Daniel E. Butz

NMR‐based metabolomics and breath studies show lipid and protein catabolism during low dose chronic T1AM treatment

3‐Iodothyronamine (T1AM), an analog of thyroid hormone, is a recently discovered fast‐acting endogenous metabolite. Single high‐dose treatments of T1AM have produced rapid short‐term effects, including a reduction of body temperature, bradycardia, and hyperglycemia in mice.

Long-term sex selective hormonal and behavior alterations in mice exposed to low doses of chlorpyrifos in utero.

Chlorpyrifos, O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, is an organophosphate insecticide known to be present in human urine. In utero exposure to chlorpyrifos may cause long-term hormonal and behavior alterations. In this study mice were exposed to 0, 1 or 5mg/kg chlorpyrifos on gestational days 17-20. In utero exposed mice were then tested in a novel foraging behavior maze and assayed for thyroid hormones. Free Thyroxine Index increased significantly in females, but not males. Learning latency and reduced learning ability was evident during training sessions 5-9 in female mice exposed to 1 or 5mg/kg chlorpyrifos. No learning deficiencies were observed in male mice. No differences were seen in behavior when using a standard radial arm maze during the nine training sessions. These data suggest that mice are susceptible to neuro-endocrine reprogramming by chlorpyrifos, and demonstrate the efficacy of the novel foraging maze as an efficient behavior assay tool.

Individual Isomers of Conjugated Linoleic Acid Reduce Inflammation Associated with Established Collagen-Induced Arthritis in DBA/1 Mice

Previously, dietary conjugated linoleic acid [(CLA), an equal mixture of cis-9, trans-11 (c9t11) and trans-10, cis-12 (t10c12) CLA isomers], was found to reduce inflammation in the murine collagen antibody-induced arthritis model, but less so in the murine collagen-induced arthritis (CIA) model, an arthritic model dependent upon acquired immunity. Because CLA is known to alter the acquired immune response, it was hypothesized that feeding CLA after the establishment of arthritis would reduce paw swelling in the CIA model. In this study, upon the establishment of arthritic symptoms, mice were randomized to the following dietary treatments: corn oil (CO) control (n = 6), 0.5% c9t11-CLA (n = 8), 0.5% t10c12-CLA (n = 6), or 1% combined CLA (1:1 c9t11:t10c12-CLA, n = 6). Paws were scored for severity of arthritis and measured for changes in thickness during an 84-d study period. Dietary c9t11- and combined-CLA similarly decreased the arthritic score (29%, P = 0.036, P = 0.049, respectively, when normalized to initial score) and paw thickness (0.11 mm, P = 0.027, P = 0.035, respectively) compared with CO. Dietary t10c12-CLA reduced the arthritic score (41%, P = 0.007 when normalized) and paw thickness (0.12 mm, P = 0.013) relative to CO. Reduced interleukin-1beta on d 7 and 21 for all CLA treatments (n = 3) relative to CO suggested that antiinflammatory effects of CLA isomers might work by common mechanisms of known pathways involved in chronic inflammation. In conclusion, dietary CLA reduced inflammation associated with CIA, and both c9t11-CLA and t10c12-CLA exhibited antiinflammatory effects.

Metabolic Evidence of Diminished Lipid Oxidation in Women With Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS), a common female endocrinopathy, is a complex metabolic syndrome of enhanced weight gain. The goal of this pilot study was to evaluate metabolic differences between normal (n=10) and PCOS (n=10) women via breath carbon isotope ratio, urinary nitrogen and nuclear magnetic resonance (NMR)-determined serum metabolites. Breath carbon stable isotopes measured by cavity ring down spectroscopy (CRDS) indicated diminished (p<0.030) lipid use as a metabolic substrate during overnight fasting in PCOS compared to normal women. Accompanying urinary analyses showed a trending correlation (p<0.057) between overnight total nitrogen and circulating testosterone in PCOS women, alone. Serum analyzed by NMR spectroscopy following overnight, fast and at 2 h following an oral glucose tolerance test showed that a transient elevation in blood glucose levels decreased circulating levels of lipid, glucose and amino acid metabolic intermediates (acetone, 2-oxocaporate, 2-aminobutyrate, pyruvate, formate, and sarcosine) in PCOS women, whereas the 2 h glucose challenge led to increases in the same intermediates in normal women. These pilot data suggest that PCOS-related inflexibility in fasting-related switching between lipid and carbohydrate/protein utilization for carbon metabolism may contribute to enhanced weight gain.

