Association of Alanine Aminotransferase and Periodontitis: A Cross-Sectional Analysis—NHANES 2009–2012

Objective. Alanine Aminotransferase is an enzyme associated with not only liver diseases, liver conditions, and metabolic syndrome, but also inflammation. Periodontitis is associated with increased cytokines and other markers of inflammation. The purpose of this study is to determine if an independent association between Alanine Aminotransferase and periodontitis exists. Methods. Data from the 2009-2010 and 2011-2012 National Health and Nutrition Surveys (NHANES) were combined. Data concerning periodontitis and Alanine Aminotransferase were extracted and analyzed with Rao Scott Chi-square and logistic regressions. Serum Alanine Aminotransferase was dichotomized at 40 units/liter, and periodontitis was dichotomized to the presence or absence of periodontitis. Results. In bivariate Chi-square analyses, periodontitis and Alanine Aminotransferase were associated (p = 0.0360) and remained significant in unadjusted logistic regression (OR = 1.30 [95% CI: 1.02, 1.65]). However, when other known risk factors of periodontitis were included in the analyses, the relationship attenuated and failed to reach significance (adjusted OR = 1.17 [95% CI: 0.85, 1.60]). Conclusion. Our study adds to the literature a positive but attenuated association of serum Alanine Aminotransferase with periodontitis which failed to reach significance when other known, strong risk factors of periodontitis were included in the analysis.


Introduction
Alanine Aminotransferase is an enzyme that is a catalyst in the transfer of -NH 2 group from L-alanine to alphaketoglutarate creating pyruvate and L-glutamate which are important reactions in the tricarboxylic acid cycle (also known as the Krebs cycle or the citric acid cycle of energy production) [1]. Alanine Aminotransferase is primarily found in hepatocyte intracellular fluid [1]. Serum levels are used to detect hepatic distress and diseases such as chronic viral infections, autoimmune hepatitis, and steatohepatitis, as well as other conditions such as celiac disease, muscle injury, hemochromatosis, and nonalcoholic fatty liver disease [1]. Serum levels also increase with extreme physical exertion, excessive alcohol intake, some medications, and metabolic syndrome [1], the clustering and synergistic effects of interrelated risk factors leading to diseases of chronic inflammation (cardiovascular disease, stroke, and/or diabetes) [2][3][4][5]. Many mechanisms and pathways may be involved in elevated serum Alanine Aminotransferase levels; however, chronic inflammation is one potential mechanism for its increase.
Periodontitis is the destruction of the soft and hard oral tissue supporting the teeth as the result of a biofilm (bacteria, fungi, and viruses), genetic susceptibility, and plaqueinduced chronic inflammation, among other risk factors [6][7][8][9][10][11][12][13]. Systemic chronic inflammation negatively impacts periodontal health. Periodontitis is a public health concern as it is a common disease affecting 46% of US adults (64.7 million individuals) [14]. Periodontitis affects oral health quality of life, the number of remaining teeth in older adults, 2 International Journal of Inflammation and glycemic control in individuals with diabetes and has social and financial impacts.
The purpose of this study is to determine if there exists an independent association of Alanine Aminotransferase and periodontitis. The theoretical basis is that Alanine Aminotransferase levels are increased with inflammation and periodontitis is a disease of chronic inflammation. Researchers using animal models indicated that such an association exists [15][16][17]. Researchers also determined that an association of periodontitis and Alanine Aminotransferase existed using a convenience sample of 2225 young Japanese male university students [18]. However, the number of participants with periodontitis was small (4.7%) in that study. The rationale for this study is that there is a need for a large, nationally representative study to determine if an association exits between periodontitis and Alanine Aminotransferase. Such knowledge could help inform decisions by medical personnel concerning dental referrals for patients with high Alanine Aminotransferase levels.
The null hypothesis for this research is that the adjusted odds ratio for the association of high levels of serum Alanine Aminotransferase (≥40 units/liter) on periodontitis is the same as the adjusted odds ratio for normal levels of serum Alanine Aminotransferase (<40 units/liter) on periodontitis.

Materials and Methods
This study was acknowledged by the West Virginia University Institutional Review Board (protocol 14010466515). The study was conducted using the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines.

Data
Source. Data used in this study were from the combined 2009-2010 and 2011-2012 National Health and Nutrition Examination Surveys (NHANES) which were collected by researchers from the Centers for Disease Control and Prevention (CDC). The NHANES surveys had a stratified, multistage probability sampling strategy. Noninstitutionalized civilian individuals were selected for the survey. They provided written informed consent. Each participant was interviewed and had a physical evaluation in NHANES MEC (Mobile Examination Center).
The periodontal examinations were completed by licensed dentists. He or she had an available halogen light, a HuFriedy™ periodontal probe, and a mouth mirror for each examination. The examination was limited to individuals who were not edentulous and who did not have heart transplants, artificial valves, congenital heart disease, rheumatic fever, hemophilia, kidney disease with dialysis, or a pacemaker.
Alanine Aminotransferase level in serum or plasma was determined by NHANES researchers using a kinetic rate method. Alanine Aminotransferase is a catalyst in the reversible reaction of L-alanine and a-ketoglutarate to pyruvate and L-glutamate. In the analysis, the pyruvate is further reduced. The rate of change in absorbance at 340 nm over time is proportional to the Alanine Aminotransferase level [19]. The details of the NHANES procedures are available at http://www.cdc.gov/nchs/nhanes.htm.

