Free haem levels in gingival crevicular fluid and their relationship to periodontal clinical parameters, smoking and subgingival microbial composition.

BACKGROUND AND OBJECTIVES: Periodontitis caused by multifactorial polymicrobial infection results in a destructive inflammatory process and loss of tooth supporting tissues. Many putative bacterial virulence factors that cause host destruction are regulated by iron and haem. Therefore, this study investigated the free haem levels in the gingival crevicular fluid (GCF) at periodontitis sites in smokers and nonsmokers and their relationship to subgingival microbial composition.
MATERIALS AND METHODS: A cross-sectional study was carried out on 78 patients with a split-mouth design who were divided into Group I A - periodontally healthy sites and Group I B - periodontally diseased sites in nonsmokers with chronic periodontitis and Group II A - periodontally healthy sites and Group II B - periodontally diseased sites in smokers. Clinical parameters recorded included a plaque and gingival index, papillary bleeding index, pocket probing depth, and clinical attachment level. The collected GCF samples were subjected to Biovision™ Hemin Colorimetric Assay Kit and subgingival plaque samples to BANA™ test.
RESULTS: Increased GCF free haem concentration and positive BANA sites were seen at periodontitis sites compared to healthy sites, in both smokers and nonsmokers group. However, no difference was found in GCF free haem levels between smokers and nonsmokers, but it was statistically significant with respect to BANA-positive sites.
CONCLUSION: Thus, this study concludes that the higher concentration of GCF free haem at diseased sites indicates that it could be used as a potential biomarker to determine active periodontal sites, also smoking and BANA results did not influence the biomarker levels. Copyright:

INTRODUCTION

Periodontitis results from a complex interaction between the bacteria-colonizing gingival crevice and immune and inflammatory responses of the host.[1] It is characterized by destructive inflammatory process resulting in loss of tooth supporting tissues.[2]

Establishment and progression of an infection depends on the ability of the microorganism to evade the hosts defensive mechanism.[2] In addition, many periodontal pathogens require heme-iron containing compounds for their growth but lack the ability to synthesize.[3] Virulence factors of many putative periodontal pathogens are regulated by iron and haem. The production of pro-inflammatory cytokines such as interleukin (IL)-1 β, tumor necrosis factor-α, IL-8, and IL-6 from macrophages is enhanced by iron and haem resulting in destruction of the host. Therefore, this study investigates free haem levels in gingival crevicular fluid (GCF) at periodontitis sites in smokers and nonsmokers and determines their relationship to subgingival microbial composition.

MATERIALS AND METHODS

Recruitment of patients was done from the outpatient department of periodontics.

Power of the study was 85%.

n = 2(Zα + Z[1−β])2/ d2

n = 36.7 (sample size), α = 0.05 β = 0.85, Zα = 1.96, Z(1−β) = 1.04, d = 0.7

The present study was approved by the Institutional Ethics Committee (Ref. No. 85/2016-17). A total of 78 patients were selected for the study and written informed consent was obtained from each patient.

Inclusion criteria

Systemically healthy individuals aged between 20 and 50 years

Healthy sites with probing pocket depth of ≤3 mm, absence of bleeding on probing, and no radiographic evidence of bone loss (healthy sites)

Chronic localized periodontitis subjects with at least 20 teeth and 2 nonadjacent probing pocket depth ≥5 mm and CAL ≥3 mm per quadrant with radiographic bone loss >20% (periodontitis sites)

Current smokers, i.e., persons who have smoked 100 cigarettes and who currently smoke every day or someday, will be selected (smokers group).[4]

Exclusion criteria

Systemic conditions that affected periodontitis or the hemoglobin level of the subject or pregnant/lactating women

Subjects with a history of periodontal treatment or who have taken antibiotics in the last 6 months

Subjects with symptoms of acute illness, orthodontic appliances, or presence of oral mucosal inflammatory conditions or salivary gland dysfunction.

A cross-sectional study was carried out on 78 patients with a split-mouth design who were divided into the following groups:

Group I A – Periodontally healthy sites in nonsmokers

Group I B – Periodontally diseased sites in nonsmokers

Group II A – Periodontally healthy sites in smokers

Group II B – Periodontally diseased sites in smokers.

