L. Gao1, W. Ge1, C. Peng1, J. Guo1, N. Chen1, L. He1
1. Department of Neurology, West China Hospital of Sichuan University, Chengdu, China
Corresponding Author: Dr Li He, Department of Neurology, West China Hospital of Sichuan University, #37 Guoxue Xiang, Chengdu, China, E-mail: email@example.com, Telephone: +86 18980601679, Fax Number: 85422327; Dr Ning Chen, Department of Neurology, West China Hospital of Sichuan University, #37 Guoxue Xiang, Chengdu, China, E-mail: firstname.lastname@example.org, Telephone: +86 18108080230, Fax Number: 85422327
J Prev Alz Dis 2022;
Published online April 22, 2022, http://dx.doi.org/10.14283/jpad.2022.39
Background: Despite reports on neuroprotective effects of dietary theobromine intake, whether dietary theobromine has beneficial effects on cognitive function is unclear.
Objectives: To investigate the association between dietary theobromine and cognitive function.
Design: A cross-sectional study.
Setting: Data were collected from the 2011-2014 cycles of the National Health and Nutrition Examination Survey conducted by the Centers for Disease Control and Prevention of the USA.
Participants: A representative American population aged ≥60 years.
Measurements: L-theobromine was treated as a log transform and dichotomous form (the highest quantile vs. others). Cognitive function was measured using four tests: Consortium to Establish a Registry for Alzheimer’s Disease Word Learning tests, Consortium to Establish a Registry for Alzheimer’s Disease delayed recall test, animal fluency test, and digit symbol substitution test. We conducted multiple regression analyses and subgroup analyses to study the association between theobromine and cognitive performance. Basic characteristics, lifestyle factors, disease history, and nutritional intake were adjusted for in these models.
Results: A total of 2,845 participants were included in the study. The highest quantile of L-theobromine intake was positively associated with sores of delayed recall, animal fluency, and digit symbol substitution tests (β, 95% confidence interval, P: 0.11, -0.00-0.30, 0.049; 0.50, 0.02-0.99, 0.043; 1.55, 0.33-2.77, 0.015; respectively) in the fully adjusted model, but not with immediate recall score (β=0.13, 95% confidence interval -0.16-0.43, P=0.361). Subgroup analyses showed that L-theobromine intake was associated with cognitive performance in the highest quantile of caffeine intake.
Conclusions: Daily theobromine intake was associated with cognitive performance in a large nationally representative population. However, further research is needed to corroborate our findings.
Key words: Theobromine, cognitive performance, American population.
Age-related cognitive decline, characterized by impairment of episodic memory, working memory, and attention, can affect the quality of life. Nutritional conditions are reportedly involved in degenerative cognitive impairment (1, 2). Since previous studies have reported a protective effect of chocolate (3, 4), and theobromine is one of its main active components (5), it could also be associated with cognitive function.
In animal experiments, dietary theobromine has been suggested to exert cognitive protection. In addition, theobromine intake is reported to be capable of crossing the blood-brain barrier in mice. Theobromine is thought to exert protective effects through regulating neurotransmitters. Mendiola-Precoma et al (6). found that theobromine intake could improve the expression of the A1 receptor. Theobromine might play a role in phosphodiesterase inhibitors to enhance motor learning skills (7).
Despite this evidence, few population-based studies have investigated the association between dietary theobromine and cognitive function. Therefore, we included a large, representative sample of American participants aged ≥60 years from the cross-sectional National Health and Nutrition Examination Survey (NHANES) dataset to study the association between daily theobromine intake and cognitive performance. We hypothesized that daily theobromine intake would be positively associated with cognitive performance.
The NHANES is a complex, stratified, multistage sampling designed cross-sectional survey conducted by the Centers for Disease Control and Prevention of the USA to assess the health and nutritional status of Americans (8). The survey cycle has been running every two years since 1999. In this study, we retrieved data from the 2011-2012 and 2013-2014 cycles. A total of 3,632 participants aged ≥60 years who were qualified as understanding English, Spanish, Korean, Vietnamese, traditional or simplified Mandarin, or Cantonese were eligible for the cognitive function questionnaire. A total of 508 participants did not answer the cognitive function questionnaire; 269 participants were not available for dietary theobromine intake. Finally, 2,854 participants were analyzed (Figure 1).
