Mithun Pal

Continuous wave external-cavity quantum cascade laser-based high-resolution cavity ring-down spectrometer for ultrasensitive trace gas detection

A high-resolution cavity ring-down spectroscopic (CRDS) system based on a continuous wave (cw) mode-hop-free (MHF) external-cavity quantum cascade laser (EC-QCL) operating at λ∼5.2  μm has been developed for ultrasensitive detection of nitric oxide (NO). We report the performance of the high-resolution EC-QCL based cw-CRDS instrument by measuring the rotationally resolved Λ-doublet e and f components of the P(7.5) line in the fundamental band of NO at 1850.169  cm-1 and 1850.179  cm-1. A noise-equivalent absorption coefficient of 1.01×10-9  cm-1  Hz-1/2 was achieved based on an empty cavity ring-down time of τ0=5.6  μs and standard deviation of 0.11% with averaging of six ring-down time determinations. The CRDS sensor demonstrates the advantages of measuring parts per billion NO concentrations in N2, as well as in human breath samples with ultrahigh sensitivity and specificity. The CRDS system could also be generalized to measure simultaneously many other trace molecular species within the broad tuning range of cw EC-QCL, as well as for studying the rotationally resolved hyperfine structures.

An EC-QCL based N2O sensor at 5.2 μm using cavity ring-down spectroscopy for environmental applications

Nitrous oxide (N2O) is an important anthropogenic greenhouse gas emitted into the atmosphere that can contribute to ozone destruction. Considering its environmental importance, the real-time monitoring and molecule-specific detection of atmospheric N2O with high sensitivity have received much attention in the 21st century. In this study, a widely tunable continuous wave (cw) external-cavity quantum cascade laser (EC-QCL)-based cavity ring-down spectroscopy (CRDS) in the mid-infrared region has been used to measure the mixing ratios of N2O in ambient air. The detection of atmospheric N2O was made using a rotationally resolved R(8e) absorption line of N2O centred at 1887.666 cm−1. Several atmospheric air samples were collected at various locations in Kolkata on seven consecutive days in different periods of the day. In situ measurements were carried out by the EC-QCL-based high-resolution cw-CRDS method. The laser-based CRDS sensor allowed us to perform direct, quantitative and selective measurements of atmospheric N2O mixing ratios at the levels of parts per billion by volume (ppbv). A significant change in N2O levels was observed in different sub-areas depending on the source of local pollution. We also observed a marked difference in N2O levels between morning and afternoon sessions of the day in a particular sub-area. The CRDS sensor for the detection of N2O allows a minimum detectable absorption coefficient of αmin = 4.8 × 10−9 cm−1 and an estimated detection limit of 4.5 ppbv at atmospheric pressure is also reported.

Mechanisms linking metabolism of Helicobacter pylori to 18O and 13C-isotopes of human breath CO2

The gastric pathogen Helicobacter pylori utilize glucose during metabolism, but the underlying mechanisms linking to oxygen-18 (18O) and carbon-13 (13C)-isotopic fractionations of breath CO2 during glucose metabolism are poorly understood. Using the excretion dynamics of 18O/16O and 13C/12C-isotope ratios of breath CO2, we found that individuals with Helicobacter pylori infections exhibited significantly higher isotopic enrichments of 18O in breath CO2 during the 2h-glucose metabolism regardless of the isotopic nature of the substrate, while no significant enrichments of 18O in breath CO2 were manifested in individuals without the infections. In contrast, the 13C-isotopic enrichments of breath CO2 were significantly higher in individuals with Helicobacter pylori compared to individuals without infections in response to 13C-enriched glucose uptake, whereas a distinguishable change of breath 13C/12C-isotope ratios was also evident when Helicobacter pylori utilize natural glucose. Moreover, monitoring the 18O and 13C-isotopic exchange in breath CO2 successfully diagnosed the eradications of Helicobacter pylori infections following a standard therapy. Our findings suggest that breath 12C18O16O and 13C16O16O can be used as potential molecular biomarkers to distinctively track the pathogenesis of Helicobacter pylori and also for eradication purposes and thus may open new perspectives into the pathogen’s physiology along with isotope-specific non-invasive diagnosis of the infection.

Isotope selective activation: a new insight into the catalytic activity of urease

Urease, a metalloenzyme, requires carbon dioxide (CO2) for its activation. But, whether this activation is isotope-specific to 12CO2 or 13CO2, is not yet known and even the potential role of CO2 in the enzymatic activity of urease is poorly understood. Here, we provide direct experimental evidence that the catalytic activity of urease exhibits a unique isotope-specific response where the 12CO2 isotope is strongly preferred over the 13CO2 isotope during its catalytic activation. Moreover, this isotope-selective activation depends on different isotopic fractionations (12C:13C) of the reaction-environment as well as the substrate urea (13C-urea and 12C-urea), where the 12CO2 isotope in the reaction medium essentially facilitates the hydrolysis of 13C-enriched urea. This deepens our understanding of the isotope-specific urease activation and its potential role in hydrolytic reaction. Our findings thus may offer novel opportunities for a better fundamental understanding of isotope-specificity in chemical reactions involving metalloenzymes.

