Immunoglobulin free light chain-κ in association with NT-proBNP levels after smoking cessation

Abstract

Background

Smoking induces inflammation in the heart and intima layer of blood vessels by activating nuclear factor kappa B, which controls the transcription of immunoglobulin free light chain (FLC)-κ. FLC levels are indicative of higher mortality in the overall population and poorer prognoses in cardiovascular diseases. This study aimed to analyze the effect of smoking cessation (SC) on the levels of FLCs and markers of inflammation and heart failure.

Research design and methods

This study enrolled first visit smokers determined to quit smoking at our hospital. Levels of various clinical parameters, including inflammatory markers, such as FLC, were measured on their first clinic visit and after 3 months of successful SC.

Results

Body-mass-index (BMI) values significantly increased from baseline to 3 months after SC (n = 76, p = 0.004). Conversely, the levels of inflammatory markers including FLC-κ, neutrophil-to-lymphocyte-ratio and NT-proBNP significantly decreased after SC (p = 0.042, p = 0.024 and p = 0.030, respectively). Proportional changes in FLC-κ levels observed after SC showed a significant correlation with those of C-reactive-protein (r = 0.349, p = 0.002) and NT-proBNP (r = 0.332, p = 0.005) levels in the successful SC group.

Conclusions

Levels of FLC-κ, a novel inflammatory and cardiovascular risk biomarker, decreased significantly after SC in association with NT-proBNP, suggesting the SC-induced reduction of cardiac load as well as a decrease in inflammation.

PLAIN LANGUAGE SUMMARY

This longitudinal study is the first to show that FLC-κ, an inflammatory and cardiovascular risk biomarker, decreases early (within 3 months) after smoking cessation. This study is the first to demonstrate that NT-proBNP, a representative biomarker for heart failure, decreases early after smoking cessation. The decrease in NT-proBNP and FLC-κ after smoking cessation is an important finding suggesting a relationship between smoking-induced inflammation and heart failure.

ARTICLE HIGHLIGHTS

• This longitudinal study is the first to show that FLC-κ, which has been associated with cardiovascular prognosis, decreases early (within 3 months) after smoking cessation.

• However, not all markers decreased early after smoking cessation, with CRP and IL-6 showing no decrease at all.

• The levels of FLC-κ, an inflammatory and cardiovascular risk biomarker, decreased after smoking cessation despite body weight gain, suggesting that its beneficial effect overcomes potential risks posed by obesity.

• This longitudinal study is the first to demonstrate that NT-proBNP, a representative biomarker for heart failure, decreases early after smoking cessation.

• Decreases in FLC-κ levels were associated with that in NT-proBNP suggesting that smoking cessation promotes a reduction in cardiac load as well as inhibits inflammation.

1. Introduction

1.1. Background

Inflammation constitutes a core part of the different stages of cardiovascular diseases (CVDs). Tobacco smoke contains numerous oxidants, and smoking is associated with expression of various inflammatory cytokines [1] and induces a systemic inflammatory reaction. Smoking is a major contributor to the onset and progression of atherosclerosis and formation of CVD with the prevailing processes of unstable plaque generation activating proinflammatory macrophages and dendritic cells due to oxidative stress [2–4].

The transcription nuclear factor kappa B (NF-κB) is an integral part of activating signal transduction in inflammatory reactions to various stimuli [5]. NF-κB is overall present in the cytoplasm in the inactive form; however, upon the stimulation by oxidative stress or other factors, it promotes the transcription of interleukin (IL)-6 and other target genes (genes participating in inflammatory and immune reactions), thereby inducing these processes. NF-κB also regulates the production of immunoglobulin free light chains (FLCs) in B- cells [6]. Smoking activates FLC-κ transcription and NF-κB in B-cells, thus leading to inflammatory processes in cells [7].

In diseases in which inflammation is considered essential in the onset or progression, FLC level (concentration in serum) is of interest as a novel prognostic indicator [8]. Abnormal levels of FLC-kappa (κ) and FLC-lambda (λ) are thought to represent impaired immunoglobulin synthesis caused by excessive inflammatory response. In addition, interest in the clinical use of FLC as an attractive therapeutic target in chronic inflammatory diseases or as a biochemical marker of disease progression or remission has been increasing [9]. In addition, FLC values are associated with death and negative CVD outcomes in the general population [10]. Abnormal FLC concentrations are suggested to predict the development of diabetic renal disease, and markedly high FLC levels in those with CVD have shown a link to a higher risk of hospitalization and death [8]. Moreover, measuring FLC values was considered a useful initial health monitoring method in all individuals [10].