All NHANES surveyors were eligible for dietary interviews. Trained interviewers used an automated data collection system under the guidance of an examination protocol. A 24-hour dietary questionnaire from the 2011-2012 and 2013-2014 cycles was used to obtain data on the daily theobromine intake of Americans. The 24-hour dietary questionnaire collected the types and amounts of all beverages and foods consumed within 24 h prior to the interview. Subsequently, energy and 64 nutrients from each beverage and food were calculated from the U.S. Department of Agriculture’s Food and Nutrient Database for Dietary Studies 2011-2014. More details are available at www.ars.usda.gov/ba/bhnrc/fsrg.
Based on previous studies, we also compared nutrients data reported to be associated with cognitive function, including total energy intake (9), protein (10, 11), lutein, zeaxanthin (13), folic acid (13–15), vitamin B12 (14, 16), vitamin B12 (14, 16), vitamin D (17, 18) magnesium (19), iron (20), zinc (20), copper (20), selenium (20), alcohol, and caffeine (21, 22). Dietary theobromine and other potentially confounding dietary nutrients were treated as continuous variables. Since the cutoff of theobromine intake quantile was 0 (25%), 0 (50%), and 43[mg/day] (75%), we used 43 as the grouping criteria.
In the 2011-2014 NHANES cycles, three cognition tests were employed: the Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) word learning and recall test, the animal fluency test, and the digit symbol substitution test (DSST). The reliability of these tests for evaluating cognitive function in Americans has been validated (23–25). The participants completed a cognitive test using online tests. The CERAD word learning and recall tests assess immediate and delayed learning abilities for new verbal information, respectively (25). The animal fluency test evaluates the component of executive function, categorical verbal fluency (26). The DSST is a performance module from the Wechsler Adult Intelligence Scale that evaluates processing speed, sustained attention, and working memory (27). Poor cognitive performance was defined as the lowest quartile of these four scores as in previous references (28).
Demographic variables, including age, sex, race, education, marital status, home status, employment status, smoking status, body mass index (BMI), and history of disease were also recorded. Age was treated as a continuous variable and stratified by 10-year age group (60-69, 70-79, and ≥80 years). Race was categorized as Mexican American, other Hispanic, non-Hispanic white, non-Hispanic black, non-Hispanic Asian, and other races including multi-racial. Education was classified as less than 9th grade, 9-11th grade (including 12th grade with no diploma), high school graduate/general education development/diploma (GED) or equivalent, some college or Associate of Arts (AA) degree, and college graduate or above. Marriage was categorized as married/living with a partner, widowed/divorced/separated, and never married. Home status referred to whether participants owned their living house or did not own their own home (other arrangements). Smoking status was defined as never (never smoked or smoked <100 cigarettes in their life), previous smoker (smoked ≥100 cigarettes in their life and currently no longer smoking), and current smoker (smoked ≥100 cigarettes in their life and currently smoking). BMI was classified as underweight
(<18.8 kg/m2), healthy and underweight weight (>=18.9-24.9 kg/m2), overweight (>=25-29.9 kg/m2), or obese (>=30 kg/m2). If participants were diagnosed with hypertension, diabetes, stroke, heart disease, or depression, the history of the disease was binarily recorded as yes or no. Heart disease was recorded positive with previous congestive heart failure, coronary heart disease, angina pectoris, and medically confirmed heart attack. Hypertension, diabetes, and stroke were defined based on previous medical diagnoses. Depression was defined as a patient health questionnaire-9 (PHQ-9) score >5.
All analyses were weighted according to the 2011-2016 NHANES analytical guidelines (29). Weighting was required to perform the NHANES analyses. The sample weight is assigned to each NHANES participant in different questionnaires to estimate the representativeness of a U.S. civilization. We used the 2-yeat weight collected from the dietary questionnaire in this study. The original weight was calculated by dividing by two in the final analyses. Continuous variables are presented as weighted mean ± SE, and categorical variables as unweighted numbers and weighted percentages. Mean levels of continuous variables were compared using Student’s t-test, and categorical variables were compared using chi-square tests between the theobromine subgroups. As shown in Supplementary Figure 1, the distribution of daily theobromine was not normal. Thus, we applied a log transformation and the original form in the later association analysis. We applied weighted regression analyses to study the association between theobromine intake and cognitive scores and poor cognitive performance, adjusting for age, sex, race, education, employment status, disease history, as well as dietary lycopene, zinc, and caffeine. Potential confounders associated with word learning test scores, recall scores, animal fluency test scores, and DSST (P <0.1) in the univariate regression model were first selected. We further selected confounders by a change of more than 10% in the effect estimation (29). We then excluded covariates with multicollinearity (variance inflation factor >5). Supplementary Tables 1 to 4 show the associations of each confounder with cognition performance, effect variation, and multicollinearity results. For the log form of theobromine, a linear regression model was applied; for impaired cognition performance, a logistic regression model was used. Daily theobromine intake was analyzed as a log transform and dichotomous in these analyses. We also conducted a subgroup analysis to study the association between theobromine intake (dichotomous form) and cognition function scores. Age subgroups, sex, race, and quantiles of caffeine intake were used as stratification factors. The subgroup analyses applied multiple linear regression analysis with adjusted variables in the full model, except for the stratification variable. Statistical significance was set at p < 0.05. All analyses were performed using the R version 4.0.0 (R Core Team (2020), Vienna, Austria) (30).