The clinical parameters recorded for each patient included gingival index (Loe and Silness), plaque index (Silness and Loe), papillary bleeding index (Muhlemann HR), probing pocket depth, clinical attachment level, and radiographic bone loss.

Probing pocket depth and clinical attachment level were measured to the nearest millimeter using University of North Carolina-15 probe and six sites were examined per tooth excluding third molars.

GCF sample collection method: The tooth selected for GCF sampling was isolated with cotton rolls to decrease salivary contamination. GCF sample collection was done by extracrevicular method using calibrated microcapillary pipettes of length 125 mm and bore size of 0.01 mm. Icebox was used to immediately store the pooled GCF samples and they were later transported to the laboratory for processing.

The collected GCF samples were subjected to Biovision™ Hemin Colorimetric Assay Kit for estimating the levels of free haem [Graph 1].

Graph 1

Human hemin standard, Abs - Absorbance, Y - Equation of standard curve, R2 - coefficient of determination

BANA™ test was done to assess the microbiological status. From the deepest probing site, the subgingival plaque samples collected were wiped onto the BANA-impregnated strip found at the lower edge of the BANA reagent card. After incubation, if blue color appeared on the upper reagent strip, the test was considered positive. If the blue color was absent, the test was considered negative, when no blue color was visible.

Statistical methods employed for analysis of data were student paired t-test, independent Student's t-test, Chi-square test, mean standard deviation, and Mann–Whitney U-test.

RESULTS

A total of 78 subjects (66 males and 12 females) in the age range of 26–54 years completed the study with no dropouts. The smokers group consisted of only males [Table 1]. At diseased sites, all clinical periodontal parameters were statistically significant (higher) compared to healthy sites (P < 0.001). The gingival index, papillary bleeding index, probing pocket depth, and clinical attachment level were significantly higher in the nonsmokers compared to the smokers group (P < 0.001) [Tables 2 and 3].

Table 1

Distribution of demographic characteristics among the study subjects

Variable	Category	Smokers	Nonsmokers	P
Age	Mean±SD	37.4±6.5	37.2±5.1	0.99 (NS)
	Range	26-54	30-47
Sex	Males (%)	39 (100.0)	27 (69.2)	<0.001 (S)
	Females (%)	0 (0.0)	12 (30.8)

S – Statistically significant (P<0.001); NS – Statistically nonsignificant (P>0.001); SD – Standard Deviation; P – Level of Significance

Table 2

Comparison of mean values of gingival, plaque, and papillary bleeding indices between smokers and nonsmokers group using independent Student’s t-test

Variables	Group	n	Mean±SD	Mean difference	P
GI	Smokers	39	1.80±0.25	−0.35	<0.001* (S)
	Nonsmokers	39	2.15±0.34
PI	Smokers	39	1.95±0.38	−0.12	0.13 (NS)
	Nonsmokers	39	2.06±0.29
PBI	Smokers	39	1.40±0.18	−1.07	<0.001* (S)
	Nonsmokers	39	2.46±0.17

*Statistically significant. S – Statistically significant (P<0.001); NS – Statistically nonsignificant (P>0.001); n – Sample size; SD – Standard deviation; GI – Gingival index; PI – Plaque index; PBI – Papillary bleeding index; P – Level of Significance

Table 3

Comparison of mean probing pocket depth, clinical attachment level (mm), and gingival crevicular fluid free haem concentration (ng/ml) between healthy and diseased sites in nonsmokers and smokers group using Student’s paired t-test

Variables	Sites	n	Nonsmokers			Smokers
			Mean±SD	Mean difference	P	Mean±SD	Mean difference	P
PPD	Healthy site	39	2.31±0.44	−3.11	<0.001* (S)	1.48±0.20	−3.79	<0.001* (S)
	Diseased site	39	5.42±0.61			5.27±0.56
CAL	Healthy site	39	1.64±0.43	−3.34	<0.001* (S)	1.47±0.18	−3.61	<0.001* (S)
	Diseased site	39	4.98±0.65			5.08±0.56
GCF free haem concentration	Healthy site	39	21.95±4.86	−4.75	<0.001* (S)	22.90±3.60	−3.08	0.005* (S)
	Diseased site	39	26.70±4.80			25.98±4.83

