Estimation of neopterin as a biomarker in biofluids of pre and post-menopausal women after initial periodontal therapy: A biochemical assay.

CONTEXT: Periodontitis (PDD) is a chronic inflammatory condition by nature which destroys the surrounding tooth structures. Menopause (pre and post) worsens the inflammatory state. Biofluids could serve as sources of biomarkers that could diagnose or indicate the intensity and extent of both conditions. AIMS: This study aimed to guestimate the various biofluids of neopterin (NP) levels in premenopause (pre-m) and postmenopausal (post-m) women with PDD after initial periodontal therapy (IPT). SETTINGS AND
DESIGN: This was an interventional cross-sectional study.
MATERIALS AND METHODS: Based on menstrual history, within the age range of 40-60 years, 30 female patients having PDD participated, who were grouped into pre-m and post-m, each group comprised 15 subjects. A biochemical test of NP levels was done at the initial and three months following IPT. Statistics used: Group comparisons and percentage decrement using independent sample t-test and paired t-test for intragroup comparison were done with the version 21, IBM SPSS software.
RESULTS: Intragroup NP analysis at different time points showed significant improvement. Intergroup comparison, however, has shown significant improvement in the premenopausal group (Group I) only.
CONCLUSIONS: Reduced levels of NP were found in pre-m and post-m groups 3 months after IPT, inferring that IPT is a paragon in periodontal therapy and NP might be a good diagnostic marker to detect PDD. Copyright:

INTRODUCTION

Plaque-laden biofilm is the initiator of PDD and host–microbial interactions which destroys the connective tissue and bone. This periodontal tissue damage is a pathological process involving the commencement of host immunity, besides the release of various biomolecules.[1] Biomarkers are such biomolecules, which act as diagnostic indicators of the extent and disease severity and they also could indicate the outcome of therapy.[2]

Neopterin (NP) is a nonconjugated pteridine, a catabolic product of guanosine triphosphate. Macrophages/monocytes synthesize it when stimulated by interferon-y performances as a crucial marker of inflammatory pathophysiological processes such as PDD.[3] NP emerges in various biofluids like plasma (PLA), saliva (SAL), and urine (URI) can be correlated to periodontal disease intensity.

Prior recognition change in inflammation facilitates preventive measures and enhances the disease prognosis. The evidence available indicates that initial periodontal therapy (IPT) lessens the inflammatory burden and aids to restore periodontal and general health.[4] Even though the body potentiates inflammatory changes in premenopause (pre-m) and postmenopause (post-menopause) one and the other, literary work states that more harmful effects occur in post-m. This study explored to ascertain the diminution of inflammation by evaluating NP values and how it responds to IPT in both types of menopausal women with PDD.

MATERIALS AND METHODS

Thirty women within the age range of 40–60 years have participated in this analysis. The sample size was approximated by setting a value at 0.05 and power 80%. (1-ß= 0.8). The least possible sample required for this survey was 26. This exploration was cross-sectional in design which was followed up for 3 months. The protocol was summarized and written approval was obtained before commencement of the procedures from all the participants. Prior institutional ethical committee approval was obtained (Reg No ECR/267/Indt/AP/2016). The clinical trial registration number is NCT02357745 (https://clinicaltrials.gov). The PDD patients were recruited as per the classification 1999 American Academy of Periodontology.[5] After a thorough gynecological examination, depending on the menstrual history and periodontal clinical criteria, required subjects were grouped into two, each containing 15 subjects: Group I 15 pre-m and Group 15 post-m women having chronic PDD. The prototype for pre-m was the monthly cycles becoming irregular and post-m were the individuals who arrested menstrual flow for a minimum of 12 months.

Inclusion criteria

Loss of attachment ≥3 mm or depth of the pocket ≥4 with moderate PDD.

Fifteen natural teeth remaining

Not using any tobacco products

No issues of systemic health in the preceding 6 months.