Smoking cessation (SC) helps reduce inflammation and the repair of damage it has caused; however, the time course and patterns of repair vary, ranging from an early onset of recovery to its slow speed. For example, the levels of some markers, such as neutrophil-to-lymphocyte ratio (NLR) [2], decrease significantly after only 3 months of SC, whereas those of others, such as C-reactive protein (CRP) [11], lower over a longer period (5–10 years), and achieving the nonsmoker level takes 10–20 years. Although higher FLC values have been reported in smokers [12], the details of this mechanism and the changes in FLC after SC have not been broadly investigated. An experiment performed in two groups of rats reported that after 8 weeks of exposure to cigarette smoke, NF-κB levels decreased in rats that were in a smoke-free condition for 8 weeks [13]. Moreover, NF-κB expression was reduced in mice after 3 months of hookah inhalation, followed by the same period of SC [14]. Therefore, the expression level of NF-κB may decrease if a short-term exposure to tobacco smoke, for example, 2–3 months, is avoided, resulting in lower FLC values. However, no studies have reported changes in FLC values associated with SC in humans. Thus, this study aimed to examine changes in FLC, an inflammation marker, after 3 months of SC.

2. Methods

2.1. Patients

This prospective cohort study was performed at the National Hospital Organization, Kyoto Medical Center, from August 2017 to October 2019. All patients who had consultations at our SC clinic during this period provided informed consent. A total of 170 patients were finally recruited, and 100 of them completed the 3-month SC programme. The patients were assessed for different parameters at the first consultation and 3 months after SC. Cardiovascular (CV) marker measurements were performed by blood analysis. In addition to these measurements, medical examination information, which had been collected by a medical interview about the current smoking state at the first visit to our SC department, body measurements, and other blood tests were performed. Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.

2.2. Ethical aspects

The participants provided informed written consent for study participation, and their identity was anonymized. The conduct of the research was in accordance with the World Medical Association Declaration of Helsinki, Act 2013 revised and Ethical Guidelines for Medical and Health Research Involving Human Subjects, the Ministry of Health, Labor, and Welfare, Dec 22, 2014, amended. The approval from the Ethics Committee at the National Hospital Organization Kyoto Medical Center (No. 22-016 and No. 14-042) was obtained.

2.3. SC clinic

Anti-smoking therapy was carried out in compliance with the Standard Procedures for Anti-Smoking Treatment (originally issued in March 2006 by the Japanese Circulation Society, Japan Lung Cancer Society and Japanese Cancer Association) [15]. The patients underwent examination at the baseline and after 2, 4, 8 and 12 weeks (3 months) and were administered transdermal nicotine patches or oral varenicline. On subsequent visits, the maintenance of SC was verified, and the involved medical personnel provided particular recommendations considering the continuation of SC. At the end of the 3-month anti-smoking therapy, the adherence to SC was checked. Abstinence was reflected in an exhaled carbon monoxide (CO) concentration of <7 parts per million (ppm) and by the patient’s affirmation of no smoking. An unsuccessful attempt was confirmed as the absence of patients during the treatment period or failure to quit smoking.

2.4. Test items

Age, sex, average cigarette intake (no. of cigarettes), length of smoking history, Fagerström Test for Nicotine Dependence (FTND) score, and medications currently being taken were collected. Nicotine dependence was assessed using FTND, a standard test evaluating physically derived addiction to nicotine [16–18]. The scores range from 0 to 10, where higher scores indicate higher severity of nicotine dependence. The following question was asked to estimate the daily consumption of cigarettes: “On average, in the past month, how many cigarettes did you smoke per day?”

At the onset of SC treatment (0 months, baseline) and at the end of it (after 3 months), body height, body weight, abdominal circumference and exhaled CO concentration were measured, and blood tests were performed.

Body mass index (BMI) was determined as the ratio of weight in kilograms to the square of height in meters. Systolic and diastolic blood pressures were measured in a sitting position after >5 min of rest and with an automatic electronic sphygmomanometer (BP-103iII; Nippon Colin, Komaki, Japan)　 [19]. A regular-sized cuff that passed relevant Japanese guidelines (arm length: 17–32 cm) was applied. At all visits, nurses measured the expiratory CO concentrations with an EC50 Micro Smokerlyzer^® (Bedfont Scientific Ltd., Kent, UK), determining the end-tidal CO electrochemically, at <2% precision [20].

2.5. Blood sampling

Blood samples were obtained three times at the first visit for screening and 3 months thereafter to evaluate changes in biochemical and general blood tests of the patients. The antecubital vein was catheterized 2–3 h after taking lunch for the analyses of the values of the CRP, leukocytes, neutrophils, lymphocytes, NLR, IL-6, LC-κ and FLC-λ.