Natural 18 O and 13 C-urea in gastric juice: a new route for non-invasive detection of ulcers

AbstractThe 13C-urea breath test (13C-UBT), developed a few decades ago, is widely used as a non-invasive diagnostic method to detect only the presence of the gastric pathogen Helicobacter pylori infection; however, the actual disease state, i.e. whether the person harbouring H. pylori has peptic ulcer disease (PUD) or non-ulcerous dyspepsia (NUD), is still poorly understood. Nevertheless, the present 13C-UBT has numerous limitations, drawbacks and pitfalls owing to the ingestion of 13C-labelled external urea. Here, we show that H. pylori is able to utilize the natural 13C and 18O-urea inherently present in the gastric juice in humans for its urease activity which has never been explored before. In vitro measurements of isotopic fractionations of gastric juice urea provide new insights into the actual state of the infection of PUD or NUD. We also provide evidence of the unusual 13C and 18O-isotopic fractionations of breath CO2 that are distinctively altered in individuals with PUD encompassing both gastric and duodenal ulcers as well as with NUD by the enzymatic activity of H. pylori in the gastric niche without oral administration of any 13C-enriched external urea. This deepens our understanding of the UBT exploiting the natural 13C and 18O-gastric juice urea in the pathogenesis of H. pylori infection, reveals the actual disease state of PUD or NUD and thus offers novel opportunities for a simple, robust, cost-effective and non-toxic global strategy devoid of any 13C-enriched urea for treating these common diseases by a single breath test. Graphical AbstractUrea breath test without any external urea

Molecular hydrogen in human breath: a new strategy for selectively diagnosing peptic ulcer disease, non-ulcerous dyspepsia and Helicobacter pylori infection

The gastric pathogen Helicobacter pylori utilizes molecular hydrogen (H2) as a respiratory substrate during colonization in the gastric mucosa. However, the link between molecular H2 and the pathogenesis of peptic-ulcer disease (PUD) and non-ulcerous dyspepsia (NUD) by the enzymatic activity of H. pylori still remains mostly unknown. Here we provide evidence that breath H2 excretion profiles are distinctly altered by the enzymatic activity of H. pylori for individuals with NUD and PUD. We subsequently unravelled the potential molecular mechanisms responsible for the alteration of H2 in exhaled breath in association with peptic ulcers, encompassing both gastric and duodenal ulcers, along with NUD. We also established that carbon-isotopic fractionations in the acid-mediated bacterial environment regulated by bacterial urease activity cannot discriminate the actual disease state i.e. whether it is peptic ulcer or NUD. However, our findings illuminate the unusual molecular H2 in breath that can track the precise evolution of PUD and NUD, even after the eradication of H. pylori infection. This deepens our understanding of the pathophysiology of PUD and NUD, reveals non-invasively the actual disease state in real-time and thus offers a novel and robust new-generation strategy for treating peptic-ulcer disease together with non-ulcer related complications even when the existing (13)C-urea breath test ((13)C-UBT) fails to diagnose.

Exploring the physiological link of breath N 2 O through nitrification and denitrification processes in human gastric juice

Over the past several decades, it has been generally believed that microbial nitrification and denitrification are not significant processes in the human gastrointestinal tract. Moreover, the underlying physiological link between exhaled nitrous oxide (N2O) and aerobic denitrification in the gastric environment is still largely unknown. In this report, we provide direct experimental evidence of the aerobic denitrification process in the human gastrointestinal tract by evaluating concentrations of dissolved N2O and its precursor nitrite ([Formula: see text]) ion in the gastric juice along with exhaled N2O concentration using a high-precision laser spectroscopy technique. Moreover, in vitro studies of gastric fluid in patients reveal a new mechanism of nitrification of ammonium ion ([Formula: see text]) followed by denitrification of [Formula: see text] leading to the formation of N2O in the gastric environment, which is eventually excreted in exhaled breath. This observation was subsequently validated under in vivo physiological conditions exploiting the urease activity of the gastric pathogen Helicobacter pylori. Consequently, our findings established a strong physiological link between exhaled N2O and bacterial infection in the stomach. This deepens our understanding of the unusual microbial denitrification in the gastric environment, providing new insight into the activities of human-associated microorganisms, which eventually affect the human physiology and health.

New Strategy for in Vitro Determination of Carbonic Anhydrase Activity from Analysis of Oxygen-18 Isotopes of CO2

The oxygen-18 isotopic (18O) composition in CO2 provides an important insight into the variation of rate in isotopic fractionation reaction regulated by carbonic anydrase (CA) metalloenzyme. This work aims to employ an 18O-isotope ratio-based analytical method for quantitative estimation of CA activity in erythrocytes for clinical testing purposes. Here, a new method has been developed that contains the measurements of 18O/16O isotope ratios during oxygen-18 isotopic exchange between 12C16O16O and H218O of an in vitro biochemical reaction controlled by erythrocytes CA and estimation of enzymatic activity of CA from the isotopic composition of CO2. We studied the enrichments of 18O-isotope of CO2 with increments of CA activities during isotopic fractionation reaction. To check the influence of subject-specific body temperature, pH, H218O, and cellular produced CO2 on this reaction, we performed the in vitro experiments in closed containers with variations of those parameters. Finally, we mimicked the exchange reaction at 5% [CO2], 5‰ [H218O], pH of 7.4, and temperature of 37 °C to create the physiological environment equivalent to that of the human body and monitored the exchange kinetics with variations of CA activities, and subsequently, we derived the quantitative relation between the 18O-isotope of CO2 and CA activity in erythrocytes. This assay may be applicable for rapid and simple quantification of carbonic anhydrase activity which is very important to prevent the carbonic-anhydrase-associated disorders in human.