Salivary annexin A1: A candidate biomarker for periodontitis.

AIM: To compare the salivary proteomic profile of periodontitis-affected (PA) parents and their offspring to periodontally healthy (PH) dyads in the pursuit of possible biomarkers for early diagnosis of this disease. MATERIALS AND METHODS: Unstimulated saliva samples collected from 17 pairs of PA or PH individuals and their children were submitted to mass spectrometric analyses followed by proteomic analyses. Primary PA fibroblasts were triggered towards having an inflammatory response, and an immunoenzymatic assay of its supernatant was performed to validate the obtained data. RESULTS: ANXA1, KRT4, GSTP1, HPX, A2M and KRT13 were lower in PA parents and their children, and IGHG1, CSTB, KRT9, SMR3B, IGHG4 and SERPINA1 were higher. ANXA1 presented the highest fold change, 7.1 times less produced in children of PA parents, and was selected as a potential biomarker for periodontitis. The in vitro assay also showed lower ANXA1 production by cells of PA patients. CONCLUSION: Before any clinical sign of periodontal loss, descendants of PA patients have an altered proteomic profile compared to PH individuals, presenting a lower abundance of ANXA1. This protein is suggested as a potential biomarker for periodontitis.

IL10 promoter rs6667202 polymorphism is functional in health but not in grade c periodontitis patients: A pilot study.

BACKGROUND AND OBJECTIVE: Previous studies have demonstrated an association between the IL10 promoter rs6667202 (C > A) single-nucleotide polymorphism (SNP) and grade C, stage 3 or 4 periodontitis (Perio4C) in the Brazilian population, where the altered A allele was detected more frequently in these patients. However, no functional analysis of this variation has yet been performed. Thus, the objective of this preliminary study was to evaluate the functionality of rs6667202 in gingival fibroblasts (GFs) of individuals with Perio4C and with periodontal health (PH) stimulated with Aggregatibacter actinomycetencomitans protein extract (AaPE). METHODS: Patients with PH and Perio4C were segregated according to their genotype (AA, AC, or CC), and a biopsy was performed to establish the culture of the GFs. After GFs exposure to AaPE at 5 µg/ml for 1.5 h, RNA was extracted to analyze IL10 expression by qPCR. Aliquots of the cell's supernatant were subjected to immunoenzymatic analysis (MAGpix) to detect interleukin-10 (IL-10). RESULTS: In PH, the genotypes AA and AC are related to less expression of IL10 (p = 0.027 and p < 0.0001) and less production of IL-10 (p = 0.002 and p = 0.001), when compared to CC. In Perio4C, there was no statistical difference between the genotypes (p > 0.05), although a lower IL-10 expression and release compared with PH CC was seen (p = 0.033 and p < 0.001). CONCLUSION: The rs6667202 SNP is functional in PH, as it decreases the expression and production of IL-10. In Perio4C, other factors may be masking its action by altering the IL-10's response.

Effects of the Pulsed Electromagnetic Fields on Experimental Periodontitis and Estrogen Deficiency.

Periodontitis is an inflammatory disease resulting from a complex polymicrobial infection that causes tissue destruction in susceptible individuals. Osteoporosis has been associated with greater clinical attachment loss in patients with periodontitis. Experimental studies have shown positive results in the treatment of osteoporosis through pulsed electromagnetic field (PEMF) stimulation. The aim of this study was to evaluate the effects of PEMF in the presence of estrogen deficiency associated with periodontitis, verifying its role in bone metabolism and in the inflammatory response. Sixty rats were divided into four groups: Sham surgery + ligature-induced periodontitis (P); Sham surgery + ligature-induced periodontitis + PEMF therapy (P + PEMF); Ovariectomy surgery + ligature-induced periodontitis (P + OVX); Ovariectomy surgery + ligature-induced periodontitis + PEMF therapy (P + OVX + PEMF). The area of bone loss in the furcation region (BL), connective tissue attachment loss (CTAL) and alveolar bone loss (ABL), BV/TV and BMD were evaluated. In addition to immunohistochemical labelling of RANKL, OPG, and TRAP and the inflammatory response of interleukin (IL)-1b, IL-6, TNF-α, IL-10, and vascular endothelial growth factor. P + OVX showed significant BL in relation to P + PEMF and the greatest CTAL and ABL. P + OVX and P + OVX + PEMF showed a significant reduction in BV/TV (%). P and P + PEMF showed a significantly lesser amount of Tb.Sp (mm) while P + OVX and P + OVX + PEMF showed a lesser of Tb.N. P + PEMF had the greatest BMD. P + OVX presented higher RANKL and lower OPG immunolabeling than other groups. P + PEMF and P + OVX + PEMF showed a reduction on all biomarkers evaluated. The application of PEMF seems to attenuate the effects of bone loss in the presence of periodontitis and ovariectomy. © 2022 Bioelectromagnetics Society.

