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Isotopes in Environmental and Health Studies

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Non-invasive diagnosis of type 2 diabetesin Helicobacter pylori infected patientsusing isotope-specific infrared absorptionmeasurements

Suman Som, Gourab Dutta Banik, Abhijit Maity, Chiranjit Ghosh, SujitChaudhuri & Manik Pradhan

To cite this article: Suman Som, Gourab Dutta Banik, Abhijit Maity, Chiranjit Ghosh, SujitChaudhuri & Manik Pradhan (2018) Non-invasive diagnosis of type 2 diabetes in Helicobacterpylori infected patients using isotope-specific infrared absorption measurements, Isotopes inEnvironmental and Health Studies, 54:4, 435-445, DOI: 10.1080/10256016.2018.1467414

To link to this article: https://doi.org/10.1080/10256016.2018.1467414

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Non-invasive diagnosis of type 2 diabetes in Helicobacterpylori infected patients using isotope-specific infraredabsorption measurementsSuman Soma, Gourab Dutta Banika, Abhijit Maitya, Chiranjit Ghosha, Sujit Chaudhurib

and Manik Pradhan a,c

aDepartment of Chemical, Biological and Macro-Molecular Sciences, S. N. Bose National Centre for BasicSciences, Salt Lake, Kolkata, India; bDepartment of Gastroenterology, AMRI Hospital, Salt Lake City, Kolkata,India; cTechnical Research Centre (TRC), S. N. Bose National Centre for Basic Sciences, Salt Lake, Kolkata, India

ABSTRACTHelicobacter pylori causes several gastrointestinal diseases and mayalso contribute to the development of type 2 diabetes (T2D). Severalstudies suggest that there might be a potential link betweenH. pylori infection and T2D, but it still remains the subject ofdebate. Here, we first report the cumulative effect of H. pyloriinfection and T2D by exploiting the excretion kinetics of 13C/12Cand 18O/16O isotope ratios of exhaled breath CO2 in response toan oral dose of 13C-enriched glucose in individuals with T2D andnon-diabetic controls (NDC) harbouring the H. pylori infection.Using a high-resolution integrated cavity output spectroscopy(ICOS) technique in the infrared region, we observed that theisotopic fractionations of 13C and 18O in breath CO2 are distinctlyaltered in H. pylori infected T2D patients as well as in H. pyloriinfected NDC. Several optimal diagnostic cut-off points of 13C and18O isotopes of breath CO2 were also determined which exhibitedthe diagnostic sensitivity and specificity of ∼97 % and thussuggesting that breath 13C and 18O isotopes might be consideredas potential biomarkers for the non-invasive assessment of thegastric pathogen prior to the onset of T2D. This may open a newdiagnostic strategy for treating these common diseases in analternative way.

ARTICLE HISTORYReceived 23 October 2017Accepted 16 February 2018

KEYWORDSBiomarker; breath analysis;carbon-13; glucose breathtest; Helicobacter pylori;isotope application indiagnostic medicine;type 2 diabetes

1. Introduction

Helicobacter pylori, the most common bacterial pathogen in the human stomach, causes awide variety of gastrointestinal disorders including gastritis, peptic ulcer disease, non-ulcerdyspepsia, gastric cancer and mucosa-associated lymphoid tissue lymphoma [1–3]. Overthe past decade, several lines of evidence suggest that the effects of H. pylori infectionmay not only be confined to the gastrointestinal tract but can also be associated withseveral other extragastric diseases such as idiopathic thrombocytophenic purpurae,cardiovascular disease, anemia, diabetes mellitus and insulin resistance [4–6]. Earlystudies demonstrated that the prevalence of H. pylori infection is much higher in type 2

© 2018 Informa UK Limited, trading as Taylor & Francis Group

CONTACT Manik Pradhan [email protected]

ISOTOPES IN ENVIRONMENTAL AND HEALTH STUDIES2018, VOL. 54, NO. 4, 435–445https://doi.org/10.1080/10256016.2018.1467414

diabetes (T2D) patients compared to non-diabetic controls (NDC), where T2D is the mostcommon metabolic disorder that is characterized by high levels of blood glucose resultingfrom insulin resistance and pancreatic β-cell dysfunction [7,8]. However, in contrast, someepidemiological studies also implicated that individuals with T2D have a higher risk ofdeveloping H. pylori infection [9,10]. Therefore, a growing body of evidence suggeststhat there is a potential link between H. pylori infection and T2D but it still remains con-troversial [6,11], and the underlying mechanism in the pathogenesis of H. pylori infectionin subjects with T2D is still unclear.