Basic characteristics of survey participants from the NHANES 2011-2012 and 2013-2014
Participants completed a cognitive questionnaire together with the first dietary intake questionnaire from the 2011-2012 and 2013-2014 NHANES. As shown in Figure 1, this study included 2,854 representative U.S. participants. As shown in Supplementary Table 5, the basic characteristics of the included and excluded participants were not significantly different.
The mean age of study participants was 69.6 years with males accounting for 48.6%. The mean scores of the CERAD word list learning test, CERAD recall test, animal fluency test, and DSST of the study participants were 19.2, 6.0, 16.7, and 45.6, respectively. The mean CERAD word list learning and recall test scores were 0.2 points higher in the subgroup of theobromine intake >= 43 mg/day than in the opposite subgroup. The animal fluency test and DSST scores were significantly higher in the theobromine intake ≥ 43 mg/day subgroup. The mean theobromine intake was 81.8 mg/day. Impaired animal fluency and the DSST were also significantly different between the two subgroups. A total of 20.6% of participants with an impaired animal fluency test were counted for theobromine intake ≥ 43 mg/day, while impaired DSST participants accounted for 18.5%. The characteristics of the other covariates are listed in Table 1. The daily nutrient intake by theobromine subgroups is shown in Table 2. Caffeine intake was significantly higher in the subgroup of theobromine intake < 43 mg/day.
1: Categorical variables were presented as the unweighted sample size (weighted percentage); Continuous variables as mean ± SE; Abbreviations: BMI, Body Mass Index; GED, General Education Development/Diploma; AA, Associate of Arts; CERAD, Consortium to Establish a Registry for Alzheimer’s disease.
Continuous variables as mean ± SE.
Association between dietary theobromine intake and cognition performance in the 2011-2012 and 2013-2014 NHANES
As shown in Supplementary Figure 1, daily theobromine intake did not follow a normal distribution; however, the log-transformed theobromine intake did. Thus, we used a log transform of daily theobromine intake to analyze the association between dietary theobromine and cognitive performance, as shown in Table 3. In the initial model, log transformation of daily theobromine intake was significantly associated with immediate recall test score (β=0.59, 95% confidence interval [CI] 0.15-1.03, P=0.012), animal fluency test score (β=0.73, 95% CI 0.19-1.26, P<0.001), and DSST score (β=1.53, 95% CI -0.00-3.05, P=0.047), but not significantly with the delayed recall test (β=0.17, 95% CI -0.04-0.39, P=0.113). In the fully adjusted model, dietary theobromine intake was significantly associated with cognitive performance. In addition, theobromine intake ≥ 43 mg/day was associated with better scores of delayed word recall test (β=0.15, 95% CI -0.00-0.30, P=0.049), animal fluency test (β=0.50, 95% CI 0.02-0.99, P=0.043), and DSST (β=1.55, 95% CI 0.33-2.77, P=0.015) in the fully adjusted model.
1: Age, sex, race, education, employment status, disease history, as well as dietary lycopene, zinc, and caffeine levels were adjusted; Abbreviations: CI, Confidential interval; CERAD, Consortium to Establish a Registry for Alzheimer’s Disease.
For poor cognitive performance, the log form of theobromine was not significantly associated with impaired cognition performance. Theobromine intake ≥ 43 mg/day was significantly inversely associated with poor animal fluency (odds ratio [OR]=0.95, 95% CI 0.91-0.99, P=0.028) and DSST performance (OR=0.95, 95% CI 0.92-0.98, P=0.003) only in the crude model (Table 4).
1: Age, sex, race, education, employment status, disease history, as well as dietary lycopene, zinc, and caffeine levels were adjusted; Abbreviations: OR, odds ratio; CI, confidence interval; CERAD, Consortium to Establish a Registry for Alzheimer’s disease.
To further analyze any non-linear associations, we conducted a curve-fitting analysis of theobromine intake and cognitive performance. A non-linear association was found, as shown in Supplementary Figure 2 and 3.
