folate, compared to unhealthy dietary pattern with low consumption of fruits and vegetables. It is important to note that consumption of several food groups that form a dietary pattern does not mean absence of other food groups in that pattern. In other words, other food groups may also have a limited contribution in a food pattern. For example, while meat is not necessarily included as a speci c item in healthy dietary patterns, it never implies complete lack of meat intake. In fact, it means that meat consumption is lower than other food group. In the case of vitamin B12, the human body only needs very low amount of this vitamin, which might be met by low consumption of meat in healthy dietary patterns. Besides, the healthy dietary pattern includes milk as a good source of vitamin B12.
Moreover, the interaction between several foods could intensify the protective effect of folate and vitamin B12 against depression. For instance, in addition to folate and vitamin B12, fruits and vegetables contain non-nutrients with bene cial effects on health in healthy dietary patterns. One notable example of nonnutrients are dietary bers, which improves both the immune and in ammatory functions by affecting the intestinal microbiota. Besides, phytochemicals, consumed along with dietary bers, can reduce the oxidative stress and eventually in ammation (29). Impaired immune function and in ammation are also observed in various disorders, including depression. Furthermore, the interaction between different foods of the unhealthy dietary patterns could also weaken the protective role of folate and vitamin B12 against depression. It is important to note that low attendance of unhealthy food items in healthy dietary patterns could also reduce the risk of depression.
The methylene tetra hydro folate reductase (MTHFR) reduces 10-Methylenetetrahydrofolate to 5methylenetetrahydrofolate, which is the main active circulatory form of folate. Furthermore, 5-Methylenetetrahydrofolate has a key role in one-carbon metabolism and is essential for methylation of homocysteine to methionine. Methionine is the prerequisite of S-adenosylmethionine and is considered as a common methyl group donor in various methylation reactions in the brain for the synthesis of neurotransmitters such as dopamine, serotonin, and noradrenalin; all play an important role in the etiology of depression (30,31).
In contrast to the previous results (32)(33)(34)(35), we observed no signi cant difference in the mean serum level of tryptophan and Trp/Caa ratio between the case and control groups, before and after adjustment for the confounding factors. Tryptophan hydroxylase is an enzyme responsible to control the serotonin synthesis. In normal condition, only half of this enzyme is saturated with tryptophan. Therefore, alteration of the tryptophan concentration in blood changes the enzyme saturation and eventually the extent of serotonin synthesis. Since valine, leucine, tyrosine, phenylalanine, and isoleucine compete with tryptophan to pass the blood brain barrier, the tryptophan/competing amino acids (Trp/Caa) ratio should rise to access the brain with more tryptophan (36). The participants of previous investigations were all either under antidepressant treatment or resistant to the treatment. However, patients of the current study were all new cases of depression with no intake of anti-depressant drugs. Therefore, it might be assumed that at the beginning of the disease, the patient's body might prevent reduced brain access to tryptophan through a compensatory mechanism. Joyce et al. compared patients with a different genetic polymorphism in the promoter region of the serotonin transporter and other depressed patients. These investigators noted that the rst group had higher Trp/Caa ratio and poorer response to serotonergic antidepressant drugs than the second group (37). Thus, changes in the Trp/Caa ratio among our depressed patients might stem from the polymorphism in their serotonin transporter gene. Due to the lack of the molecular analysis, the possible relationship of genetic polymorphisms in depression-related genes with clinical status or biochemical parameters was not discussed in the current study.
Findings show that the patients were less educated than healthy people. It is documented that high educated people are more probably to choose healthier food, and have more health checks and better health outcomes (38,39). This issue can also be seen in high-risk groups such as pregnant mothers or immigrants who are more at risk for depression (6).
Considering the study limitations, the following two factors can be mentioned. Similar to all case-control studies, the temporal relationship between depression and dietary patterns was not realized. Furthermore, due to the nancial restrictions biochemical measurements were not conducted for all participants.
To decrease recall bias, which is a limitation of case-control studies, we selected new cases among depressed patients and to minimize selection bias, another limitation of case-control studies, we selected control group from the area in which patient individuals were inhabited. To distribute confounders in two groups homogenously in addition to age and gender the participants were matched according to socioeconomic status, as well.
As a case-control study, our information can help individuals to understand the differences of dietary patterns between patients with depression and healthy individuals. We assessed the relationship between dietary patterns and depression before and after adjusting for many confounders and depression risk factors. All adjusted confounders were similar to the confounders of other studies (40)(41)(42)(43), except for the dietary pattern, which was adjusted in just one study (44). In one investigation on non-new cases of depression, the effect of anti-depression drugs was adjusted as a confounder (45). Nevertheless, the patients of our study were all new cases with no drug use history so we controlled this confounder statistically.

Conclusion
The healthy dietary pattern is related to the risk of depression via increasing the serum level of folate and vitamin B12. However, the unhealthy dietary pattern is related to the risk of depression via decreasing the

Consent for publication
Not applicable.

Availability of data and material
The dataset supporting the conclusions of this article can be made available upon request after approval by the authors.

Competing Interest
The authors declare that they have no con ict of interest.

Funding
This study was funded by the Research Deputy of Tehran University of Medical Sciences (TUMS) by the grant number 19374-161-03-91. TUMS led all aspects of the study, including the design, analysis, interpretation of data, and writing of the manuscript.
Authors' contributions MK contributed to conception and design; contributed to analysis and interpretation; drafted the manuscript; gave nal approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy. GS contributed to conception and design, contributed to analysis and interpretation; gave nal approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy. MA critically revised the manuscript; gave nal approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy. FR contributed to conception and design; contributed to analysis and interpretation; gave nal approval, and agreed to be accountable for all aspects of work ensuring integrity and accuracy. AM and MH gave nal approval; and agreed to be accountable for all aspects of work ensuring integrity and accuracy. All authors read and approved the nal version of the manuscript.