Changes in the natural abundance of 13CO2/12CO2 in breath due to lipopolysacchride-induced acute phase response.

The natural abundance of carbon-13 in blood proteins increases during the cachectic state and may be a biomarker for disease status. We hypothesized a corresponding drop in the relative abundance of 13C in breath CO2. Using the lipopolysacchride (LPS)-induced endotoxemia model of the acute cachectic state, we demonstrated that the acute phase response causes shifts in the stable isotopes of carbon in exhaled CO2 (13CO2/12CO2 delta value) shortly after administration of LPS while glucocorticoid treatment does not. Mice were injected with LPS and stable isotopes of blood amino acids and carbon in exhaled CO2 were monitored. An increase in the relative isotopic mass of serum alanine, proline and threonine was observed at 3 h after LPS injection. Breath delta values began dropping immediately after administration of LPS, and were 4-5 delta values lower than those of the control animals by 2.5 h after injection. A corresponding drop in delta value was not observed with dexamethasone treatment. Thus protein synthesis during the acute phase response probably caused the fractionation of stable isotopes observed in the plasma amino acids and in exhaled breath 13CO2 delta values. The exhaled breath 13CO2 delta value may be a valuable real-time biomarker of cachexia associated with an acute phase response due to endotoxemia.

Breath carbon stable isotope ratios identify changes in energy balance and substrate utilization in humans

Rapid detection of shifts in substrate utilization and energy balance would provide a compelling biofeedback tool for individuals attempting weight loss. As a proof of concept, we tested whether the natural abundance of exhaled carbon stable isotope ratios (breath δ13C) reflects shifts between negative and positive energy balance. Volunteers (n=5) consumed a 40% energy-restricted diet for 6 days followed by 50% excess on day 7. Breath was sampled immediately before and 1 h and 2 h after breakfast, lunch and dinner. Exhaled breath δ13C values were measured by cavity ring-down spectroscopy. Using repeated measures analysis of variance (ANOVA) followed by Dunnett’s contrasts, pre-breakfast breath values on days 2–6 were compared with day 1, and postprandial day 7 time points were compared with pre-breakfast day 7. Energy restriction diminished pre-breakfast breath δ13C by day 3 (P<0.05). On day 7, increased energy intake was first detected immediately before dinner (−23.8±0.6 vs −21.9±0.7‰, P=0.002 (means±s.d.)), and breath δ13C remained elevated at least 2 h post dinner. In conclusion, when shifting between negative and positive energy balance, breath δ13C showed anticipated isotopic changes. Although additional research is needed to determine specificity and repeatability, this method may provide a biomarker for marked increases in caloric intake.

Optical imaging of mitochondrial redox state in rodent models with 3-iodothyronamine.

This study used an optical technique to measure the effects of treating low (10 mg/kg) and high (25 mg/kg) doses of 3-iodothyronamine (T1AM) on the metabolism in the kidney and heart of mice. The ratio of two intrinsic fluorophores in tissue, (NADH/FAD), called the NADH redox ratio (NADH RR), is a marker of the metabolic state of the tissue. A cryofluorescence imaging instrument was used to provide a quantitative assessment of NADH RR in both kidneys and hearts in mice treated with 3-iodothyronamine. We compared those results to corresponding tissues in control mice. In the kidneys of mice treated with a high dose T1AM, the mean values of the maximum projection of NADH RR were 2.6 ± 0.6 compared to 3.20 ± 0.03 in control mice, indicating a 19% ( ± 0.4) significant increase in oxidative stress (OS) in the high dose-treated kidneys (P = 0.047). However, kidneys treated with a low dose of T1AM showed no difference in NADH RR compared to the kidneys of control mice. Furthermore, low versus high dose treatment of T1AM showed different responses in the heart than in the kidneys. The mean value of the maximum projection of NADH RR in the heart changed from 3.0 ± 0.3 to 3.2 ± 0.6 for the low dose and the high dose T1AM-treated mice, respectively, as compared to 2.8 ± 0.7 in control mice. These values correspond to a 9% (±0.5) (P = 0.045) and 14% (±0.5) (P = 0.008) significant increase in NADH RR in the T1AM-treated hearts, indicating that the high dose T1AM-treated tissues have reduced OS compared to the low dose-treated tissues or the control tissues. These results suggest that while T1AM at a high dose increases oxidative response in kidneys, it has a protective effect in the heart and may exert its effect through alternative pathways at different doses and at tissue specific levels.

