Breathing new life into clinical testing and diagnostics: perspectives on volatile biomarkers from breath.

Human breath offers several benefits for diagnostic applications, including simple, noninvasive collection. Breath is a rich source of clinically-relevant biological information; this includes a volatile fraction, where greater than 1,000 volatile organic compounds (VOCs) have been described so far, and breath aerosols that carry nucleic acids, proteins, signaling molecules, and pathogens. Many of these factors, especially VOCs, are delivered to the lung by the systemic circulation, and diffusion of candidate biomarkers from blood into breath allows systematic profiling of organismal health. Biomarkers on breath offer the capability to advance early detection and precision medicine in areas of global clinical need. Breath tests are noninvasive and can be performed at home or in a primary care setting, which makes them well-suited for the kind of public screening program that could dramatically improve the early detection of conditions such as lung cancer. Since measurements of VOCs on breath largely report on metabolic changes, this too aids in the early detection of a broader range of illnesses and can be used to detect metabolic shifts that could be targeted through precision medicine. Furthermore, the ability to perform frequent sampling has envisioned applications in monitoring treatment responses. Breath has been investigated in respiratory, liver, gut, and neurological diseases and in contexts as diverse as infectious diseases and cancer. Preclinical research studies using breath have been ongoing for some time, yet only a few breath-based diagnostics tests are currently available and in widespread clinical use. Most recently, tests assessing the gut microbiome using hydrogen and methane on breath, in addition to tests using urea to detect Helicobacter pylori infections have been released, yet there are many more applications of breath tests still to be realized. Here, we discuss the strengths of breath as a clinical sampling matrix and the technical challenges to be addressed in developing it for clinical use. Historically, a lack of standardized methodologies has delayed the discovery and validation of biomarker candidates, resulting in a proliferation of early-stage pilot studies. We will explore how advancements in breath collection and analysis are in the process of driving renewed progress in the field, particularly in the context of gastrointestinal and chronic liver disease. Finally, we will provide a forward-looking outlook for developing the next generation of clinically relevant breath tests and how they may emerge into clinical practice.

The Sodium/Iodide Symporter (NIS): Molecular Physiology and Preclinical and Clinical Applications.

Active iodide (I-) transport in both the thyroid and some extrathyroidal tissues is mediated by the Na+/I- symporter (NIS). In the thyroid, NIS-mediated I- uptake plays a pivotal role in thyroid hormone (TH) biosynthesis. THs are key during embryonic and postembryonic development and critical for cell metabolism at all stages of life. The molecular characterization of NIS in 1996 and the use of radioactive I- isotopes have led to significant advances in the diagnosis and treatment of thyroid cancer and provide the molecular basis for studies aimed at extending the use of radioiodide treatment in extrathyroidal malignancies. This review focuses on the most recent findings on I- homeostasis and I- transport deficiency-causing NIS mutations, as well as current knowledge of the structure/function properties of NIS and NIS regulatory mechanisms. We also discuss employing NIS as a reporter gene using viral vectors and stem cells in imaging, diagnostic, and therapeutic procedures.

Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms.

Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.

Na+ coordination at the Na2 site of the Na+/I- symporter.

The sodium/iodide symporter (NIS) mediates active I(-) transport in the thyroid-the first step in thyroid hormone biosynthesis-with a 2 Na(+): 1 I(-) stoichiometry. The two Na(+) binding sites (Na1 and Na2) and the I(-) binding site interact allosterically: when Na(+) binds to a Na(+) site, the affinity of NIS for the other Na(+) and for I(-) increases significantly. In all Na(+)-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na(+) ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues-S66, D191, Q194, and Q263-are also involved in Na(+) coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I(-) These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

Breath Biopsy® to Identify Exhaled Volatile Organic Compounds Biomarkers for Liver Cirrhosis Detection.

