Preparation of [1'-13 C]citric acid as a probe in a breath test to evaluate tricarboxylic acid cycle flux.

[1'-13 C]Citric acid (1) was efficiently prepared from dimethyl 1,3-acetonedicarboxylate in two steps as a probe for a breath test. The synthetic method was selected because of the yield and reproducibility. Compound 1 was orally administrated to rats, and the time course of the increase of 13 CO2 /12 CO2 ratios (Δ13 CO2 ) in their breath was successfully followed, indicating the metabolism of 1. Thus, the 13 C-breath test using 1 is a promising method to evaluate tricarboxylic acid (TCA) cycle flux.

Effects of ethanol and sex on propionate metabolism evaluated via a faster 13C-propionate breath test in rats.

BACKGROUND: Alcoholism is regarded as a risk factor for vitamin B12 (VB12) deficiency. Because VB12 serves as a coenzyme of methylmalonyl-CoA mutase, a key enzyme in propionate metabolism, the 13C-propionate breath test (PBT) has been studied as a non-invasive diagnostic modality for VB12 deficiency. However, the conventional PBT requires 2 h, which is inconvenient in clinical practice. We hypothesized that a faster PBT can be used to evaluate propionate metabolism and is more easily adaptable for clinical practice. AIM: To evaluate a faster PBT for assessing the effects of long-term ethanol consumption on propionate metabolism in ethanol-fed rats (ERs). METHODS: ERs were obtained by replacing standard drinking water (for control rats, CRs) with 16% ethanol solution in descendants of F344/DuCrj rats. Faster PBT was performed by administering 13C-propionate aqueous solution to male and female ERs and CRs by inserting a metal tubule from the mouth to the stomach; exhaled gas was collected in a bag to measure its 13CO2/12CO2 isotope ratio via infrared isotope spectrometry. Serum VB12 and alanine transaminase (ALT) levels were measured via chemiluminescence immunoassay and the lactate dehydrogenase-ultraviolet method, respectively. We evaluated statistical differences in mean body weight, change in 13CO2 (Δ13CO2‰), peak Δ13CO2‰, and serum VB12 and ALT, between males and females and between ERs and CRs using the t-test and Mann-Whitney U test for normally and non-normally distributed variables, respectively. RESULTS: Males weighed significantly more than females (P < 0.001); CRs weighed significantly more than ERs (P < 0.008). Δ13CO2 reached a peak (Cmax) at 20 min and 30 min in females and males, respectively, decreasing after 20-30 min without rebound in all groups. Males had significantly higher Cmax and Δ13CO2 at 15-45 min than females (P < 0.05; for all pairs). Propionate metabolism was enhanced in male ERs relative to male CRs, whereas metabolism did not differ markedly between ERs and CRs for females. Males had higher serum VB12 levels than females, without prominent differences between the ER and CR groups. Male CRs had notably higher ALT levels than male ERs. Thus, chronic ethanol consumption may trigger fatty acid production via intestinal bacteria and changes in gut microbiome composition. CONCLUSION: Faster PBT shows that 16% ethanol consumption promotes propionate metabolism without inducing liver injury. This PBT may be used clinically to evaluate gut flora status.

Evaluation of the One-Hour ¹³C-Propionate Breath Test in 49 Patients from a Single Center in Japan to Detect Vitamin B12 Deficiency.

BACKGROUND Given the unavailability of reliable biomarkers for vitamin B12 (VB12) deficiency in clinical settings, the usefulness of the ¹³C-propionate breath test (PBT), utilizing VB12 as a coenzyme of methylmalonyl-CoA in propionate metabolism, as a diagnostic modality for VB12 deficiency has been studied. However, a collection time of 2 h reduces its convenience. Hence, we evaluated the effectiveness of 1-h PBT for detecting VB12 deficiency in 49 patients with suspected VB12 deficiency. MATERIAL AND METHODS We collected 100-200 mL breath gas every 10 min until 1 h after the administration of 1 g of ¹³C-propionate from 49 patients (31 men, 18 women; median age, 70 years) with clinically suspected VB12 deficiency and calculated the ¹³CO2 recovered in the breath per hour as the recovery rate (RR [%dose/h]) from ¹³CO2/¹²CO2 using infrared isotope spectrometry. We compared the RRs between groups: (1) with serum VB12 levels ≥145 pg/mL and <145 pg/mL, (2) with mean corpuscular volume ≤100 fL and >100 fL, and 3) pre- and post-VB12 supplementation. RESULTS The RRs peaked within 30 min. The RRs at 20 min (RR20) and 30 min (RR30) were significantly lower in macrocytotic patients (41.28 vs 50.07, p=0.026 and 37.82 vs 43.93, P=0.003). The RR30 was higher in the supplemented patients (41.93 vs 32.84, P=0.024). There was no significant difference in RRs between the patients with normal and low serum VB12 levels. CONCLUSIONS The 1-h PBT can be a diagnostic modality for VB12 deficiency because 1 h is a sufficient collection time.