*Statistically significant. S – Statistically significant (P<0.001) (P<0.005); n – Sample size; SD – Standard deviation; PPD – Probing pocket depth; CAL – Clinical attachment level; GCF – Gingival crevicular fluid; mm – Millimeter; ng/ml – Nanogram/milliliter; P – Level of Significance

The mean GCF free haem levels at healthy and diseased sites for Group I were 21.95 ± 4.86 (ng) and 26.70 ± 4.80 (ng) and for Group II, they were 22.90 ± 3.60 (ng) and 25.98 ± 4.83 (ng). A statistically significant difference was observed (P < 0.001) [Table 3].

The mean GCF free haem level at healthy sites for Group I was 21.95 ± 4.86 (ng) and for Group II, it was 22.90 ± 3.60 (ng). Statistically insignificant difference was seen between the groups (P = 0.33) [Table 4]. The mean GCF free haem level at diseased sites for Group I was 26.70 ± 4.80 (ng) and for Group II, it was 25.98 ± 4.83 (ng). Statistically insignificant difference was seen between the groups (P = 0.51) [Table 4].

Table 4

Comparison of mean gingival crevicular fluid free haem concentration (ng/ml) at healthy and diseased sites between smokers and nonsmokers group using independent Student’s t-test

Sites	Group	n	Mean±SD	Mean difference	P
Healthy sites	Smokers	39	22.90±3.60	0.95	0.33 (NS)
	Nonsmokers	39	21.95±4.86
Diseased sites	Smokers	39	25.98±4.83	−0.72	0.51 (NS)
	Nonsmokers	39	26.70±4.80

S – Statistically significant (P<0.05); NS – Statistically nonsignificant (P>0.001); n – Sample size; SD – Standard deviation; ng – Nanogram; ml – Milliliter; P – Level of Significance

The percentage of positive sites for BANA test was more at the diseased sites compared to healthy sites in both the groups. Statistically insignificant difference was seen in Group I (P = 0.30) and was statistically significant for Group II (P = 0.001) [Table 5]. There was a statistically significant difference between the groups at healthy sites (P = 0.04) [Table 6] and there was no difference at diseased sites (P = 0.34) [Table 6].

Table 5

Comparison of N-benzoyl-DL-arginine-2-naphthylamide test expressions between healthy and diseased sites in smokers and nonsmokers using McNemar’s test

Group	BANA test	Healthy site, n (%)	Diseased site, n (%)	P
Smokers	Positive	10 (25.6)	28 (71.8)	<0.001* (S)
	Negative	29 (74.4)	11 (28.2)
Nonsmokers	Positive	19 (48.7)	24 (61.5)	0.30 (NS)
	Negative	20 (51.3)	15 (38.5)

*Statistically significant. S – Statistically significant (P<0.001); NS – Statistically nonsignificant (P>0.001), n – Sample size, BANA – N-benzoyl-DL-arginine-2-naphthylamide; P – Level of Significance

Table 6

Comparison of N-benzoyl-DL-arginine-2-naphthylamide test expressions at healthy and diseased sites between smokers and nonsmokers using Chi-square test

Sites	BANA test	Smokers, n (%)	Nonsmokers, n (%)	P
Healthy sites	Positive	10 (25.6)	19 (48.7)	0.04* (S)
	Negative	29 (74.4)	20 (51.3)
Diseased sites	Positive	28 (71.8)	24 (61.5)	0.34 (NS)
	Negative	11 (28.2)	15 (38.5)

*Statistically significant. S – Statistically significant (P<0.001); NS – Statistically nonsignificant (P>0.001); n – Sample size; BANA – N-benzoyl-DL-arginine-2-naphthylamide; P – Level of Significance

At healthy sites, a very weak correlation was found between plaque index, probing pocket depth, and GCF free haem concentration in nonsmokers group and between gingival index, papillary bleeding index, and GCF free haem concentration in smokers group [Table 7]. A negative correlation was found between gingival index, papillary bleeding index, clinical attachment level, and GCF free haem concentration in nonsmokers group and between plaque index, clinical attachment level, pocket probing depth, and GCF free haem concentration in the smokers group [Table 7].