Background and Aims: The prevalence of chronic liver disease in adults exceeds 30% in some countries and there is significant interest in developing tests and treatments to help control disease progression and reduce healthcare burden. Breath is a rich sampling matrix that offers non-invasive solutions suitable for early-stage detection and disease monitoring. Having previously investigated targeted analysis of a single biomarker, here we investigated a multiparametric approach to breath testing that would provide more robust and reliable results for clinical use. Methods: To identify candidate biomarkers we compared 46 breath samples from cirrhosis patients and 42 from controls. Collection and analysis used Breath Biopsy OMNI™, maximizing signal and contrast to background to provide high confidence biomarker detection based upon gas chromatography mass spectrometry (GC-MS). Blank samples were also analyzed to provide detailed information on background volatile organic compounds (VOCs) levels. Results: A set of 29 breath VOCs differed significantly between cirrhosis and controls. A classification model based on these VOCs had an area under the curve (AUC) of 0.95±0.04 in cross-validated test sets. The seven best performing VOCs were sufficient to maximize classification performance. A subset of 11 VOCs was correlated with blood metrics of liver function (bilirubin, albumin, prothrombin time) and separated patients by cirrhosis severity using principal component analysis. Conclusions: A set of seven VOCs consisting of previously reported and novel candidates show promise as a panel for liver disease detection and monitoring, showing correlation to disease severity and serum biomarkers at late stage.

Exogenous Volatile Organic Compound (EVOC®) Breath Testing Maximizes Classification Performance for Subjects with Cirrhosis and Reveals Signs of Portal Hypertension.

Background: Cirrhosis detection in primary care relies on low-performing biomarkers. Consequently, up to 75% of subjects with cirrhosis receive their first diagnosis with decompensation when causal treatments are less effective at preserving liver function. We investigated an unprecedented approach to cirrhosis detection based on dynamic breath testing. Methods: We enrolled 29 subjects with cirrhosis (Child-Pugh A and B), and 29 controls. All subjects fasted overnight. Breath samples were taken using Breath Biopsy® before and at different time points after the administration of 100 mg limonene. Absolute limonene breath levels were measured using gas chromatography-mass spectrometry. Results: All subjects showed a >100-fold limonene spike in breath after administration compared to baseline. Limonene breath kinetics showed first-order decay in >90% of the participants, with higher bioavailability in the cirrhosis group. At the Youden index, baseline limonene levels showed classification performance with an area under the roc curve (AUROC) of 0.83 ± 0.012, sensitivity of 0.66 ± 0.09, and specificity of 0.83 ± 0.07. The best performing timepoint post-administration was 60 min, with an AUROC of 0.91, sensitivity of 0.83 ± 0.07, and specificity of 0.9 ± 0.06. In the cirrhosis group, limonene bioavailability showed a correlation with MELD and fibrosis indicators, and was associated with signs of portal hypertension. Conclusions: Dynamic limonene breath testing enhances diagnostic performance for cirrhosis compared to static testing. The correlation with disease severity suggests potential for monitoring therapeutic interventions. Given the non-invasive nature of breath collection, a dynamic limonene breath test could be implemented in primary care.

Breath-Taking Perspectives and Preliminary Data toward Early Detection of Chronic Liver Diseases.

The gold standard method for chronic liver diseases diagnosis and staging remains liver biopsy, despite the spread of less invasive surrogate modalities based on imaging and blood biomarkers. Still, more than 50% of chronic liver disease cases are detected at later stages when patients exhibit episodes of liver decompensation. Breath analysis represents an attractive means for the development of non-invasive tests for several pathologies, including chronic liver diseases. In this perspective review, we summarize the main findings of studies that compared the breath of patients with chronic liver diseases against that of control subjects and found candidate biomarkers for a potential breath test. Interestingly, identified compounds with best classification performance are of exogenous origin and used as flavoring agents in food. Therefore, random dietary exposure of the general population to these compounds prevents the establishment of threshold levels for the identification of disease subjects. To overcome this limitation, we propose the exogenous volatile organic compounds (EVOCs) probe approach, where one or multiple of these flavoring agent(s) are administered at a standard dose and liver dysfunction associated with chronic liver diseases is evaluated as a washout of ingested compound(s). We report preliminary results in healthy subjects in support of the potential of the EVOC Probe approach.

Liver Impairment-The Potential Application of Volatile Organic Compounds in Hepatology.

Liver diseases are currently diagnosed through liver biopsy. Its invasiveness, costs, and relatively low diagnostic accuracy require new techniques to be sought. Analysis of volatile organic compounds (VOCs) in human bio-matrices has received a lot of attention. It is known that a musty odour characterises liver impairment, resulting in the elucidation of volatile chemicals in the breath and other body fluids such as urine and stool, which may serve as biomarkers of a disease. Aims: This study aims to review all the studies found in the literature regarding VOCs in liver diseases, and to summarise all the identified compounds that could be used as diagnostic or prognostic biomarkers. The literature search was conducted on ScienceDirect and PubMed, and each eligible publication was qualitatively assessed by two independent evaluators using the SANRA critical appraisal tool. Results: In the search, 58 publications were found, and 28 were kept for inclusion: 23 were about VOCs in the breath, one in the bile, three in urine, and one in faeces. Each publication was graded from zero to ten. A graphical summary of the metabolic pathways showcasing the known liver disease-related VOCs and suggestions on how VOC analysis on liver impairment could be applied in clinical practice are given.