FLC-κ and FLC-λ were measured using the following method. After serum separation, the blood samples were stored at −80 °C until analysis. These free light chains were quantified using the Diazyme assay kit (distributed by Roche) on a Roche analyzer. Sample measurements were performed simultaneously using the same reagent lot. While the assays are suitable in clinical laboratories for research, they are not widely used in daily clinical practice. Roche Diagnostics Japan kit, where enhanced immune turbid metric assay was applied for FLC binding to a specific antibody and mounted on latex particles, causing agglutination. The extent of turbidity induced by agglutination could be established optically and correlated with the amount of FLC-κ/λ in the sample. The interpolation of the received signals of 6‑point calibration with a known concentration was performed to calculate FLC κ/λ concentrations.

2.6. Statistical analysis

SPSS Statistics version 17.0 (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses by a professional statistician. The normality of distribution was evaluated with the Shapiro–Wilk test. Clinical parameters assessed before and after SC were compared using paired t-tests for parametric data or the Wilcoxon signed-rank test for non-parametric ones. Besides, the correlation between the change in the percentage of FLC-κ and clinical parameters between unsuccessful and successful cases was evaluated with Spearman’s rank correlation coefficient.

3. Results

3.1. Participants

Among the 100 patients who consented to study participation and completed the 3-month SC therapy, 4 were eliminated from the sample because of missing data. Out of the 96 study participants, 76 [55 men and 21 women] successfully quit smoking and 20 [16 men and 4 women] did not achieve SC (Figure 1).

Figure 1.

Flow chart of study participants.

In these patients, the mean age was 60.6 ± 13.2 years; average number of cigarettes smoked, 19.1 ± 7.3 cigarettes/day; length of smoking in medical history, 39.5 ± 12.7 years; and the FTND score, 6.3 ± 2.3 points. During the SC therapy, 32 (42%) patients were administered antihypertensive agents, 27 (36%) received statins, and 12 (16%) took medications for diabetes mellitus.

Table 1 shows the comparative analysis of the data gathered between the indicated time points. Expired CO concentrations significantly decreased from the first visit to 3 months after SC (from 17.0 to 3.0 ppm, p < 0.0001) in both groups (successful and unsuccessful SC).

Table 1.

Data on patients before and after 3 months of successful and unsuccessful smoking cessation.

	Successful patients (n = 76)						Unsuccessful patients (n = 20)
	Before		After		p-value		Before		After		p-value
BMI (kg/m2)	23.8 ± 3.9		24.2 ± 4.1		0.004	a	25.2 ± 3.0		25.3 ± 2.8		0.517	a
SBP (mmHg)	131.0	[119.3, 145.8]	133.0	[118.0, 145.0]	0.519	b	130.5	[120.3, 141.8]	141.0	[119.5, 148.8]	0.603	b
DBP (mmHg)	78.0	[69.0, 87.0]	74.5	[66.3, 89.8]	0.349	b	79.5	[71.8, 90.5]	81.0	[72.5, 86.3]	0.722	b
WBC (×103/mm3)	6.3	[5.4, 7.8]	6.2	[5.5, 7.6]	0.248	b	6.2	[5.4, 7.3]	6.1	[4.9, 7.3]	0.262	b
Neutrophils (×103/mm3)	3.9	[2.9, 4.9]	3.5	[2.9, 4.4]	0.079	b	3.4	[2.8, 4.7]	3.2	[2.6, 4.5]	0.296	b
Lymphocytes (×103/mm3)	1.8	[1.6, 2.2]	1.9	[1.5, 2.5]	0.037	b	1.9	[1.6, 2.3]	2.0	[1.4, 2.3]	0.191	b
Neutrophil/lymphocyte ratio	2.0	[1.6, 3.0]	1.9	[1.4, 2.4]	0.024	b	1.8	[1.4, 2.6]	1.9	[1.4, 2.5]	1.000	b
FLC-κ (mg/L)	27.5	[20.7, 37.8]	24.7	[17.4, 34.4]	0.042	b	24.2	[19.6, 30.8]	23.1	[17.3, 27.5]	0.178	b
FLC-λ (mg/L)	21.6	[16.5, 30.5]	20.0	[14.4, 30.2]	0.165	b	21.7	[15.9, 26.1]	18.7	[15.8, 21.7]	0.059	b
FLC-κ/ FLC-λ	1.2	[1.1, 1.4]	1.2	[1.1, 1.4]	0.010	b	1.1	[1.0, 1.3]	1.2	[1.0, 1.3]	0.526	b
IL-6 (pg/mL)	2.8	[1.7, 4.2]	2.6	[1.9, 4.2]	0.408	b	3.2	[2.6, 4.0]	3.2	[2.6, 4.9	0.469	b
CRP (mg/dL)	0.06	[0.06, 0.12]	0.08	[0.06, 0.18]	0.428	b	0.10	[0.06, 0.22]	0.10	[0.06, 0.14]	0.149	b
NT-proBNP	63.5	[23.4, 121.3]	44.4	[20.6, 92.8]	0.030	b	57.0	[32.6, 88.1]	46.8	[26.7, 80.0	0.550	b
CO (ppm)	17.0	[11.0, 26.8]	3.0	[2.0, 4.8]	<0.001	b	21.5	[10.5, 30.5]	8.0	[3.5, 20.8]	0.003	b