Alteration of Breath Hydrogen and Methane in Ethanol-Fed Rats.

Chronic alcohol consumption can cause dysbiosis, but it is difficult to determine the effect of alcohol on the structure and activity of gastrointestinal tract microbiota. We therefore designed a noninvasive hydrogen breath test (HBT) to investigate the alteration in the chemical profile of gut microbiota in ethanol-fed rats. Thirteen F344/DuCri rats were fed on a commercial mash food with 16% ethanol solution drinking fluid from 4 weeks of age. HBTs were carried out on six 8-week-old and seven 24-week-old ethanol-fed rats. As controls, HBTs were carried out on sixteen 8-week-old, six 24-week-old, and five 48-week-old male rats. Six 24-week- old male rats were examined twice at 1-week intervals. HBTs were performed after fasting for 24 hr. Rats were orally administrated 4 mL/kg of 65% lactulose solution and housed in an animal chamber. The expired air was collected in a breath-sampling bag at 10-min intervals for 180 min. The hydrogen (H2) and methane (CH4) levels in the breath sample were measured using a breath analyzer and were expressed.as parts- per million (ppm). Elevated H2 and CH4 levels were more frequent in male rats. Maximal values of H2 and CH4 were highest in 8-week- old rats, followed by 48-week-old and 24-week-old rats. No ethanol-fed rat exhaled more than 2 ppm of H2 or CH4 until 180 min after the oral administration of lactulose, unlike the controls. This alteration was more obvious than that of aging or gender differentiation. We conclude that there is a close association between chronic ethanol consumption and H2 and CH4 production. An asymptomatic heavy drinker might have dysbiosis that involves gut microbiota with lower fermentation performance.

Evaluation of remnant liver function using 13C-breath tests in a rat model of 70% partial hepatectomy.

BACKGROUND/AIMS: A 13C breath test was applied to evaluate the early stage of postoperative liver function after hepatectomy. It was examined in comparison with functional and morphological recovery. METHODOLOGY: Rats were subjected to 70% partial hepatectomy (H group) and control group (S group). Expired air, blood and remnant liver tissue were collected at 12, 24, 48 and 72h, and 7 and 14 days postoperatively. Phenylalanine and leucine were used as the 13C-labeled substrates. The 13CO2 was collected until 120min. The mean values of the H and S groups were compared using the highest mean level of 13CO2 excreted in breath (Cmax). The liver regenerative ability was evaluated by Ki-67. RESULTS: In the 13C-phenylalanine breath test, the Cmax value tended to be lower for the H group 12h postoperatively (p=0.06). Cmax was significantly lower for the H group 24h postoperatively (p<0.05). Both groups showed equal recovery at 48h postoperatively. In the 13C-leucine breath test, both groups had no significant difference after hepatectomy. The peak of Ki-67 positive rate is estimated to be at 52.8h postoperatively, meaning that the time difference was only approximately 5h between the peak time of Ki-67 positive rate and the 13C-phenylalanine breath test. CONCLUSIONS: These results strongly support that the 13C-phenylalanine breath test can become a non-invasive indicator to evaluate the early stage of postoperative liver function after hepatectomy.

[Mechanism of alcohol consumption-mediated Th2-polarized immune response].