Elimination criteria

Smokers (present and former)

Any lesions or malignancies/tumors

People using longstanding steroids and on any other medications for underlying systemic diseases

Subjects on hormone treatment (HRT)

Persons who have undergone any dental procedures within the last 6 months

Patients with systemic diseases that may alter the status of periodontium were not accepted in the study design.

A sum of 30 women was examined, and collection of samples was done at 0 and 3 months following IPT to standard the NP levels. Criteria for choosing the subjects have been interpreted [Figure 1]. Sample collecting day instructions were given, not to use any rinses.

Figure 1

Patient selection criteria – Consort flow chart. n – number of patients; IPT – Initial Periodontal Therapy

Collecting samples and procedure

SAL, URI, and PLA samples were acquired from all the participants for the analysis of NP at pre- and 3-months posttreatment. All the processed samples were concealed from exposure of light and preserved at − 20°C up until testing. Plasma: Peripheral venous blood 5 ml from the antecubital vein was drawn by venipuncture which was preserved in purple capped tubes Ethylene diaminetetraacetic acid (EDTAtubes, CML Biotech (Pvt) India). Collected blood at 3000×g for 10 min was centrifuged (REMI Lab Equipment, Meerut). The supernatant PLA was collected into a neat clean tube (Fisherman, Technologies Pvt. Gujarat) with a pipette (Pasteur Pipettes [Recombigen Laboratories Private Limited Samay Pur, New Delhi). Samples were preserved at −20°C till assayed.

Saliva: One-milliliter unstimulated SAL was collected by passive drool method into a sterilized tube (Polypropylene tubes, Thermo Scientific-Lab). Patients were advised to allow pooling of SAL intraorally followed by tilting their head frontward to pool the SAL. Eight hours before sample collection, patients were instructed not to chew food and prohibit brushing. Strict instructions were given that, 48 hours prior to the procedure, no dental examination or treatment would be performed. Subjects were demanded, not to cleanse with any mouthrinses on the sample collection day. Clear SAL was obtained by centrifugation of samples.

Urine: The very first URI in the morning was acquired and shielded out of the light; tin foil was wrapped immediately and preserved at −20°C until measure.

No food restrictions were given for obtaining URI. Dilution of samples was done as per the protocol before assessment.

Biochemical/immunological analysis

It was performed using NP ELISA Kit (Alpha Diagnostics, USA). For estimation of NP levels, samples ultracentrifuged for 10 min at 10,600×g at 4°C. 200μl of ethanol was blended to 100 μl of the sample for 60 s to precipitate proteins and vortexes. For biochemical analysis, the supernatant was collected from all specimens.

Statistics

By using version 21 IBM SPSS software (SPSS v. 21, IBM, Chicago, IL), the results were analyzed. Percentage decrement and intergroup comparison were executed by independent sample t-test and paired t-test was accomplished for valuating between the two groups. P < 0.05 kept it as significant.

RESULTS

A statistically expressive diminution of NP was found in the premenopausal category, which was assessed from the initial treatment phase 3 months after the procedure. URI NP concentration reduced to 265.57 ± 54.57 from 304.91 ± 60.92. The salivary NP levels also reduced to 6.69 ± 1.24 from 13.63 ± 1.55 and the NP levels in PLA reduced to 6.95 ± 0.95 from 11.72 ± 1.51 3 months post treatment [Table 1].

Table 1

Comparison of the premenopausal group at baseline and 3 months

Premenopausal	n	Mean±SD	Mean difference (95% CI)	t	df	P
Urine (μmol/mol creatinine)
Baseline	15	304.91±60.92	39.34 (3.96-74.73)	2.39	14	0.032*
3 months	15	265.57±54.57
Saliva (nmol/L)
Baseline	15	13.63±1.55	6.93 (5.67-8.19)	11.81	14	<0.001*
3 months	15	6.69±1.24
Plasma (nmol/L)
Baseline	15	11.72±1.51	4.77 (3.77-5.77)	10.20	14	<0.001*
3 months	15	6.95±0.95

Paired t-test. *P<0.05 statistically significant. P>0.05 statistically NS. n – Number; SD – Standard deviation; CI – Confidence interval; df – Degrees of freedom; t – t-statistic; nmol/L – Nanomoles per liter; μmol – Micromole; P – Probability

The post-m group also showed noticeable progress of the measured parameter from the initial therapy to post-treatment of 3 months. There was a decline in NP levels 290.45 ± 52.48 from 391.22 ± 61.98 in URI, 7.05 ± 1.41 from 15.61 ± 1.83 in SAL, and to 7.23 ± 1.09 from 13.52 ± 2.51 in PLA [Table 2].