However, genome-wide sequence data suggest that H. pylori utilizes glucose as theprimary energy source and metabolizes it both in oxidative and fermentative pathways[12]. Carbon dioxide (CO2) is produced as a major by-product of the glucose catabolism,which is then transported through the bloodstream to the lungs, and is afterwardsexcreted in exhaled breath. Accumulated evidence indicates that during colonization,H. pylori plays a vital role in glucose homeostasis through the regulation of gastric hor-mones and has the ability to alter the glucose metabolism by inducing chronic inflam-mation, which further leads to the development of T2D [6,13]. However, to ourknowledge, no studies have focused till date on the glucose metabolism for the assess-ment of the cumulative role of H. pylori and T2D. Therefore, the primary aim of thepresent study was to unravel the role of T2D in the glucose metabolism of H. pylori infectedindividuals by monitoring isotopic breath 13CO2 in response to 13C-enriched glucose,which may directly contribute to the early detection of H. pylori infection and its compli-cacies prior to the onset of T2D.

Furthermore, recent evidence suggests that oxygen-18 (18O) isotopes of body water(H2

18O) and oxygen-16 (16O) isotopes of 12C16O2 are rapidly interchanged during the respir-ation process catalyzed by the enzymatic activity of carbonic anhydrase (CA), a ubiquitousmetalloenzyme present in the human body as well as in H. pylori [14]. It is also noteworthythat alteration of the glucose metabolism is strongly associated with changes in CA activityin patients with T2D [15–17]. All these activities, therefore, suggest that simultaneousmoni-toring of 18O/16O and 13C/12C isotope ratios of breath CO2 associated with the glucosemetabolism may potentially be linked with H. pylori infection and T2D and thus might actas potential markers for the non-invasive assessment of H. pylori infection in complicationwith T2D without any endoscopic biopsy tests and blood sample measurements. However,there has been so far no direct experimental evidence to support this concept and, there-fore, it still remains a tantalizing but unproven hypothesis. Therefore, another aim of thepresent study was to explore a new non-invasive methodology which can precisely diag-nose T2D in theH. pylori infected subjects. Thismay have an immense impact on identifyingany possible coexistence of T2D in H. pylori infected individuals.

In this study, we first report the cumulative effect of H. pylori infection and T2D in theglucose metabolism by exploiting the isotopic fractionations of carbon-13 (13C) andoxygen-18 (18O) isotopes of CO2 in human exhaled breath. Using a laser-based high-pre-cision integrated cavity output spectroscopy (ICOS) technique in infrared (IR) absorptionregion, we have investigated the excretion kinetics of 18O and 13C isotopes of breathCO2 in individuals with T2D or non-diabetic controls harbouring H. pylori infection.Finally, we determined numerous diagnostic parameters such as optimal diagnostic cut-off values of breath 12C18O16O and 13C16O16O, diagnostic sensitivity and specificitylevels to gain a better insight into the diagnostic efficacy of the present methodology.

436 S. SOM ET AL.

2. Materials and methods

2.1. Subjects

In the present study, we enrolled 131 individuals (n = 73 male and n = 58 female) withdifferent gastrointestinal disorders such as chronic gastritis, dyspepsia, peptic ulcerdisease. The subjects were classified into three distinct groups: H. pylori positive withT2D (Hp(+)T2D(+), n = 37), H. pylori positive without T2D (Hp(+)T2D(−), n = 51) andH. pylori negative without T2D (Hp(−)T2D(−), n = 43) depending on both gold standardinvasive and non-invasive methods, i.e. endoscopy, biopsy-based rapid urease test (RUT)and 13C-urea breath test (13C-UBT). The 13C-UBT indicates individuals with positiveH. pylori infection with δDOB