Alcohol consumption impairs Th1-mediated cellular immune responses and enhances serum IgE levels. It has been reported that the elevated IgE levels are associated with a Th2 polarization response, but the mechanisms for enhancing Th2 polarization by the ethanol treatment remain to be elucidated. The aim of this review is to present and discuss the mechanism of Th2 polarization response by alcohol. IL-12 production by APCs such as monocytes, macrophages, and dendritic cells (DCs) preferentially leads to Th1 polarization. Acute ethanol consumption results in a significant decrease in IL-12 production in LPS-stimulated DCs and a CD40/CD40L interaction between CD40 on the DCs and CD40 ligand expressed on activated T cells. This suggests that Th2 polarization by ethanol is caused by impaired IL-12 production from APCs. In contrast, the induction of IL-10 by LPS is enhanced by ethanol treatment, suggesting that elevated IL-10 may play a role in ethanol-induced suppression of IL-12. However, ethanol inhibited IL-12 production in LPS-stimulated DCs devoid of IL-10 (IL-10/DC), suggesting that down-regulation of IL-12 by ethanol is independent of the IL-10 levels. Furthermore, several studies report that PGE2, cAMP and linolic acid, and endogenous lipid mediators released in inflammatory conditions, also inhibit IL-12 production. These inhibitory effects are similar to the IL-12 inhibition by ethanol. In addition, increase in the levels of these lipid mediators is induced by ethanol treatment. Alternatively, cytokine signaling studies indicate that IL-12 production by DCs is negatively regulated by PI3K and GSK-3, but positively regulated by p38 MAPK, mTOR, and NF-kappa B. Thus, it seems possible that ethanol may interact on the upstream of IL-12 producing a signal pathway. In fact, ethanol alters the stability of cell membrane, and suppresses clustering of TLR4 and recruitment of signaling molecules into lipid rafts, where it associates with the Ser/Thr kinase and the adaptor proteins, and forms a signaling complex. Down-regulation of lipid raft signaling is results in the impaired IL-12 production leading to the Th1 polarization, and causes CD4+ T cells to differentiation toward the Th2 lineage.

Is the metabolism of toast alcohol beverages influenced by a meal taken five minutes later?

Alcohol is commonly consumed with, or soon after, a meal that affects gastric emptying and thus, absorption and metabolism of alcohol. The aim of this study is to evaluate the effect of liquid test meal ingested soon after, alcohol ingestion on alcohol metabolism, as is common in the social setting. First, a 100 mL of water containing of 80 mg of 13C-ethanol was administrated orally in 7 healthy subjects, and 200 mL of liquid meal (200 kcal) was administrated 5 min after alcohol ingestion. Breath samples were taken at baseline and at 10-min interval for 150 min. Next, 13C-ethanol breath test was performed without ingestion of liquid meal. Cmax was decreased after ingestion of liquid meal, whereas Tmax was unchanged. The descending gradient of 13CO2 excretion curves after ingestion of liquid meal is parallel to that without liquid meal, while the ascending gradient of 13CO2 excretion curves in the first 10 min had a tendency to be greater after ingestion of liquid meal than without ingestion of liquid meal. A significant delay in breath 13CO2 excretion after ingestion of liquid meal was found and the AUC values were lower after ingestion of liquid meal in each time point, indicating that liquid meal ingested soon after alcohol ingestion may not affect elimination but absorption and/or metabolism of a small amount of alcohol.

Omic analyses unravels global molecular changes in the brain and liver of a rat model for chronic Sake (Japanese alcoholic beverage) intake.

The effects of chronic administration of Sake (Japanese alcoholic beverage, Nihonshu) on brain and liver of female F334 (Fisher) rats were surveyed via global omic analyses using DNA microarray, 2-DE, and proton nuclear magnetic resonance. Rats weaned at 4 wk of age were given free access to Sake (15% alcohol), instead of water. At 13 months of age, and 24 h after withdrawal of Sake supply, rats were sacrificed, and the whole brain and liver tissues dissected for analyses. In general, molecular changes in brain were found to be less than those in liver. Transcriptomics data revealed 36 and 9, and 80 and 62 up- and down-regulated genes, in the brain and liver, respectively, with binding and catalytic activity gene categories the most prominently changed. Results suggested Sake-induced fragility of brain and liver toxicity/damage, though no significant abnormalities in growth were seen. At protein level, a striking decrease was found in the expression of NADH dehydrogenase (ubiquinone) Fe-S protein 1 in brain, suggesting attenuation of mitochondrial metabolism. In liver, results again suggested an attenuation of mitochondrial function and, in addition, glycoproteins with unknown function were induced at protein and gene levels, suggesting possible changes in glycoprotein binding in that organ. Metabolomic analysis of brain revealed significant increases in valine, arginine/ornithine, alanine, glutamine, and choline with decreases in isoleucine, N-acetyl aspartate, taurine, glutamate, and gamma aminobutyric acid. Our results provide a detailed inventory of molecular components of both brain and liver after Sake intake, and may help to better understand effects of chronic Sake drinking.

Removal of ovarian hormones affects the ageing process of acetate metabolism.