Table 2

Comparison of the postmenopausal group at baseline and 3 months

Postmenopausal	n	Mean±SD	Mean difference (95% CI)	t	df	P
Urine (μmol/mol creatinine)
Baseline	15	391.22±61.98	100.77 (47.63-153.91)	4.07	14	0.001*
3 months	15	290.45±52.48
Saliva (nmol/L)
Baseline	15	15.61±1.83	8.56 (7.50-9.62)	17.27	14	<0.001*
3 months	15	7.05±1.41
Plasma (nmol/L)
Baseline	15	13.52±2.51	6.29 (4.99-7.58)	10.44	14	<0.001*
3 months	15	7.23±1.09

Paired t-test. *P<0.05 statistically significant. P>0.05 statistically NS. n – Number; SD – Standard deviation; CI – Confidence interval; df – Degrees of freedom; t – t-statistic; nmol/L – Nanomoles per liter; μmol – Micromole; P – Probability

Within the two groups, the NP values significantly reduced post treatment. URI, SAL, and PLA P values were observed to be 0.001, 0.003, and 0.02 correspondingly [Table 3]. However, no statistical significance was seen when compared between two groups after IPT at 3 months [Table 4]. The URI and SAL values post IPT were encouragingly expressive (P < 0.05, URI: 0.048, and SAL: 0.04). However, no statistical significance was noticed in PLA NP levels post IPT [Table 5 and Graph 1].

Table 3

Comparison between two groups at baseline

Baseline	n	Mean±SD	Mean difference (95% CI)	t	df	P
Urine (μmol/mol creatinine)
Premenopause	15	304.91±60.92	86.31 (40.35-132.27)	3.84	28	0.001*
Postmenopause	15	391.22±61.98
Saliva (nmol/L)
Premenopause	15	13.63±1.55	1.97 (0.71-3.24)	3.20	28	0.003*
Postmenopause	15	15.61±1.83
Plasma (nmol/L)
Premenopause	15	11.72±1.51	1.80 (0.25-3.35)	2.38	28	0.02*
Postmenopause	15	13.52±2.51

Independent sample t-test. *P<0.05 statistically significant. P>0.05 statistically NS. n – Number; SD – Standard deviation; CI – Confidence interval; df – Degrees of freedom; t – t-statistic; nmol/L – Nano moles per liter; μmol – Micromole; P – Probability

Table 4

Comparison between two groups at 3 months

3 months	n	Mean±SD	Mean difference (95% CI)	t	df	P
Urine (μmol/mol creatinine)
Premenopause	15	265.57±54.57	24.88 (−15.16-64.92)	1.27	28	0.21 (NS)
Postmenopause	15	290.45±52.48
Saliva (nmol/L)
Premenopause	15	6.69±1.24	0.35 (−0.64-1.34)	0.73	28	0.47 (NS)
Postmenopause	15	7.05±1.41
Plasma (nmol/L)
Premenopause	15	6.95±0.95	0.28 (−0.48-1.05)	0.75	28	0.46 (NS)
Postmenopause	15	7.23±1.09

Independent sample t-test. *P<0.05 statistically significant. P>0.05 statistically NS. n – Number; SD – Standard deviation; CI – Confidence interval; df – Degrees of freedom; t – t-statistic; nmol/L – Nano moles per liter; μmol – Micromole; NS – Not significant; P – Probability