Data are presented as mean ± SD or median [interquartile range].

p value: a, paired t test; b, Wilcoxon signed rank test.

BMI, body mass index: WC, waist circumference: SBP, systolic blood pressure: DBP, diastolic blood pressure: HbA1c, hemoglobin A1c: LDL-C, low-density lipoprotein cholesterol: HDL-C, high-density lipoprotein cholesterol: TG, triglyceride: UA, uric acid: Cre, creatinine: WBC, white blood cell: FLC-κ, Free Light chain kappa: FLC-λ: free light chain lambda: IL-6, interleukin-6: CRP, C-reactive protein: NT-proBNP, N-terminal pro brain natriuretic peptide: CO, carbon monoxide.

3.2. Changes of CV markers and metabolic parameters

Among patients with successful SC, comparing baseline values and those taken 3 months after SC, BMI significantly increased (from 23.8 to 24.2 kg/m², p = 0.004). Conversely, FLC-κ levels and FLC-κ/FLC-λ significantly decreased from baseline to 3 months (from 27.5 to 24.7 mg/L, p = 0.042; from 1.2 to 1,2, p = 0.010), respectively)). FLC-λ did not change before and after SC in either group. No significant changes were observed in FLC-λ in both groups.

Among patients with successful SC, in the leukocyte fraction, the lymphocyte count significantly increased (1.9–2.0 × 103/mm³, p = 0.042), and the neutrophil count tended to drop (4.1–3.8 × 103/mm³, p = 0.068) after therapy compared with those before it. The NLR significantly decreased from baseline to 3 months (from 2.0 to 1.9, p = 0.024) only in this group. NT-proBNP levels decreased from baseline to 3 months (from 63.5 to 44.4 mg/L, p = 0.030) in this group.

In contrast, no significant changes were observed in these parameters among patients with unsuccessful SC (n = 20).

As shown in Table 2, percent changes in FLC-κ levels from baseline to 3 months significantly correlated with those of white blood cell counts (ρ = 0.233, p = 0.048), CRP (ρ = 0.349, p = 0.002) and NT-proBNP (ρ = 0.322, p = 0.005) levels only among patients with successful SC. Percent changes in FLC-κ significantly correlated with percent change IL-6 in both groups.

Table 2.

Correlation between percent change in FLC-K and clinical parameters from pre- to post- smoking cessation: Comparison between patients succeeded and unachieved in 3 months of smoking cessation.

	% change FLC-κ in successful patients (n = 76)		% change FLC-κ in unachieved patients (n = 20)
	ρ	p-value	ρ	p-value
% change BMI	0.228	0.056	0.127	0.615
% change SBP	−0.093	0.438	0.073	0.773
% change DBP	0.064	0.593	0.149	0.556
% change WBC	0.233	0.048	−0.068	0.777
% change Neutrophils	0.108	0.363	−0.057	0.811
% change Lymphocytes	0.197	0.095	−0.056	0.816
% change Neutrophil/lymphocyte ratio	−0.049	0.679	−0.114	0.631
% change IL-6	0.321	0.005	0.483	0.031
% change CRP	0.349	0.002	0,060	0.803
% change NT-proBNP	0.322	0.005	0.435	0.056
% change CO	0.151	0.193	−0.034	0.887

ρ:Spearman’s rank correlation coefficient.

4. Discussion

In this study, we demonstrate for the first time that FLC levels significantly decreased in a relatively short time (3 months) after SC. This may be a sensitive marker of reduced oxidative stress during SC. The decrease in FLC-κ exhibited a weak p-value (0.04) and may not be clinically significant in the short term. However, given that FLC-κ is a long-term prognostic marker [8,21] and is closely related to CVD prognosis, even a small decrease may reflect a reduction in cardiovascular events 5 to 10 years later.