BACKGROUND: Despite a close association between gastrointestinal motility and sex hormones, it has been unknown whether ovarian hormones affect absorption and metabolism of nutrients. The aim of this study is, therefore, to evaluate metabolism of acetate in rats with age and the influence of ovariectomy on its change. METHODS: Fourteen female rats of the F344 strain were used, and 13C-acetate breath test was performed at 2, 7 and 13 months of age. Seven rats were ovariectomized at three weeks of age (ovariectomy group) and the remaining seven rats were studied as control group. After 24-hr fasting, rats are orally administrated 1ml of water containing sodium (13)C-acetate (100mg/kg) and housed in an animal chamber. The expired air in the chamber is collected in a breath-sampling bag using a aspiration pump. The (13)CO(2) concentration is measured using an infrared spectrometer for 120 min and expressed as delta per mil. RESULTS: The breath (13)CO(2) excretion increased with time and peaked 30 min in control rats. In ovariectomized rats, thee peak time of (13)CO(2) excretion was prolonged to 40 min at 7 and 13 months of age. Cmax was significantly higher at 2 months of age but lower at 4 months of age in ovariectomized rats than in control rats. Those of two groups became equal at 7 months of age. CONCLUSIONS: From the viewpoint of acetate metabolism, removal of ovarian hormones might make rats to be precocious ones and accelerate ageing.

Influence of chronic ethanol consumption on extra-pancreatic secretory function in rat.

BACKGROUND: The usefulness of the typical direct methods involving duodenal intubation, such as the secretin and secretin-cholecystokinin tests, in the diagnosis of exocrine pancreatic dysfunction is widely accepted. However, these diagnostic tests tend to be avoided because of their technical complexity and the burden on patients. Recently, a simple breath test was developed for assessment of exocrine pancreatic function employing 13C-dipeptide [i.e., benzoyl-L-tyrosyl-[1-13C] alanine (Bz-Tyr-Ala)]. Although alcohol abuse causes pancreatic damage in humans, this has been unclear in rats. AIMS: The aim of the study is to evaluate the effect of ethanol exposure beginning at an early age on extra-pancreatic secretory function in rats. MATERIALS AND METHODS: Twelve female rats of the F344 strain aged 12 months were used. Seven rats were fed on a commercial mash food with 16% ethanol solution (Japanese Sake) as drinking-fluid since at 29 days of age (ethanol group). The remaining five rats were fed on a nutrient-matched isocaloric diet with water as drinking-fluid (control group). After 24-hr fasting, rats are orally administrated 1cc of water containing sodium 13C-dipeptide (5 mg/kg) and housed in an animal chamber. The expired air in the chamber is collected in a breath-sampling bag using a tube and aspiration pump. The 13CO2 concentration is measured using an infrared spectrometer at 10-min interval for 120 min and expressed as delta per mil. RESULTS: The breath 13CO2 level increased and peaked at 20 min in both two groups. In general, 13CO2 excretion peaked rapidly and also decreased sooner in ethanol rats than in control rats. The mean value of the maximal 13CO2 excretion is 34.7 per mil in ethanol rats, greater than in control rats (31.4 per mil), but the difference did not reach the statistically significance. CONCLUSION: Chronic ethanol feeding beginning at an early age does not affect extra-pancreatic secretory function in rats.

ALDH2 and CYP2E1 genotypes, urinary acetaldehyde excretion and the health consequences in moderate alcohol consumers.

Deficiencies in mitochondrial low-Km aldehyde dehydrogenase (ALDH2) activity, and consequently high blood acetaldehyde levels, have been suggested to relate to various diseases in Japanese, including esophageal cancer. In the present study, 200 men aged 35-59 years randomly selected from an occupational population were analyzed for the association of ALDH2 genotypes and cytochrome P450-2E1 (CYP2E1) genotypes with the urinary excretion of acetaldehyde (which is bound to some chemicals in the urine) and with common alcohol-related health consequences. Urinary acetaldehyde excretion was increased, reflecting increased alcohol consumption even in this moderate alcohol-consuming population. Neither the ALDH2 nor the CYP2E1 genotypes showed significant influence on the elevation of urinary acetaldehyde excretion. Neither these genotypes nor urinary acetaldehyde concentration significantly affected blood pressure, serum aspartate aminotransferase and gamma-glutamyl transferase activities, or serum HDL-cholesterol and lipid peroxide concentrations. It was concluded that acetaldehyde accumulates in moderate alcohol consumers irrespective of ALDH2 and CYP2E1 genotype, and that the implications of these genotypes and acetaldehyde accumulation in terms of common alcohol-related health consequences were obscure. The results also suggest that the carcinogenicity of acetaldehyde on esophageal mucosa depends greatly upon repeated exposure to high blood acetaldehyde, even through transient rather than chronic exposure.