Breath Biopsy Assessment of Liver Disease Using an Exogenous Volatile Organic Compound-Toward Improved Detection of Liver Impairment.

INTRODUCTION: Liver cirrhosis and its complication - hepatocellular carcinoma (HCC) - have been associated with increased exhaled limonene. It is currently unclear whether this increase is more strongly associated with the presence of HCC or with the severity of liver dysfunction. METHODS: We compared the exhaled breath of 40 controls, 32 cirrhotic patients, and 12 cirrhotic patients with HCC using the Breath Biopsy platform. Breath samples were analyzed by thermal desorption-gas chromatography-mass spectrometry. Limonene levels were compared between the groups and correlated to bilirubin, albumin, prothrombin time international normalized ratio, and alanine aminotransferase. RESULTS: Breath limonene concentration was significantly elevated in subjects with cirrhosis-induced HCC (M: 82.1 ng/L, interquartile range [IQR]: 16.33-199.32 ng/L) and cirrhosis (M: 32.6 ng/L, IQR: 6.55-123.07 ng/L) compared with controls (M: 6.2 ng/L, IQR: 2.62-9.57 ng/L) (P value = 0.0005 and 0.0001, respectively) with no significant difference between 2 diseased groups (P value = 0.37). Levels of exhaled limonene correlated with serum bilirubin (R = 0.25, P value = 0.0016, r = 0.51), albumin (R = 0.58, P value = 5.3e-8, r = -0.76), and international normalized ratio (R = 0.29, P value = 0.0003, r = 0.51), but not with alanine aminotransferase (R = 0.01, P value = 0.36, r = 0.19). DISCUSSION: Exhaled limonene levels are primarily affected by the presence of cirrhosis through reduced liver functional capacity, as indicated by limonene correlation with blood metrics of impaired hepatic clearance and protein synthesis capacity, without further alterations observed in subjects with HCC. This suggests that exhaled limonene is a potential non-invasive marker of liver metabolic capacity (see Visual abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A388).

An extremely high dietary iodide supply forestalls severe hypothyroidism in Na+/I- symporter (NIS) knockout mice.

The sodium/iodide symporter (NIS) mediates active iodide (I-) accumulation in the thyroid, the first step in thyroid hormone (TH) biosynthesis. Mutations in the SLC5A5 gene encoding NIS that result in a non-functional protein lead to congenital hypothyroidism due to I- transport defect (ITD). ITD is a rare autosomal disorder that, if not treated promptly in infancy, can cause mental retardation, as the TH decrease results in improper development of the nervous system. However, in some patients, hypothyroidism has been ameliorated by unusually large amounts of dietary I-. Here we report the first NIS knockout (KO) mouse model, obtained by targeting exons 6 and 7 of the Slc5a5 gene. In NIS KO mice, in the thyroid, stomach, and salivary gland, NIS is absent, and hence there is no active accumulation of the NIS substrate pertechnetate (99mTcO4-). NIS KO mice showed undetectable serum T4 and very low serum T3 levels when fed a diet supplying the minimum I- requirement for rodents. These hypothyroid mice displayed oxidative stress in the thyroid, but not in the brown adipose tissue or liver. Feeding the mice a high-I- diet partially rescued TH biosynthesis, demonstrating that, at high I- concentrations, I- enters the thyroid through routes other than NIS.

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine.

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.

A locus on mouse chromosome 2 is involved in susceptibility to congenital hypothyroidism and contains an essential gene expressed in thyroid.

We report here the mapping of a chromosomal region responsible for strain-specific development of congenital hypothyroidism in mice heterozygous for null mutations in genes encoding Nkx2-1/Titf1 and Pax8. The two strains showing a differential predisposition to congenital hypothyroidism contain several single-nucleotide polymorphisms in this locus, one of which leads to a nonsynonymous amino acid change in a highly conserved region of Dnajc17, a member of the type III heat-shock protein-40 (Hsp40) family. We demonstrate that Dnajc17 is highly expressed in the thyroid bud and had an essential function in development, suggesting an important role of this protein in organogenesis and/or function of the thyroid gland.