NMR Metabolomics Show Evidence for Mitochondrial Oxidative Stress in a Mouse Model of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is associated with metabolic and endocrine disorders in women of reproductive age. The etiology of PCOS is still unknown. Mice prenatally treated with glucocorticoids exhibit metabolic disturbances that are similar to those seen in women with PCOS. We used an untargeted nuclear magnetic resonance (NMR)-based metabolomics approach to understand the metabolic changes occurring in the plasma and kidney over time in female glucocorticoid-treated (GC-treated) mice. There are significant changes in plasma amino acid levels (valine, tyrosine, and proline) and their intermediates (2-hydroxybutyrate, 4-aminobutyrate, and taurine), whereas in kidneys, the TCA cycle metabolism (citrate, fumarate, and succinate) and the pentose phosphate (PP) pathway products (inosine and uracil) are significantly altered (p < 0.05) from 8 to 16 weeks of age. Levels of NADH, NAD(+), NAD(+)/NADH, and NADH redox in kidneys indicate increased mitochondrial oxidative stress from 8 to 16 weeks in GC-treated mice. These results indicate that altered metabolic substrates in the plasma and kidneys of treated mice are associated with altered amino acid metabolism, increased cytoplasmic PP, and increased mitochondrial activity, leading to a more oxidized state. This study identifies biomarkers associated with metabolic dysfunction in kidney mitochondria of a prenatal gluococorticoid-treated mouse model of PCOS that may be used as early predictive biomarkers of oxidative stress in the PCOS metabolic disorder in women.

Changes in breath carbon isotope composition as a potential biomarker of inflammatory acute phase response in mechanically ventilated pediatric patients

Sepsis is a leading cause of mortality in intensive care units. Animal studies have shown exhaled breath carbon isotope delta values (BDVs, i.e., 13CO2/12CO2 delta value) to be a marker for the inflammatory acute phase response (APR). The purpose of this study was to determine the baseline variability of BDVs in mechanically ventilated pediatric patients with and without systemic inflammatory response syndrome (SIRS) and to correlate the BDV with clinical course over time. The study was an observational pilot study in a pediatric intensive care unit at an urban, tertiary care childrens hospital. Seventeen mechanically ventilated pediatric patients underwent measurement of exhaled BDVs every 8 hours for 72 hours. The BDV was not statistically different between SIRS, No-SIRS and SIRS with shock. The mean BDV was significantly lower in subjects with active sepsis or trauma/post-op status compared to subjects with No-Infection/Trauma/Surgery (No-ITS) or septic shock. Trend analysis over time revealed that the No-ITS and ITS in recovery groups had positive slopes. Subjects who developed infections during the study and subjects who underwent shock had a negative trend over time. These results indicate that the BDV does not correlate well with the SIRS status. However, when patients are classified based on their inflammatory APR the BDV correlates with the severity of systemic inflammation. When monitored over time, changes in the BDV may correlate with changes in physiology related to fractionation during the APR to infection, trauma or due to altered macronutrient oxidation during episodes of septic shock.

The emerging role of carbon isotope ratio determination in health research and medical diagnostics

Variations in the isotopic signature of carbon in biological samples (e.g. breath, blood and tissues) can be used to monitor shifts in whole body metabolism. As a conservative recorder of our diet, changes in the isotopic signature of carbon in biological samples provide an objective means to distinguish dietary patterns and the relationship with diseases. In addition, metabolic discrimination of carbon within the body can be informative regarding changes in the bodys metabolic fuel usage during situations where shifts in the macronutrient oxidation ratio are expected. Therefore, changes in the isotopic signature over time have proven to be a tremendously powerful and sensitive means of detecting and measuring changes in steady-state systems. As such, this review focuses on how a naturally occurring ratio of stable isotopes of carbon (13C/12C) can be used as a biomarker for nutritional and metabolic status, altered macronutrient metabolism, and health and disease.