Eligible Study Population.
The researchers for this study used a subset of the available data from the NHANES 2009-2012 surveys. Eligible participants were aged 30-69 years and had complete data on periodontitis and serum Alanine Aminotransferase. The eligible sample included 5,758 participants.

Dependent Variable.
The periodontal variable was the dependent variable for this study. Periodontitis was dichotomized as a yes/no variable to establish an adequate sample size. Even though several years of NHANES data were combined, there were too few participants in the categories of "mild periodontitis" and "severe periodontitis" who also had Alanine Aminotransferase data to use those categories as separate categories. Instead, the categories were no periodontitis (the reference group) and periodontitis (mild, moderate, or severe) using the definition of periodontitis for population-based surveillance supported by the CDC in partnership with the American Academy of Periodontology [20]. Their more detailed definition of periodontitis includes "mild periodontitis" in which there are at least 2 interproximal areas in which the attachment loss is at least 3 mm and there are at least 2 interproximal areas on different teeth in which the probing depth is at least 4 mm or at least one area with a probing depth of 5 mm; "moderate periodontitis" in which there are at least 2 interproximal areas on different teeth in which the attachment loss is at least 4 mm or there are at least 2 interproximal areas on different teeth in which the probing depth is at least 5 mm; and "severe periodontitis" in which there are at least 2 interproximal areas which are on different teeth in which the attachment loss is at least 6 mm and there is at least 1 interproximal area in which the probing depth is at least 5 mm [20].

Independent Variable and Other Variables.
The keyindependent variable was serum Alanine Aminotransferase. It was dichotomized at <40 units/liter (the reference group) and ≥40 units/liter. The cut-point at ≥40 units/liter is routinely used and has been previously reported in peerreviewed literature to determine normal and high levels of Alanine Aminotransferase in adults [21][22][23].
The other variables that were included in this study to account for confounding or to further explain the variance for periodontitis included sociodemographic and behavioral variables: sex, race/ethnicity, education, age, income to poverty ratio, smoking, body mass index, insurance coverage, presence of diabetes mellitus, and alcohol consumption. The categories for the additional variables were sex (male versus female), race/ethnicity, (non-Hispanic black, Mexican American, and other versus non-Hispanic white), education (high school, less than high school, and some college versus college degree and above), age (45-54, 55 years and above versus 30-44 years), family income to poverty ratio (less than 2 versus 2 and above), smoking status (current, former smoker versus never smoker), alcohol consumption (moderate, heavy versus no consumption), body mass index (25)(26)(27)(28)(29)30 and above versus 0-24), insurance coverage (no versus yes), and diabetes (yes versus no).

Statistical Analyses.
Due to the complex study design, the analysis accounted for clustering, stratification, selected sample population, and sample weights. Descriptions of the sample population, bivariate (Rao Scott Chi-square) analyses with periodontitis, and logistic regression analyses were completed. In logistic regression model development, significant variables from the bivariate analyses (at < 0.05) were included in the model. The statistics package that was used was SAS 9.3® (Cary, NC). Table 1 has the results of the sample description. There were 5758 eligible participants, 50.1% of whom were female. Considering race/ethnicity, 68.1% of participants were non-Hispanic white, 10.6% of participants were non-Hispanic black, and 8.4% of participants were Mexican American. There were 41.9% of participants who were aged 30-44 years; 28.7% of participants who were 45-54 years; and 29.4% of participants who were 55-69 years. There were 64.2% of participants who attended some college courses, technical school courses, or above. Most had an income to poverty ratio above 2.0 (69.9%). There were 56.1% of participants who were never smokers and 20.5% of participants who never used alcohol. There were 27.0% of participants who had a body mass index of 0-24, 36.0% of participants who had a body mass index of 25-29, and 37.0% of participants who had a body mass index of 30 and above. There were 12.1% of participants who had ≥40 units/liter Alanine Aminotransferase and 39.9% of participants who had periodontitis.

Results
The Chi-square bivariate analyses and standard errors are presented in Table 2. There was a significant relationship ( = 0.0360) of serum Alanine Aminotransferase levels ≥40 U/L and periodontitis. Other significant relationships (at < 0.0001) were with periodontitis and sex; race/ ethnicity; age; education; family income to poverty ratio; alcohol consumption; smoking status; insurance coverage; and diabetes mellitus. Body mass index was also significant at = 0.0151. Table 3 has the results of the logistic regression of serum Alanine Aminotransferase on periodontitis. The odds ratio, in unadjusted logistic regression, for periodontitis associated with higher levels of serum Alanine Aminotransferase was 1.30 (95% confidence interval: 1.02, 1.65).
An Adjusted Model was created which included sex (male versus female), race/ethnicity, (non-Hispanic black, Mexican American, and other versus non-Hispanic white), education (high school, less than high school, and some college versus college degree and above), age (45-54, 55 years and above versus 30-44 years), family income to poverty ratio (less than 2 versus 2 and above), smoking status (current, former smoker versus never smoker), alcohol consumption (moderate, heavy versus no consumption), body mass index (25-29, 30 and above versus 0-24), insurance coverage (no versus yes), and diabetes (yes versus no). The adjusted odds