13 C≥ 3 ‰ at 30 min [3]. Moreover, we also categorized diabeticand non-diabetic subjects in accordance to the standard outlines of the American DiabeticAssociation (ADA) [18]. Individuals with T2D were determined with glycosylated hemo-globin (HbA1C)≥ 6.5 % and 2h-oral glucose tolerance test (2h-OGTT)≥ 200 mg dL−1,whereas individuals with HbA1C < 5.7 % and 2h-OGTT < 140 mg dL−1 were consideredas non-diabetic controls. All the clinical parameters are given in Table 1. Subjects wereexcluded from the study if they had any previous history of gastric surgery, hypertension,asthma, anemia, smoking and alcohol consumption. Moreover, subjects taking any medi-cation that may alter the glucose metabolism, proton pump inhibitors or H2 receptorantagonists in four weeks prior to endoscopy and 13C-UBT were also excluded from thepresent study. The current study was performed in accordance with the guidelinesapproved by the Ethical Committee Review Board of AMRI Hospital, Salt Lake, Kolkata,India (Study no.: AMRI/ETHICS/2013/1). The study was also approved by the administrationof S. N. National Centre, Kolkata, India (Ref. no.: SNB/PER-2-6001/13-14/1769). Informedwritten consents were taken from all the subjects prior to the breath test.

2.2. Breath sample collection

All subjects completed their endoscopic examination and 13C-UBT 1–2 days prior to theglucose breath test (GBT). All subjects were instructed to wash their mouths before theGBT to prevent any kind of contact from orally administered test meal with oral cavity bac-teria, and also restricted their physical movement during the experiment period. After anovernight fasting (∼10–12 h), a baseline breath sample was collected in a breath collectionbag (QUINTRON, USA, SL No.QT00892). Then a test meal containing 75 mg U-13C6 labelledD-glucose (CIL-CLM-1396-CTM, Cambridge Isotope Laboratories, Inc., USA) with 75 gnormal glucose dissolved in 250 ml water was given to the subject for oral administration,and, subsequently, breath samples were collected in 30 min intervals up to 180 min. The

Table 1. Clinical characteristics of the subjects (data expressed as mean ± SD).Parameter Hp(−)T2D(−) Hp(+)T2D(−) Hp(+)T2D(+) p value

No. of Subjects 43 51 37Age (years) 47 ± 10 46 ± 7 50 ± 9.5 >0.05Weight (kg) 61 ± 7.6 62 ± 7.1 65 ± 8.3 >0.05Fasting blood glucose (mg dL−1) 97 ± 10.1 101 ± 6.4 159 ± 11.4 <0.052h-post dose blood glucose (mg dL−1) 117 ± 14.1 120 ± 10.7 211 ± 31.3 <0.05HbA1C (%) 5.24 ± 0.19 5.31 ± 0.27 10.7 ± 1.1 <0.05

Note: p < 0.05 indicates a statistically significant difference.

ISOTOPES IN ENVIRONMENTAL AND HEALTH STUDIES 437

13C/12C and 18O/16O isotope ratios of CO2 in breath samples were measured using a laser-based high-resolution ICOS. Details of the ICOS technique are given below. Figure 1 rep-resents the steps and analytical protocol followed in the present study.

2.3. Integrated cavity output spectrometer (ICOS) for isotopic measurement ofbreath samples

For simultaneous measurements of 13C/12C and 18O/16O isotope ratios in exhaledbreath CO2, we employed a laser-based high-precision CO2 isotope analyzer (CCIA36-EP, LGR, USA) exploiting the off-axis integrated cavity output spectroscopy (OA-ICOS)

Figure 1. Flow diagram representing the used methodology in the present study.