Childhood Hypophosphatasia Associated with a Novel Biallelic ALPL Variant at the TNSALP Dimer Interface.

The goal of this study was to perform a clinical and molecular investigation in an eight-year-old female child diagnosed with hypophosphatasia (HPP). The proband and her family were evaluated by medical and dental histories, biochemical analyses, radiographic imaging, and genetic analysis of the tissue-nonspecific alkaline phosphatase (ALPL) gene. A bioinformatic analysis was performed to predict the structural and functional impact of the point mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) molecule and to define their potential contribution to the phenotype. We identified a novel combination of heterozygous ALPL missense variants in the proband, p.Ala33Val and p.Asn47His, compatible with an autosomal recessive mode of inheritance and resulting in skeletal and dental phenotypes. Computational modeling showed that the affected Asn47 residue is located in the coil structure close to the N-terminal α-helix, whereas the affected Ala33 residue is localized in the N-terminal α-helix. Both affected residues are located close to the homodimer interface, suggesting they may impair TNSALP dimer formation and stability. Clinical and biochemical follow-up revealed improvements after six years of ERT. Reporting this novel combination of ALPL variants in childhood HPP provides new insights into genotype-phenotype associations for HPP and specific sites within the TNSALP molecule potentially related to a childhood-onset HPP and skeletal and dental manifestations. Beneficial effects of ERT are implicated in skeletal and dental tissues.

Subgingival endotoxin and lipoteichoic acid modulate cytokine production in diabetic subjects: A Case-control Study.

BACKGROUND: Periodontal disease and diabetes mellitus (DM) are highly prevalent and interrelated diseases, resulting in altered host response microbiota. Thus, this study aimed to evaluate the impact of DM on local levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and their relationship with cytokines and matrix metalloproteinases' (MMPs) profile. METHODS: This case-control study included diabetic (n = 15) and non-diabetic (n = 15) subjects presenting Stage 3-4, Grade C, Periodontitis. Gingival crevicular fluid (GCF) was collected, and LPS and LTA levels were analyzed by enzyme-linked immunosorbent assay (ELISA), while IFN-γ, IL-10, IL-17, IL-1ß, IL-4, MMP-2, and MMP-9 were measured by LUMINEX/MAGpix. Mann-Whitney and Spearman's correlation tests were used to compared and to correlate variables (p < 0.05). RESULTS: Higher levels of LTA, LPS, IL-10, IL-1ß, and MMP-2 (p < 0.05) and lower levels of IL-17 were found in the DM group (p < 0.05). Non-diabetic subjects presented higher LPS, IFN-γ, IL-17, and MMP-2 levels and lower IL-10 concentration (p < 0.05). No significant correlation was seen between LPS and cytokine profile in non-diabetic. Local levels of LTA were positively correlated with IL-17 and MMP-2 and negatively with IL-10. CONCLUSION: LTA and LPS drove the inflammatory profile through the modulation of cytokines and MMPs in a different manner in DM and non-diabetic subjects.

Connective tissue graft associated or not with low laser therapy to treat gingival recession: randomized clinical trial.

BACKGROUND: To evaluate the treatment of gingival recession with a connective tissue graft (CTG) alone or in combination with low-level laser therapy (CTG + L). METHODS: Forty patients presenting 40 Miller Class I and II gingival recessions were included. The defects were randomly assigned to receive either CTG (n = 20) or CTG + L (n = 20). A diode laser (660 nm) was applied to the test sites immediately after surgery and every other day for 7 days (eight applications). RESULTS: The mean percentage of root coverage was 91.9% for the test group and 89.48% for the control group after 6 months (p > 0.05). The test group presented more complete root coverage (n = 13, 65%) than the control group (n = 7, 35%) (p = 0.04). Dentine sensitivity decreased significantly after 6 months in both groups (p < 0.001). The two groups showed improvement in aesthetics at the end of treatment. CONCLUSIONS: Low-level laser therapy may increase the percentage of complete root coverage when associated with CTG.