Table 5

Intergroup comparison of change from baseline to 3 months

Change from baseline to 3 months	n	Mean±SD	Mean difference (95% CI)	t	df	P
Urine (μmol/mol creatinine)
Premenopause	15	39.34±63.90	61.43 (0.45-122.40)	2.06	28	0.048*
Postmenopause	15	100.77±95.96
Saliva (nmol/L)
Premenopause	15	6.93±2.27	1.62 (0.05-3.20)	2.11	28	0.04*
Postmenopause	15	8.56±1.92
Plasma (nmol/L)
Premenopause	15	4.77±1.80	1.52 (−0.04-3.08)	1.99	28	0.056 (NS)
Postmenopause	15	6.29±2.33

Independent sample t-test. *P<0.05 statistically significant. P>0.05 statistically NS. n – Number; SD – Standard deviation; CI – Confidence interval; df – Degrees of freedom; t – t-statistic; nmol/L – Nanomoles per liter; μmol – Micromole; NS – Not significant; P – Probability

Graph 1

(a and b) Comparison of all parameters in both the groups at baseline and 3 months

DISCUSSION

When the host resistance mechanism was interacted by plaque microorganisms, PDD originates. All the diseased sites contain mononuclear cells, primarily lymphocytes, and transmigratory mononuclear phagocytes.[6] In established PDD lesions, migrated host defense cells cause major tissue destruction by activation of host molecules such as macrophages/monocytes, lymphocytes, fibroblasts, and other cell types.[7]

The long-established diagnostic measurements of PDD like alveolar bone height assessment with radiographs, clinical attachment levels, plaque scores, probing depths, and so forth are known to have their advantages and drawbacks. Moreover, all the above-mentioned tests give us a depiction of the history of disease rather than its future ramifications. The traditional measures show impairments from previous circumstances of the knockdown and need a 2–3 mm threshold change before the identification of a site. Advances in the diagnostic research in the oral together with periodontal disease are moving toward more objective attributes such as biomarkers to calculate the periodontal risk.[8]

Advancements in molecular techniques including genomic hybridization techniques, polymerase chain reaction, and DNA hybridization have their pros and cons in quantitative evaluation, periodontal pathogens are grouped into numerous clusters though they are not site-specific.

The main problem in periodontal diagnosis is determining the early onset of PDD by biomarkers.[9]

Various studies proved that when estradiol hormones are deficient, they have numerous unpredictable consequences on systemic health along with periodontium and alveolar bone. Eventual cell death might be caused due to altered immune activity like apoptotic factors expression and antigen presentation and they also encompass other factors like polymorph nuclear leukocytes with boosted chemotaxis, excessive production of prostaglandin E2 which are intensified by progesterone in the sulcus of the gingiva.[1011] Fifty percent of interleukin 6 (IL-6) production of gingival fibroblasts was also decreased by progesterone.[12] Estrogen controls the stratified squamous epithelium cytodifferentiation and production and upkeep of collagen. Due to the hormonal impact on these cells, Connective tissue, and epithelial cells set to fight against the microbial insult to increase the tissue repairing property.[13] By reason, the detrimental outcome of estrogen and progesterone during menopause, the microcirculatory system will deteriorate. The reduced proportions produce concomitant traits such as periocytes and endothelial cells venule swelling, platelets and granulocyte cling to walls of the vessel, intensified vascular permeability, perivascular mast cells interruption, microthrombi formation, and vascular accretion.[14] Pre- and postmenopause changes were considered in this procedure, as the evidence available represents that the onset of menopause has diversified imperilment on the all over body besides with the mouth orifice. Estrogen directly affects the oral mucosa, thereby altering the women's periodontal health during menopause as of its diminished levels.[15] Post-m women exhibit low salivary flow rates than menstruating women.[16] Often, postmenopausal women have a severe form of PDD and the reason behind this is, the cells-like osteoblasts and fibroblasts of periodontium containing estrogen receptors are severely affected in women with menopause symptoms, fluctuating hormone levels which thus affect periodontal health.[17] Scardina and Messina used video capillaroscopy and[18] examined possible oral microcirculatory changes in menopausal women[18] and found appreciating variations in vascular parameters like decrease in loop diameter, increase in vessel tortuosity, and lessening in periodontal mucosal density indicating a predisposed inflammation in post-m females. The landmark studies opined that the menopausal onset was linked with a reduced status of systemic inflammation, which began to slowly increase, as the hormonal levels diminish further and as the inflammatory state intensified, Due to the elevated serum proinflammatory mediators like IL-6, IL-1, or tumor necrosis factor α in a post-menopause stage.[19202122] Premenopausal women are further prone to osteopenia than postmenopausal women. However, prevalence is more in post menopause stage.