438 S. SOM ET AL.

technique [19–21]. In brief, the ICOS system consists of a high-finesse optical cavity (∼59cm) formed by two high reflectivity mirrors (R∼ 99.98 %) at the two ends of the cavity,which offers an effective optical path length of ∼3 km. A continuous-wave distributedfeedback diode laser (cw-DFB) operating at ∼2.05 μm is used to probe the absorptionsfeatures of 12C16O16O, 12C18O16O and 13C16O16O at the wave-numbers of 4878.292,4878.006 and 4877.572 cm−1, respectively, which correspond to the R(34), P(32) andP(12) rotational lines of the (2ν1 + ν3) vibrational combinational band of CO2 molecule.The isotopic enrichments of 13C16O16O and 12C18O16O are usually expressed as δ13C(‰) and δ18O (‰) notation with respect to the international standard Pee DeeBelemnite (PDB), and they are represented as δ13C = (Rsample/Rstandard− 1) × 1000 andδ18O = (Rsample/Rstandard − 1) × 1000, respectively, where RSample is the 13C/12C and18O/16O ratio of the sample and RStandard are the international standard PDB values, i.e.0.0112372 and 0.0020672, respectively. The time-dependent isotopic enrichments of 13Cand 18O with respect to the baselines were expressed as the delta-over-baseline (DOB)values, i.e. δDOB

13 C or δDOB18 O.

d13DOBC = (d13C)t=t min–(d13C)t=0 min

and

d18DOBO = (d18O)t=t min–(d18O)t=0 min

2.4. Statistical analysis

We utilized both the one-way ANNOVA test for parametric variables and theMann–Whitney test for non-parametric variables for the analysis of experimental data. Atwo sided p value < 0.05 was considered for the statistical significance of data. Receiveroperating characteristic (ROC) curves were used to obtain an optimal diagnostic cut-offpoint of δDOB

13 C and δDOB18 O values associated to the glucose metabolism for clinical

validation. All data were analyzed using Origin Pro 8.0 software (Origin Lab Corporation,USA) and Analyse-it Method Evaluation software (Analyse-it Software Ltd, UK, version2.30). All data were presented as mean ± SD.

3. Results and discussion

Here, we first explored the potential role of the major metabolite, CO2 and its three mostabundant stable isotopic species in the pathogenesis of T2D in H. pylori infected individ-uals in response to the glucose metabolism. To investigate this, we studied thetime-dependent excretion kinetics of both 13C and 18O isotopic fractionations ofexhaled breath CO2 using the ICOS method (Figure 2(a)) after ingestion of an oral doseof 13C-enriched glucose in three different group of patients with Hp(+)T2D(+), Hp(+)T2D(−) and Hp(−)T2D(−). It was observed that the subjects with Hp(+)T2D(−) exhibitedsignificantly higher isotopic enrichments of δDOB

13 C values in exhaled breath with timecompared to both groups of patients with Hp(−)T2D(−) and Hp(+)T2D(+) during the 3 hglucose metabolism.

ISOTOPES IN ENVIRONMENTAL AND HEALTH STUDIES 439

Early reports suggest that H. pylori can utilize glucose in both oxidative and fermenta-tive pathways, and the metabolism follows biphasic characteristics involving an initial slowphase of metabolism followed by a rapid phase [20,22] which is most likely involved incausing the significantly higher enrichments of δDOB

13 C values for the Hp(+)T2D(−) groupwith a change of about 0.41 ‰/min compared to a change of 0.32 ‰/min for theHp(−)T2D(−) group of patients within 30–120 min. But for the group of patients withHp(+)T2D(+), there was a significantly lower isotopic enrichment of δDOB

13 C values inresponse to the 13C-glucose metabolism with a change of about 0.17 ‰/min within thestipulated time. It is quite well known that both H. pylori infection and T2D are inflamma-tory diseases and produce oxidative stress [23–25] leading to β-cell dysfunction and def-icits in insulin secretion, which directly affects the glucose metabolism. Recent evidencealso demonstrates that patients with H. pylori infection accompanied with T2D exaggeratemore oxidative stress and chronic inflammation than the non-diabetic controls withH. pylori infection [26], causing further depletion of isotopic 13C in exhaled breath CO2

for the Hp(+)T2D(+) group with respect to the groups of Hp(−)T2D(−) and Hp(+)T2D(−).We also observed statistically significant differences (p < 0.05) of δDOB

13 C values in excretionkinetics among the three distinct groups of patients (Figure 2(b)). Taken together, our find-ings suggest that isotopic fractionations of 13C in exhaled breath CO2 are strongly associ-ated with the glucose metabolism in H. pylori infected patients and subsequently explorethe role of T2D in the alteration of the glucose metabolism in H. pylori infected individuals.