Immune response characterization of primary gingival fibroblasts from Grade C periodontitis patients.

BACKGROUND: Grade C, Stage 3-4 Periodontitis (Perio4C) is a rapidly destructive disease caused by an unequilibrated immune response that starts after the primary contact of the periodontopathogens with the gingival tissue. However, it is still unclear how this imbalanced response initiates and what is the role of the connective tissue cells in the progression of this disease. Thus, this study aims to assess the local immune response of Perio4C patients through the exposure of primary gingival fibroblast cells (GFs) with Aggregatibacter actinomycetemcomitans protein extract (AaPE) and the quantification of the inflammatory cytokines interleukin (IL)-4, IL-17, tumor necrosis factor (TNF)-α, IL-1ß, interferon (IFN)-γ, and IL-10 super-family members (IL-10, IL-19, and IL-24) secreted by them. METHODS: Gingival biopsies from nine periodontally health (PH) and eight Perio4C patients were harvested, and the primary culture of GFs was obtained. The cells were exposed to AaPE (5 and 20 µg/ml) and 12-myristate 13-phorbol acetate and ionomycin - calcium salt (PMA). The supernatant was collected after 1.5 and 3 h, and a cytokine panel was evaluated. RESULTS: Clustering analysis indicated dissimilar and stimuli-dependent cytokine production between Perio4C and PH subjects. Perio4C GFs presented lower production of IL-4, TNF-α, IFN-γ, IL-17, IL-10, IL-24, and IL-19, while IL-1ß levels were similar to the PH group, leading to a disruption in the pro-/anti-inflammatory cytokine ratio (p < 0.05). IL-1ß and IL-10 super-family were the most discriminative representants for PH and Perio4C, respectively. CONCLUSION: GFs from individuals with Perio4C tended to hypo-respond to stimulation with AaPE, producing lower concentrations of some pro- and anti-inflammatory molecules, trending to develop a pro-inflammatory extracellular environment.

Comparison of conventional and contemporary root canal disinfection protocols against bacteria, lipoteichoic acid (LTA), and lipopolysaccharide (LPS).

This study devised a dual-species biofilm model to investigate bacteria, lipoteichoic acid (LTA), and lipopolysaccharide (LPS) simultaneously, and compared the efficacy of conventional and contemporary disinfection protocols. Seventy single-rooted mandibular premolars were included. Fourteen teeth were negative control, and 56 teeth were infected with 3-week-old E. faecalis and E. coli GFP biofilm. Fourteen/56 teeth were positive control, with seven teeth processed for CLSM analysis and seven teeth sampled with paper points (PPs) and cryogenically ground for bacterial, LTA, and LPS analyses. Forty-two teeth were randomly divided into three groups: GWS (GentleWave system) + MIT (minimally invasive technique), PUI (passive ultrasonic irrigation) + CIT (conventional instrumentation technique), and XP-EF (XP-endo Finisher) + CIT (All, n = 14). Samples were collected before (s1) and after disinfection (s2) with PPs and after cryogenically ground (s3). CFUs were counted, and LTA and LPS were quantified with LTA-ELISA and LAL assay, respectively. XP-EF was as effective as PUI (p > 0.05). GWS + MIT was the most effective disinfection protocol against bacteria, LTA, and LPS (p < 0.05). In conclusion, PUI, XP-EF, and GWS were highly effective against bacteria, LTA, and LPS, with GWS being the most effective.

Diagnostic reproducibility of the 2018 Classification of Gingival Recession Defects and Gingival Phenotype: A multicenter inter- and intra-examiner agreement study.