This specifies that the adversely affected osteoid mass persists in both stages. These factors along with estrogen deficiency seen with the inception of menopause can make women more osteoporotic.[23]

SAL, URI, and PLA were used as indicators in this report. Unstimulated SAL was well thought of as a marker, the reason being the existence of biomarkers in high concentration for easy detection during assays. SAL, a beneficial diagnostic tool, aids in detecting any oral or systemic disease. It is perfect for the preliminary identification of diseases as it contains multiple biomarkers that could be utilized for various assays.[24252627] Majority of the SAL and URI biological markers might be present in blood too. However, this explanation is not applicable for all the biomarkers like as the conjoined steroid hormone SAL and serum concentrations are not often correlated. On the contrary, the SAL/PLA ratio of dehydroepiandrosterone sulfate and glucose is comparable.

The cause for this inconsistency is because the oral orifice is the gateway of hormone passage. Limited studies gave a definite association of PLA with NP. Therefore, in this presentation, PLA samples were also taken to assay the NP ratio.[28293031]

Early morning first void, URI samples were examined due to their high accuracy, concentration, and easy collection for detection. Furthermore, it is relatively free of nutritive and physical influences.[32] NP is metabolized mainly by the kidney. Therefore, any changes in NP levels in biofluids like serum are commonly witnessed in the URI. Owing to that diseases related to cellular immunity can easily be interrelated in both the fluids.[333435] Here, the correlation of three biofluids such as SAL, URI, and PLA NP concentrations were interpreted in pre-m and post-m women at the initial and 3 months post IPT. When the scrutiny of intragroup was done, a denoted decline was constituted in all parameters after IPT within 3 months from the baseline. Evaluation of intergroup disclosed that only SAL and urinary NP measures were reduced absolutely. However, insignificant decrement was viewed in PLA. These study conclusions are partly matching with by Ozmeric et al.[4] where they related NP value of GCF, URI, and SAL in aggressive PDD and observed an appreciating decrease in salivary NP levels and nonsignificant outcomes in the URI. Pradeep et al.[36] detected a raise in NP values in GCF of persons without PDD signs compared to PDD patients and commensurate results were seen by Arjun Kumar et al.[37] Mahendra et al.[38] also had given a similar conclusion when the source was SAL, Concluded that elevated NP levels were found in conditions with severe inflammation. Prasanna et al.[39] found an appreciated difference of SAL NP succeeding PDD treatment in the premenopausal condition when distinguished with post-m. This current study outcome is also in harmony with the above study consequences. To conclude, when related mutually, nearby an encouraging correlation is observed in NP values in both the situations.

Considered study limitations are the duration of the post-m period was not regulated. Macrophage or monocyte activation was not directly interrelated with the NP levels of SAL, PLA, and URI.

CONCLUSIONS

Any stage of life periodontal diseases can occur but is found most prevalent in elderly people. Women at menopause stages are more vulnerable to various PDD. The function of biomarkers is well acknowledged in life science, diagnostic prognosis along drug discovery. Earlier detection of disease advancement and efficacy of the remedial treatment is the eminence of biomarkers. To anticipate the appropriate role of biomarkers in routine practice, it is required to know the fundamental mechanism. Measuring the biomarkers present in biofluids may straightforwardly recognize the preexpression of a disorder entity and intercept the further evolution of the condition thus improving the treatment outcome.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.