We next investigated the oxygen-18 fractionations of breath CO2 by studying the time-dependent excretion dynamics of δDOB

18 O values in response to 13C-labelled glucose, andthe results are depicted in Figure 3(a). The 18O-enrichments in exhaled breath CO2 weresignificantly higher for the Hp(+)T2D(+) group compared to the Hp(−)T2D(−) andHp(+)T2D(−) groups, while no such significant enrichments of δDOB

18 O values in exhaledbreath CO2 were observed for the Hp(−)T2D(−) group. Several lines of evidence [27,28]suggest that H. pylori encodes two distinct form of CA, i.e. α-CA and β-CA, which promotes

Figure 2. Comparisons of excretion kinetics of δDOB13 C values among the three groups of subjects,

i.e. H. pylori positive with T2D (Hp(+)T2D(+)), H. pylori positive without T2D (Hp(+)T2D(−)) andH. pylori negative without T2D (Hp(−)T2D(−)) in response to the 13C-enriched glucose metabolism.(a) Significant enrichment of 13C isotopes in exhaled breath for subjects with Hp(+)T2D(−) (n = 51)compared to both Hp(−)T2D(−) (n = 43) and Hp(+)T2D(+) (n = 37); (b) Marked statistical difference(p < 0.05) of δDOB

13 C values among the three group of subjects at 120 min (*indicates p < 0.05).

440 S. SOM ET AL.

a rapid exchange of 18O isotopes of body water (H2O18) with 16O isotopes of 12C16O2 [29].

Moreover, CA is also present in the human body, and it is well documented thatthe catalytic activity of CA in erythrocytes (red blood cells) is enhanced in patients withT2D [15,16,30]. Therefore, these activities are possibly attributed to the marked enrichmentof 18O-isotopic fractionations with a change of about 0.03 ‰/min in breath CO2 for theHp(+)T2D(+) group of patients within 30–120 min.

However, due to only bacterial CA activity in the glucose-mediated bacterial environ-ment, the Hp(+)T2D(−) group exhibited comparatively higher isotopic enrichments ofδDOB18 O with a change of about 0.01 ‰/min compared to the change of −0.007 ‰/min

of the Hp(−)T2D(−) group within 30–120 min. It is also noteworthy that erythrocytes CAactivity in the human body for CO2 hydration/bicarbonate dehydration is much higherthan the bacterial CA activity [31], which is likely to be the effect of the observation ofcomparative results of δDOB

18 O for the different categories of patients. In view of theabove results, our findings suggest that the monitoring of 18O isotopes in breath maydistinctively track the coexistence of gastric pathogen H. pylori and T2D and thus mightbe considered as a potential biomarker for the non-invasive detection of T2D inH. pylori infected patients and also unveil a missing link between 18O-isotopic exchangein breath CO2 and the pathogenesis of T2D in H. pylori infected patients, which hasnever been explored before.

We finally determined several optimal diagnostic cut-off points of δDOB13 C and δDOB

18 Ovalues in breath at 120 min associated with the 13C-glucose metabolism to assess theclinical validity of the present methodology and also to precisely track the gastricpathogen and T2D in a non-invasive way. We utilized ROC curve analysis by plottingtrue positive rate (sensitivity) against false positive rate (1-specificity) as shown inFigure 4(a,b). Individuals with δDOB

13 C > 35.62‰ at 120 min were considered as H. pyloripositive without the complication of T2D with sensitivity and specificity of ∼97.6 and97.2 %, respectively. Individuals with δDOB

13 C < 24.11 ‰ were considered as H. pylori

Figure 3. Exploration of excretion kinetics plot of δDOB18 O values in response to the 13C-enriched glucose

metabolism up to 180 min. (a) Significant enrichment of 18O isotopes of breath CO2 for the HP(+)T2D(+)group (n = 37) compared to both HP(+)T2D(−) (n = 51) and HP(−)T2D(−) (n = 43) group due to theenhanced carbonic anhydrase activity; (b) Significant differences (p < 0.05) of δDOB

18 O values amongthe three groups at 120 min (*indicates p < 0.05).

ISOTOPES IN ENVIRONMENTAL AND HEALTH STUDIES 441

positive with T2D, whereas individuals were suggested to be H. pylori negative withoutT2D with 35.62 ‰≤ δDOB