BACKGROUND: The aim of this study was to investigate the inter- and intra-examiner agreement among international experts on the diagnosis of gingival recession defects using the 2018 Classification of Gingival Recession Defects and Gingival Phenotype as proposed in the 2017 World Workshop. METHODS: Standardized intraoral photographs from 28 gingival recession defects were evaluated twice by 16 expert periodontists. Recession type (RT), recession depth (RD), keratinized tissue width (KTW), gingival thickness (GT), detectability of the cemento-enamel junction (CEJ), and presence of root steps (RS) were recorded and used for the analysis. Intra- and inter-examiner agreements were calculated for individual variables and for the overall classification. Intraclass correlation coefficient with 95% CI was used for RD and KTW; Kappa with 95% CI was used for GT, CEJ, and RS; quadratic weighted Kappa with 95% CI was used for RT. RESULTS: Overall intra- and inter-examiner agreements were highest for KTW (0.95 and 0.90), lowest for GT (0.75 and 0.41), with the other variables in between (RD: 0.93 and 0.68, RS: 0.87 and 0.65, RT: 0.79 and 0.64, CEJ: 0.75 and 0.57). Overall intra- and inter-examiner agreements for the matrix were 62% and 28%, respectively. Significant effects existed between one variable's measurement and other variables' agreements. CONCLUSIONS: The 2018 Classification of Gingival Recession Defects and Gingival Phenotype is clinically reproducible within the examiners, and when the variables forming the matrix are analyzed individually. The between-examiner agreement for the complete matrix showed lower reproducibility. The agreement was highest for KTW and RD, and least for GT.

Open flap debridement compared to repeated applications of photodynamic therapy in the treatment of residual pockets: A randomized clinical trial.

BACKGROUND: The aim of the present study was to compare repeated applications of antimicrobial photodynamic therapy (aPDT) to open flap debridement (OFD) in the treatment of residual periodontal pockets in non-furcation sites. METHODS: Forty-six subjects with a diagnosis of Stage III or IV Grade C periodontitis, that had been previously treated, participated in the study.  Residual pockets were divided between two groups: (1) aPDT group: received ultrasonic periodontal debridement followed by immediate application of aPDT, and repeated on1st, 2nd, 7th, and 14th days; and (2) OFD group: treated by modified papilla preservation technique, where granulation tissue and visible calculus were removed with hand curettes and an ultrasonic device. Clinical, immunological, and microbiological parameters were evaluated before and after treatment. RESULTS: Both treatments were effective reducing clinical parameters of disease. OFD resulted in a greater mean probing pocket depths (PPD) reduction in deep pockets (p = 0.001). However, aPDT resulted in a lower occurrence of gingival recession (GR), dentin hypersensitivity (DH) and analgesic intake. Reduction in Porphyromonas gingivalis was observed in both groups. Only the OFD group had a significant reduction in Aggregatibacter actinomycetemcomitans. aPDT group had greater increase in interleukin 10 (IL-10) levels and a greater reduction of interleukin 1 beta (IL-1ß) at 14 days when compared to the OFD group (p < 0.05). CONCLUSION: OFD was superior in reducing PPD in deep pockets compared to the aPDT. However, OFD resulted in greater GR.  Both treatments lowered P. gingivalis levels but only OFD reduced levels of A. actinomycemtemcomitans.

Omega-3 PUFA and aspirin as adjuncts to periodontal debridement in patients with periodontitis and type 2 diabetes mellitus: Randomized clinical trial.

BACKGROUND: Supplementation with omega-3 polyunsaturated fatty acids (ω-3 PUFA) and low-dose aspirin (ASA) have been proposed as a host modulation regimen to control chronic inflammatory diseases. The aim of this study was to investigate the clinical and immunological impact of orally administered ω-3 PUFA and ASA as adjuncts to periodontal debridement for the treatment of periodontitis in patients type 2 diabetes. METHODS: Seventy-five patients (n = 25/group) were randomly assigned to receive placebo and periodontal debridement (CG), ω-3 PUFA + ASA (3 g of fish oil/d + 100 mg ASA/d for 2 months) after periodontal debridement (test group [TG]1), or ω-3 PUFA + ASA (3 g of fish oil/d + 100 mg ASA/d for 2 months) before periodontal debridement (TG2). Periodontal parameters and GCF were collected at baseline (t0), 3 months after periodontal debridement and ω-3 PUFA + ASA or placebo for TG1 and CG (t1), after ω-3 PUFA + ASA (before periodontal debridement) for TG2 (t1), and 6 months after periodontal debridement (all groups) (t2). GCF was analyzed for cytokine levels by multiplex ELISA. RESULTS: Ten patients (40%) in TG1 and nine patients (36%) in TG2 achieved the clinical endpoint for treatment (less than or equal to four sites with probing depth ≥ 5 mm), as opposed to four (16%) in CG. There was clinical attachment gain in moderate and deep pockets for TG1. IFN-γ and interleukin (IL)-8 levels decreased over time for both test groups. IL-6 levels were lower for TG1. HbA1c levels reduced for TG1. CONCLUSION: Adjunctive ω-3 and ASA after periodontal debridement provides clinical and immunological benefits to the treatment of periodontitis in patients with type 2 diabetes.