Discussion
In this study we evaluated the relationship of Alanine Aminotransferase on periodontitis in a large, nationally representative sample of 5758 participants. The results of this study included an initial significant association of Alanine Aminotransferase with periodontitis (odds ratio 1.30, 95% CI, 1.02, 1.65), which remained positive but attenuated and failed to reach significance with the addition of other variables (adjusted odds ratio 1.17, 95% CI, 0.85, 1.60). Serum Alanine Aminotransferase was dichotomized at <40 units/liter (the reference group) and ≥40 units/liter in this study, although other researchers have used lower cut-points, such as ≥30 units/liter [24], and some researchers have used a higher level such as ≥41 units/liter [18]. When a cut-point of ≥30 units/liter was used with these data, the adjusted odds ratio International Journal of Inflammation 5 The results of this study are similar to the results of researchers studying salivary Alanine Aminotransferase in a sample of 50 participants in Romania. They determined that salivary Alanine Aminotransferase was not statistically different among individuals who had periodontitis as compared with individuals who did not have periodontitis [25]. The study in Romania differed from this current study in the source of Alanine Aminotransferase (saliva rather than blood or serum) and study population size.
It has been suggested that changes in enzymatic activity indicate metabolic change in inflammation and that Alanine Aminotransferase release increases with cell injury and death. Researchers in a study of 49 older adult Japanese patients with chronic periodontitis found Alanine Aminotransferase to be associated with periodontal inflammation [26]. Several of the same researchers conducted an 18-month longitudinal study of 85 patients with chronic periodontitis in four Japanese university hospitals and found that salivary Alanine Aminotransferase in combination with the ratio of Porphyromonas gingivalis to total bacteria had a sensitivity of 0.4 and a specificity of 0.96 for the prediction of the progression of periodontitis [27]. Salivary Alanine Aminotransferase was increased in individuals with periodontitis disease, as compared with controls in a study of 40 patients in India [28]. Researchers found a correlation of salivary Alanine Aminotransferase and periodontitis in a sample of 60 participants [28]. These existing studies differed from the current study in that they were small and the researchers used salivary Alanine Aminotransferase for analyses.
In a study conducted in Japan with serum Alanine Aminotransferase, a positive association of elevated serum Alanine Aminotransferase and pocket depth was present only in males with low alcohol consumption and at least 3 components of the metabolic syndrome (waists at or above 90 cm in males and 80 cm in females; triglycerides at or above 150 mg/dL or treatment for triglycerides; high density lipoprotein below 40 mg/dL in males or below 50 mg/dL for females; systolic blood press at or above 130 mmHg or diastolic blood pressure at or above 85 or medication for high blood pressure; and fasting glucose at or above 100 mg/dL or treatment for elevated glucose) [29].
Researchers who conducted a systematic review with the association of Alanine Aminotransferase on mortality indicated that Alanine Aminotransferase had a complex and inconsistent relationship, which was more predictive in older adults with extremely low levels of Alanine Aminotransferase [30]. The method of collection of the Alanine Aminotransferase was not discussed in the review of the 12 articles selected for the meta-analysis [30].

Strengths, Limitations, and Sources of Bias.
Research concerning Alanine Aminotransferase and periodontitis is lacking. Alanine Aminotransferase collection methods differ among researchers and researchers have used different cutpoints for Alanine Aminotransferase as well as different definitions for periodontitis.
Our study strengths are its large, nationally representative sample; the use of definitions for population-based surveillance of the CDC in partnership with the American Academy of Periodontology; standardized blood testing procedures for Alanine Aminotransferase; and standardized/calibrated examiners. Since this study is large, many variables were included in the multivariable analysis which 6 International Journal of Inflammation are also independently related to periodontitis or may confound the relationship of Alanine Aminotransferase and periodontitis, an option that is not available for small studies.
Our study limitations are the biases associated with selfreporting of education, income, insurance status, alcohol consumption, and the reporting of diabetes mellitus. It is possible that self-reports may be influenced by participants' desires for providing a socially desirable response. However, it would be expected that social desirability bias would likely result in reports which would tend to direct the outcome to supporting the null hypothesis that the odds ratio for the association of high Alanine Aminotransferase and periodontitis is the same as the odds ratio for normal levels of Alanine Aminotransferase and periodontitis. Another limitation was not having enough participants to further refine periodontitis as "mild," "moderate," and "severe."

Conclusion
Our study adds to the literature a positive but attenuated association of periodontitis and Alanine Aminotransferase which failed to reach significance when other known, strong factors of periodontitis were included in the analysis.

Disclosure
The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the paper.

Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.