Table 7

Correlation coefficients to assess relationship between gingival crevicular fluid free haem concentration and clinical parameters in healthy sites

Variables	Smokers		Nonsmokers
	r	P	r	P
GI	0.05	0.75	−0.05	0.75
PI	−0.08	0.65	0.01	0.96
PBI	0.01	1.00	−0.01	0.97
PPD	−0.08	0.64	0.06	0.71
CAL	−0.04	0.80	−0.12	0.47

Correlation coefficient range: 0.0 – No correlation; 0.01-0.20 – Very weak correlation; 0.21-0.40 – Weak correlation; 0.41–0.60 – Moderate correlation; 0.61-0.80 – Strong correlation; 0.81–1.00 – Very strong correlation. GI – Gingival index; PI – Plaque index; PBI – Papillary bleeding index; PPD – Probing pocket depth; CAL – Clinical attachment level; r – Correlation coefficient range; P – Level of Significance

At healthy sites, there was no statistically significant difference between the GCF free haem levels and BANA results in smokers (P = 0.98) and nonsmokers (P = 1.00) [Table 8]. At diseased sites also, there was no statistically significant difference between the GCF free haem levels and BANA results in smokers (P = 0.87) and nonsmokers (P = 0.46) [Table 9].

Table 8

Comparison of mean gingival crevicular fluid free haem concentration (ng/ml) based on N-benzoyl-DL-arginine-2-naphthylamide test in smokers and nonsmokers among healthy subjects using independent student’s t-test

Group	Marker	BANA	n	Mean±SD	Mean difference	P
Smokers	Free haem concentration	Positive	10	22.86±3.91	−0.04	0.98
		Negative	29	22.91±3.56
Nonsmokers	Free haem concentration	Positive	19	21.95±4.25	0.00	1.00
		Negative	20	21.95±5.50

n – Sample size; SD – Standard deviation; BANA – N-benzoyl-DL-arginine-2-naphthylamide; ng – Nano gram; ml – Millilitre; P = 0.001

Table 9

Comparison of mean gingival crevicular fluid free haem concentration (ng/ml) based on N-benzoyl-DL-arginine-2-naphthylamide test in smokers and nonsmokers among diseased subjects using independent student’s t-test

Group	Marker	BANA	n	Mean±SD	Mean difference	P
Smokers	Free haem concentration	Positive	28	25.89±5.45	−0.29	0.87
		Negative	11	26.19±2.88
Nonsmokers	Free haem concentration	Positive	24	27.15±4.97	1.18	0.46
		Negative	15	25.97±4.59

n – Sample size; SD – Standard deviation; BANA – N-benzoyl-DL-arginine-2-naphthylamide; ng – Nanogram; ml – Milliliter; P = 0.001

At diseased sites, a very weak correlation was found between gingival index, papillary bleeding index, clinical attachment level, pocket probing depth, and GCF free haem concentration in nonsmokers group, and between gingival index, plaque index, papillary bleeding index, and GCF free haem concentration in smokers group [Table 10]. A negative correlation was found between plaque index, probing pocket depth, clinical attachment level, and GCF free haem concentration in nonsmokers group and between probing pocket depth, clinical attachment level, and GCF free haem concentration in the smokers group [Table 10].

Table 10

Correlation coefficients to assess relationship between gingival crevicular fluid free haem concentration and clinical parameters in diseased sites

Variables	Smokers		Nonsmokers
	r	P	r	P
GI	0.02	0.92	0.16	0.33
PI	0.12	0.47	−0.17	0.32
PBI	0.12	0.47	0.30	0.06
PD	−0.15	0.37	0.01	0.98
CAL	−0.16	0.34	0.01	0.99