Texture analysis of cone-beam computed tomography images assists the detection of furcal lesion.

BACKGROUND: The aim of this study was to apply texture analysis (TA) to cone-beam computed tomography (CBCT) scans of patients with grade C periodontitis for detection of non-visible changes in the image. METHODS: TA was performed on CBCT scans of 34 patients with grade C periodontitis. Axial sections of CBCT were divided into three groups as follows: Group L (lesion) in which there is a furcal lesion with periodontal bone loss; Group I (intermediate) in which the border of the furcal lesion has normal characteristics; and Group C (control) in which the area is healthy. Eleven texture parameters were extracted from the region of interest. Mann-Whitney U test was used to assess the differences in the texture between the three groups as follows: L versus I; L versus C, and I versus C. RESULTS: Statistically significant differences (P <0.05) were observed in almost all parameters in the intergroup analyses (i.e., L versus I and L versus C). However, statistical differences were smaller in groups I versus C in which only entropy of sum, entropy of difference, mean of sum, and variance of difference were statistically different (P < 0.05). CONCLUSION: TA can potentially provide prognostic information to improve the diagnostic accuracy in the grading of the tissue around the furcal lesion, thus potentially accelerating the treatment decision-making process.

Novel rare frameshift variation in aggressive periodontitis: Exomic and familial-screening analysis.

BACKGROUND: Aggressive periodontitis (AgP), currently periodontitis grade C, presents early onset, rapid progression, and a poorly established genetic association. Thus, this study aimed to identify genetic variants associated with AgP via whole exome sequencing (WES) through a familial screening approach. METHODS: WES was performed in two nuclear families, including a proband and a parent affected by AgP and an unaffected parent and sibling. Common variants among affected individuals, excluding those common to healthy people, from each family, composed the data set associated with AgP. In silico analysis evaluated the impact of each variant on protein structure and protein-protein interactions. Moreover, identified deleterious variants were validated in a populational analysis (n = 96). RESULTS: The missense single nucleotide variations (SNVs) rs142548867 in EEFSEC (c.668C>T), rs574301770 in ZNF136 (c.466C>G), and rs72821893 in KRT25 (c.800G>A) and the frameshift indels rs37146475 in GPRC6A (c.2323-2324insT) and c.1366_1372insGGAGCAG in ELN were identified in AgP and have a predicted functional impact on proteins. In silico analysis indicated that the indel in GPRC6A generates a loss of the C-terminal tail of the Gprca protein. Furthermore, this SNV was significantly associated with AgP in a population-based investigation. CONCLUSION: Novel frameshift variation in GPRC6A (c.2323-2324insT) was identified as a potential genetic alteration associated with AgP occurrence.

Two different antibiotic protocols as adjuncts to one-stage full-mouth ultrasonic debridement to treat generalized aggressive periodontitis: A pilot randomized controlled clinical trial.

BACKGROUND: To assess the clinical and microbiological responses of amoxicillin + metronidazole (AMX + MET) versus clarithromycin (CLM) as adjuncts to one-stage full-mouth ultrasonic debridement (FMUD) in the treatment of generalized aggressive periodontitis (GAgP). METHODS: For this parallel, double-masked, pilot randomized clinical trial, 46 patients with GAgP were selected and randomly assigned into two groups: AMX+MET group (n = 23): FMUD associated with AMX (500 mg three times a day) and MET (400 mg three times a day) for 7 days; and CLM group (n = 23): FMUD associated with CLM (500 mg twice a day) for 7 days. Clinical parameters were evaluated at baseline, 3, and 6 months post-treatment. The levels of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum from subgingival biofilm were determined by quantitative polymerase chain reaction. RESULTS: Both treatments significantly improved all clinical parameters compared with baseline and promoted a significant reduction of A. actinomycetemcomitans and P. gingivalis counts (P > 0.05). CLM succeeded in decreasing T. forsythia at 6 months (P < 0.05), but no antibiotic was able to reduce F. nucleatum. There was no difference between the two protocols regarding the reported adverse effects (P > 0.05). CONCLUSIONS: The results suggest that CLM is not superior than AMX + MET in the treatment of GAgP. However, this antibiotic led to good clinical outcomes and may be a possible alternative to AMX+MET in the treatment of severe periodontitis in young patients. Future studies with larger sample sizes are needed to confirm this statement (NCT02969928).