Furthermore, higher FLC have been reported to independently correlate with the mortality in patients with autoimmune diseases, cancer, CVD, diabetes and chronic kidney diseases [22,23], as well as in the overall population [24]. Therefore, a decrease in FLC levels after SC may lead to improvements in survival rates. Further long-term observations are needed to prove this hypothesis.

In addition, the levels of inflammatory markers, such as FLC-κ and N/L ratio, decreased despite a significant weight gain after SC. Similarly, we previously reported that oxidized low-density lipoprotein levels decreased gradually from baseline to 3 months and 1 year after SC despite a gradual weight gain during this period [25]. This proves that the beneficial effects of SC outweigh the disadvantages of weight gain and the potential vascular risks of obesity associated with it.

SC helps reduce inflammation and repair damages provoked by smoking; however, the time course regarding functional restoration after SC is characterized by variability in recovery speed and onset. CRP, an inflammatory marker, reflects the risk of CVD development. In this study, NLR declined significantly in a relatively short period (3 months) after SC, which is consistent with the outcomes of our previous study [2]. In contrast, levels of CRP and IL-6, cytokines thought to take a long time to deteriorate, did not decrease significantly, which is in accordance with the previous report [11]. In addition to IL-1β and IL-6, other cytokines play an essential role in the onset of inflammation. Differences in reactivity to cytokines may be related to those explained by the interaction of CRP and IL-6 and FLC and NLR. Thus, to our knowledge, this study demonstrated for the first time that FLC levels decreased within a relatively short period of 3 months after SC. Accordingly, the FLC level may be a sensitive marker of the influence of inflammation. FLC-κ is reportedly related to prognosis. However, various molecules participate in inflammation, and the specific markers involved in each inflammatory pathway causing a cardiovascular disease (CVD) are still unknown. Further investigation is required to determine the inflammatory pathways for which FLC are markers.

The sympathetic nervous system is activated in patients with chronic heart failure, where peripheral vascular resistance and afterload are increased. Nicotine promotes the constriction of blood vessels and the increase in heart rate, thus elevating the burden on the heart [26]. NT-proBNP is a cardiac stress marker, particularly for the left ventricle, used to diagnose and assess the severity of heart failure. Regarding the relationship between NT-proBNP and smoking, cross-sectional studies have reported that NT-proBNP are significantly higher in active smokers than in nonsmokers and lower in past smokers than in current smokers and that NT-proBNP negatively correlates with a longer time since SC [27]. In this longitudinal study, we reported for the first time that NT-proBNP levels decrease after SC. These results suggest that it reduces cardiac load. FLC also independently predicts mortality in patients hospitalized for decompensated heart failure [28]. Furthermore, the rate of changes in FLC-κ before and after SC correlated with that in NT-proBNP, suggesting that FLC-κ levels reflect inflammation and cardiac burden.

This study has some limitations. First, the comparison of data before and after SC showed that the confidence intervals overlapped significantly. Adding more samples is needed in future studies. Second, it is a single-center study, and the observation period covered only 3 months after SC, which is relatively short. Thus, a long-term FLC observation is warranted. Third, further studies are needed to investigate whether the decrease in FLC levels after SC correlates with a lower frequency of complications, namely, CV events.

Further research is required to prove whether a reduction in inflammation, as well as in the CV biomarker FLC-κ, cardiac overload and heart failure biomarker NT-proBNP connected with SC, represent the hard endpoints, for example, reduction in hospitalization rates due to heart failure and CV deaths. The association mechanism between FLC-κ and NT-proBNP therefore needs to be further elucidated.

5. Conclusion

In smokers, FLC-κ levels, a novel biomarker of inflammation and CV risk, decreased after SC despite body weight gain, suggesting that the positive effects of CS surpass the risk of weight gain. NT-proBNP levels in association with FLC-κ levels also decreased with SC, suggesting that they are strongly related to cardiac overload as well as inflammation.

Funding Statement

This work was supported in part by a Grant-in-Aid for Clinical Research from the National Hospital Organization and Grants-in-Aid for Scientific Research by Japan Society for the Promotion of Science (19K20139). The funder played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contributions

Study conception and design were contributed by MK, KH and AM, acquisition of data was made by YT, analysis of data was made by HW and HY, and interpretation of data was made by SM, AY, MF, YK, YS, TM, MA, MA, NS-A, MA, TM, and TN. Drafting of manuscript was made by MK, and critical revision was made by KH.

Disclosure statement

The authors declare that there is no conflict of interest.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.