13 C≤ 24.11 ‰, and these corresponded to the similar diagnosticsensitivity and specificity. Conversely, with a different cut-off value of δDOB

18 O > 2.78‰,the subjects were considered as H. pylori positive with T2D with a sensitivity of ∼100 %and a specificity of ∼96.9 %, and individuals with δDOB

18 O < 0.31 ‰ were taken into con-sideration as H. pylori negative without T2D with 92.3 % sensitivity and 100 % specificity(Table 2). Taken together, these findings suggest that 13C and 18O isotopic fractionationsof breath CO2 in response to the glucose metabolism are potentially linked to the patho-genesis of H. pylori infection and consequently suggest a broad clinical efficacy for theaccurate assessment of T2D in H. pylori infected patients, opening a new route for treatingthese common diseases. However, the present study suffers from an intrinsic drawbacknot having pre-diabetic patients and T2D patients without H. pylori infection, i.e. Hp(−)T2D(+), which might alter the diagnostic sensitivity of the present study, and hence

Figure 4. ROC curve analysis for an optimal diagnostic cut-off value for assessment of T2D in H. pyloriinfected patients at 120 min. (a) Individuals with δDOB

13 C > 35.62 ‰ considered as HP(+)T2D(−) group ofsubjects with ∼97.6 % sensitivity and δDOB

13 C values <24.11 ‰ indicating the presence of HP(+)T2D(+)group of subjects with ∼94.4 % sensitivity at 120 min. (b) δDOB

18 O > 2.78 ‰ accounting for the presenceof the HP(+)T2D(+) group with 100 % sensitivity.

Table 2. Different optimal diagnostic parameters corresponding to the cut-off values of δDOB13 C and

δDOB18 O for the assessment of T2D in H. pylori infected patients at 120 min.

GroupCut-off point of δDOB

13 C(‰)

Sensitivity(%) Specificity PPV NPV Accuracy

Hp(+)T2D(−) vs Hp(−)T2D(−) 35.62 97.6 97.2 98 97 97.4Hp(−)T2D(−) vs Hp(+)T2D(+) 24.11 94.4 96 96 94 95.08

Cut-off point of δDOB18 O

(‰)Hp(+)T2D(+) vs Hp(+)T2D(−) 2.78 100 96.9 96 100 98.27Hp(+)T2D(−) vs Hp(−)T2D(−) 0.31 92.3 100 100 94 96.15

Note: PPV and NPV indicates positive predictive value and negative predictive value, respectively.

442 S. SOM ET AL.

further in-depth study is required to validate the diagnostic efficacy of the presentmethodology for the non-invasive diagnosis of H. pylori infected T2D individuals in asingle breath test.

4. Conclusions

In conclusion, the analysis of 18O and 13C isotopes of breath CO2 through excretion kineticsrevealed the alteration of the glucose metabolism in H. pylori infected T2D individuals.Subsequently, we established that 18O and 13C isotopes in response to 13C-enrichedglucose ingestion may possibly track the coexistence of H. pylori infection and T2D andthus might be considered as potential biomarkers for the non-invasive assessment ofthe gastric pathogen prior to the onset of T2D. While there are several important gapsin our study for understanding the molecular mechanisms underlying the pathogenesisof the diseases that should be explored in future research, but our current results mayopen new perspectives into the isotope specific molecular detection of T2D associatedwith H. pylori infection. This work represents an advance in biomedical science becauseit demonstrates that 18O and 13C isotopes of breath CO2 are potentially linked withH. pylori infection and T2D and thus may open up a new route for non-invasive assessmentof T2D in H. pylori infected subjects.

Acknowledgements

M. Pradhan acknowledges the ‘Innovation Award’ (2017) USA by the World India Diabetes Foun-dation (WIDF). S. Som and C. Ghosh acknowledge S. N. Bose Centre for Ph.D fellowship, whereasG. D. Banik and A. Maity acknowledge the Department of Science & Technology (DST, India) forINSPIRE fellowship. We heartily thank all the volunteers who participated in the present study.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

The study was funded by the Research Society for the Study of Diabetes in India (RSSDI) (RSSDI/HQ/Grant/2015/190).

ORCID

Manik Pradhan http://orcid.org/0000-0001-5900-1700

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