Does supplemental photodynamic therapy optimize the disinfection of bacteria and endotoxins in one-visit and two-visit root canal therapy? A randomized clinical trial.

AIM: To evaluate the effectiveness of supplemental photodynamic therapy (PDT) in optimizing the removal of bacteria and endotoxins from primarily infected root canals after one-visit and two-visit treatments. METHODOLOGY: Twenty-four primarily infected root canals with apical periodontitis were selected and randomly divided into one-visit (n=12) and two-visit treatment groups (n=12). Chemo-mechanical preparation (CMP) was performed by using the single-file reciprocating technique+2.5% NaOCL and a final rinse with 17% EDTA. The photosensitizer agent (methylene blue 0.1mg/mL) was applied to root canals for 60s before application of laser with a potency of 60mW and energy density of 129J/cm2 for 120s after CMP in the one-visit treatment and after 14-day inter-appointment medication with Ca(OH)2+Saline solution (SSL) in the two-visit treatment. Samples were collected before and after root canal procedures. Endotoxins were quantified by chromogenic limulus amebocyte lysate assay. Culture techniques were used to determine bacterial colony-forming unit counts. RESULTS: Bacteria and endotoxins were detected in 100% of the initial samples, with median values of 1.97×105 CFU/mL and 24.983EU/mL, respectively. The CMP using single-file reciprocating technique was effective in the reduction of bacteria and endotoxins (All, p<0.05). The supplemental PDT was effective in reducing bacterial load in the one-visit (p<0.05) but not in the two-visit treatment after use of Ca(OH)2 medication for 14days (p>0.05). In the two-visit group, after 14days of inter-appointment medication with Ca(OH)2, a significant reduction in the median levels of endotoxins was found in comparison to CMP alone (from 1.041 to 0.094EU/mL) (p<0.05). Despite the type of treatment, the supplemental PDT was not effective against endotoxins (p>0.05). CONCLUSIONS: The photodynamic therapy optimized the disinfection of bacteria from root canals in one-visit but not for two visit treatment modality with the accomplishment of calcium hydroxide medication. Despite the type of treatment, the supplemental PDT was not effective against endotoxins.

Concentrations of metronidazole in human plasma and saliva after tablet or gel administration.

OBJECTIVES: The aim of this study was to determine the pharmacokinetic profile of metronidazole (Mtz) tablet and to compare Mtz gel and tablet concentrations in both blood plasma and saliva. METHODS: In this randomized cross-over study with a 1-week washout period, 13 volunteers randomly received one (a single oral dose of 750 mg Mtz (Flagyl®--tablet) and 2) 3 g of 15% Mtz benzoate gel (applied by using a dental tray). The HPLC with ultraviolet detection was used to quantify plasma and saliva concentrations of Mtz. The pharmacokinetic parameters (PPs) areas under the curves from 0 to 48 h (AUC0-48) and from 0 to infinity (AUC0-∞), the maximum plasma concentration (C(max)), the time to C(max), volume of distribution and renal clearance were determined for Mtz tablet. KEY FINDINGS: Considering the Mtz tablet, plasma showed higher Mtz concentration from 6 to 24 h after drug administration and the highest values concerning AUC0-48 h and AUC0-∞ than those obtained in saliva (P < 0.05). No significant differences were observed between plasma and saliva concentrations for Mtz gel. CONCLUSIONS: The study showed that some PPs were higher in plasma (P < 0.05) than in saliva concerning Mtz tablet. Gel formulation had similar Mtz bioavailability in plasma and saliva resulting in systemic absorption.