Subgroup association between dietary theobromine intake and cognition performance in the 2011-2012 and 2013-2014 NHANES
Figures 2 to 5 present subgroup analyses of the association between theobromine intake ≥ 43 mg/day and word learning test score, recall score, animal fluency test score, and DSST score, respectively. The association between theobromine and cognitive performance was stable. Theobromine intake tended to be associated with better cognitive performance in the higher caffeine intake subgroups.
CERAD, Consortium to Establish a Registry for Alzheimer’s Disease.
CERAD, Consortium to Establish a Registry for Alzheimer’s Disease.
In this study, we found that daily theobromine intake was associated with CERAD-delayed recall and animal fluency scores, as well as DSST scores in a representative American population. However, dietary theobromine intake was not significantly associated with the CERAD-immediate recall score. This study could be applied to adults aged ≥60 years in the U.S.
Previous studies have explored different dietary patterns that affect cognitive function. Fernández-Fernández et al (31). reported that an LMN diet rich in polyunsaturated fatty acids and polyphenols derived from dried fruits and cocoa could enhance cognitive reserve function in mice. Theobromine and caffeine are both active methylxanthine components of cocoa (32). Theobromine accounts for a relatively high quantity compared to caffeine. A randomized controlled trial (RCT) focusing on the psycho-pharmacology of methylxanthines showed a protective effect on cognitive function (32). In a Portuguese prospective cohort study, 531 participants aged ≥65 years with normal cognitive function were followed up for a median of 48-month to detect the association between chocolate intake and cognitive impairment as measured by the Mini-Mental State Examination (4). In this study, researchers reported that long-term chocolate intake was inversely associated with cognitive decline. Another RCT in Japan also investigated the effect of chocolate intake on cognitive function (3). The intervention group received dark chocolate daily for 30 days. The modified Stroop color word test and digital cancellation test were conducted to test the association between dark chocolate intake and cognitive function, finding that dark chocolate has a beneficial role in cognitive function.
We selected a large, representative American population aged ≥60 years to further study the association between dietary theobromine and cognitive performance and found a significant association between daily theobromine intake and DSST score. However, Mitchell et al. reported that theobromine (700 mg) alone or a combination of theobromine (700 mg) and caffeine (120 mg) has no effect on DSST scores in 29 healthy female participants (33). In the caffeine intake over 102 mg/day subgroups, theobromine intake over 43 mg/day was significantly associated with improvement in the DSST score compared to the subgroup without theobromine intake. Thus, a higher theobromine intake and a larger study sample size might be necessary to observe a positive association between dietary theobromine and DSST scores.
Islam et al. (34) reported that theobromine could improve the working memory of rats through the CaMKII/CREB/BDNF pathway. The DSST is a working memory task that reflects the execution ability. Thus, dietary theobromine intake may exert cognitive protection through similar mechanisms. However, more detailed experiments will be required in future studies.
There are several limitations to our study. First, owing to the intrinsic limitations of the cross-sectional design, our study cannot conclude a causal association between theobromine intake and cognitive performance. Rigorous, prospective cohort studies or RCTs are required to validate our results. Second, we excluded participants with unavailable daily dietary data or incomplete cognitive test scores, which could have biased our findings. In Supplementary Table 1, we compare the basic characteristics of the included and excluded participants, finding that the majority were balanced between these two groups. Third, because of the questionnaire design, we could not analyze side effects related to theobromine intake, such as heart rate and blood pressure. Thus, we could not estimate the negative effects of theobromine intake. Fourth, this study did not analyze the genetic interactions of dietary theobromine and cognition performance because of lacking genetic data. Further research on the use of theobromine in older adults is warranted.
The highest quantile of daily theobromine intake was associated with the CERAD-delayed recall score, animal fluency score, and DSST score in U.S. adults aged ≥60 years. Along with increased caffeine intake, daily theobromine tended to be associated with better cognitive performance. Further studies are needed to confirm our findings.
Funding: This work was funded by the Sichuan Science and Technology Program (2019YFH0196) and the National Key Research and Development Program of China (2018YFC1311400, 2018YFC1311401).
Ethics approval and consent to participate: The National Center for Health Statistics institutional review board (NCHS IRB/ERB) approved the ethics protocol of the 2009-2014 NHANES (NCHS IRB/ERB Protocol #2011-17). All participants consented to participate in the NHANES survey with a consent form.
Availability of data and materials: Data analyzed in this study could be downloaded at: https://wwwn.cdc.gov/nchs/nhanes/Default.aspx.
Competing interests: All authors declare no competing interests.
Author contribution: H.L. and C.N. contributed to the conception and design of the study. G.L.J. collected, analyzed, and interpreted the data. G.L.J. wrote the manuscript. All authors have reviewed and revised the manuscript.
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