Correlation coefficient range: 0.0 – No correlation; 0.01-0.20 – Very weak correlation; 0.21-0.40 – Weak correlation; 0.41-0.60 – Moderate correlation; 0.61-0.80 – Strong correlation; 0.81–1.00 – Very strong correlation. S – Statistically significant (P<0.05); NS – Statistically nonsignificant (P>0.05); GI – Gingival index; PI – Plaque index; PBI – Papillary bleeding index; PD – Pocket depth, CAL – Clinical attachment level; r – Correlation coefficient range; P – Level of Significance

DISCUSSION

Bacteria-induced chronic inflammatory disease such as periodontitis affects the supporting structures of the teeth. The ultimate outcome would be alveolar bone loss and exfoliation of the involved teeth if left untreated.[5]

Virulent microorganisms capable of causing disease are required for the initiation and the progression of periodontitis. The establishment and progression of periodontal disease is dependent on the ability of a pathogen to colonize and proliferate within an environmental niche in the host.[6] Furthermore, essential nutrients like iron are required for the growth of microorganism.[7]

While bacteria are responsible for initiating periodontitis, the host response to the pathogens is critical for the disease progression. Over many years, many studies have addressed potential mechanisms linking smoking and periodontitis, including molecular and genetic factors in addition to impairment of wound healing.[8]

Assessment of periodontal disease type, its location, and severity can be determined from useful periodontal diagnostic procedures. This information can also be used to plan the treatment for the patient and during the follow-up period of the disease.

Assessment of clinical parameters and radiographs which are our traditional diagnostic methods provides only a historical perspective and not a current appraisal of disease status.[3]

Therefore, it is essential to determine the disease stage of the patient to plan a good treatment. Hence, in this regard, we require diagnostic procedures which are rapid, sensitive, and at the same time very specific. Since biomarkers vary in healthy and diseased conditions, they are being evaluated to provide the necessary information.[9]

In this context, except for a pilot study by Liu et al. in 2016, till date, no studies have investigated the role of free haem in GCF.[3] Earlier studies have found that increased hemoglobin has potentiated the virulence effects of periopathogens, thereby increasing the levels of inflammatory cytokines.[6]

The porphyrin class which forms the nonprotein part of hemoglobin and some other biological molecules contains iron in the form of hem.

Hemoproteins are porphyrin-containing metalloproteins that have hem as their prosthetic group.[10] Hemoglobin, the red pigment in the blood, contains hem which can also be found in other hemoproteins which are biologically important.[11] In this context, our study was designed to investigate the GCF levels of free haem in subjects with chronic periodontitis along with microbiological examination. Smokers were also included to find out if there was any influence of smoking on the free hem levels.

Therefore, in our study, we included two groups which are Group I (chronic periodontitis in nonsmokers) and Group II (chronic periodontitis in smokers) from who samples were collected at both healthy and diseased sites.

In our study, the mean age was 37.2 years for nonsmokers and 37.4 years for smokers which were not statistically significant. With respect to gender, there were 30.8% females and 69.2% males in Group I, whereas there were 100% males in Group II. Hence, there was a statistically significant gender difference between the groups. This could probably be explained due to the higher percentage of male smokers compared to females and also, female's acceptance of smoking is less. In our study, the diagnosis of smoking was based on patients self-report and was classified as only smokers and nonsmokers.

We used microcapillary pipettes to collect GCF samples to avoid any nonspecific attachment of the analyte to filter paper and thus avoid any false detection of the marker. The collected GCF samples were subjected to Biovison Hemin Assay Kit for estimating the levels of free hem. When samples were added to the BioVision's Hemin Assay Kit, it converted a colorless probe to a strongly colored compound by utilizing peroxidase activity in the presence of hemin to provide a simple and exquisitely sensitive assay. Absorbance was measured at 450 nm. Even minute amounts of hemin in the range of 5–160 pg (10–250 fmol) could be quantitated.

The GCF free haem levels were statistically significantly higher at periodontitis sites compared to healthy sites in both the groups. This is in accordance with the study carried out by Liu et al.[3] This could be explained by the fact that many periodontal pathogens require haem-iron containing compounds for their growth but lack the ability to synthesize it. This haem also helps in electron transport by providing protoporphyrin source.[7]

In the study by Liu et al., the values for GCF free haem are in the range of 0.00–275.96 nM at healthy sites and 0.00–15254.92 nM at diseased sites. In our study, at healthy sites, the values for GCF free haem have been in the range of 11.20–29.59 ng in nonsmokers group and 17.91–29.67 ng in smokers group. At diseased sites, the values for GCF free haem have been in the range of 18.80–37.59 ng in nonsmokers group and 21.38–38.97 ng in smokers group. In the study by Liu et al., 570 nm optical density is used to determine the concentrations photometrically, whereas in our study, we have used 450 nm optical density.

The other probable reasons could be due to variations in the sample size, demographic data, method of sample collection, duration of storage of the samples, assay procedure, oral microbial flora, and regulating factors such as hem oxygenase–I levels.

Breakdown of hemin-containing proteins such as hemoglobin and myoglobin results in free hemin. It can be detected in various body fluids such as saliva, urine, and GCF under various pathological states including periodontitis. Very minute concentrations of free haem is present in the cells (<1 μM ≈650 ng/ml) exerting regulatory functions such as repression of nonspecific δ-aminolevulinate synthase expression and induction of microsomal hemin oxygenase-1.

The microbiological examination showed more positive sites for BANA test at periodontitis sites compared to healthy sites in both the groups which was statistically significant. The percentage of positive sites compared to the nonsmokers group was statistically more in smokers group. Similar results were seen in a study by Zambon et al.,[12] van Winkelhoff et al.,[13] and Haffajee et al.[14] who found that the Tannerella forsythia, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis levels were higher in smokers group as compared to nonsmokers group using immunofluorescence technique. Specific virulence factors of T. forsythia, A. actinomycetemcomitans, and P. gingivalis have been identified to be hemolytic.

However, studies carried out by Stoltenberg et al.,[15] Hanes et al.,[16] Bergström and Boström,[17] and Boström et al.[18] found no statistically significant difference between smokers and nonsmokers with respect to prevalence of the subgingival bacteria. Some of these studies have given more importance to bacterial virulence factors as compared to bacterial count alone.

BANA test is positive when there is increase in the levels of hemolytic bacterias such as P. gingivalis, T. denticola, and T. forsythia (red complex).

Therefore, an increase in GCF free haem levels can be observed as total hemolytic activity increases at diseased sites due to red blood cell lysis.

The GCF free haem levels between smokers and nonsmokers were not statistically significant. This finding is in contrast to the findings seen in a study by Liu et al.[3] who found an inverse relationship between smoking and GCF free haem concentration. This could be probably related to the increased hemoxygenase I (HO-I) protein expression in smokers which will break down hem into bilirubin and carbon monoxide. Similarly, Gayatri et al.[19] and Chang et al.[20] found increased expression of HO-I in the gingival tissue samples taken from smokers compared with periodontitis and healthy tissue.

A negative correlation was found between probing pocket depth, clinical attachment level, and GCF free haem concentration in smokers group at diseased sites (Group IIB). The increased amount of GCF as the pocket depth increases could have probably been responsible for this. A very weak correlation was seen between probing pocket depth, clinical attachment level, and GCF free haem in Group I.

SUMMARY AND CONCLUSION

In both smokers and nonsmokers, the GCF free haem concentration was statistically significantly higher at periodontitis sites compared to healthy sites. There was no statistically significant difference in the GCF free haem concentrations between smokers and nonsmokers. The BANA positive sites were statistically significantly higher at diseased sites compared to healthy sites in both smokers and nonsmokers. In the smokers group, the BANA-positive sites were statistically significantly higher compared to nonsmokers.

Thus, this study concludes that the higher concentration of GCF free haem at diseased sites indicates that it could be used as a potential biomarker to determine active periodontal sites, also smoking and BANA results did not influence the biomarker levels.

Further studies with even larger sample size, inclusion of other forms of periodontal diseases, and interventional studies are recommended to further ascertain that GCF free haem levels could be used as a risk indicator for predicting the occurrence of periodontitis in healthy subjects and their progression.

Financial support and sponsorship

This study was financially supported by Rajiv Gandhi University of Health Sciences.

Conflicts of interest

There are no conflicts of interest.