Using the Noninvasive 13C-Sucrose Breath Test to Measure Intestinal Sucrase Activity in Swine

The sucrose breath test (SBT) is a simple noninvasive technique used currently to determine intestinal absorptive function in humans and rodents. However, to date, the test has not been adapted for use in swine. During weaning, intestinal sucrase activity in piglets temporarily declines in response to stressors and is commonly used as a marker of the intestinal response to weaning. Here we assessed the sucrose dose needed for using the SBT in piglets. Six randomly allocated piglets were orogastrically gavaged with ¹³C-labeled sucrose at a dose of 2 g/kg; breath samples were collected for measurement of ¹³CO₂ on days 0 (approximately 17 h after weaning), 5, and 10 after weaning. The resultant SBT value (cumulative dose at 90 min) was decreased by 46% on day 5 after weaning relative to baseline levels, consistent with temporal changes in gastrointestinal sucrase activity associated with weaning. We conclude that a sucrose dose of 2 g/kg is satisfactory to conduct SBT studies in piglets. With further development, the SBT may provide a new tool to noninvasively monitor digestive function in weaned piglets, to assess the effects of nutritional strategies on intestinal health, and as an indicator of gut integrity and function in swine models of human gastrointestinal disease.Abbreviations: SBT, sucrose breath testThe activity of the digestive brush border enzymes, specifically sucrase, develops gradually in the small intestine as piglets mature.^3,9 Sucrase activity is absent in the newborn piglet, with levels increasing slowly during the first 4 d postnatally.^3,7,9 Sucrase activity is measurable in all segments of the small intestine by 7 d of age^9,16 and continues to increase thereafter, reaching a plateau at approximately 14 d of age.⁷ However, this pattern of sucrase activity is adversely affected by the weaning process, with the abrupt weaning typical of commercial pig production resulting in decreased villus height, increased crypt depth, and significantly reduced intestinal sucrase activity.¹³Studies in rats^{1,2,6,11,22,23} and humans²¹ have demonstrated that total intestinal sucrase activity can be determined accurately and noninvasively by using the recently developed sucrose breath test (SBT). The release of ¹³CO₂ from ¹³C-labeled substrates forms the basis for several noninvasive gut function tests, including those to assess gastric emptying,¹⁷ and gastrointestinal transit.⁸ The SBT relies on the detection of ¹³CO₂ in expired breath after oral ingestion of an appropriate substrate. For the SBT, the substrate used is sucrose, which is metabolized to glucose and fructose by sucrase. After transit through the hepatic and respiratory systems, ¹³CO₂ is released. The expression of sucrase decreases in a proximal-to-distal gradient along the small intestine.¹⁵ Therefore, the SBT provides an integrated index of the total activity of sucrase throughout the small intestine.The SBT quantifies ¹³CO₂ in the expired breath after oral ingestion of ¹³C-sucrose to provide a marker of total intestinal sucrase activity.^2,20 This technique has demonstrated diagnostic application in humans to monitor the development of chemotherapy-induced intestinal mucositis.²¹ Moreover, the SBT has been used in rats to determine the potential of diverse nutraceutical agents^1,22,23 and probiotics¹⁹ to modify small intestinal absorption. Pigs are an excellent omnivore model on which to base relevant nutritional models to investigate the mechanisms underlying chronic human gastrointestinal diseases.¹⁴ However, to date, the SBT has not been adapted for use in pigs, in which it might become a valuable tool to assess longitudinal changes in intestinal sucrase activity in response to weaning, dietary manipulations, and disease processes. This technique therefore might be applied in the biomedical field in addition to the animal production research environment. In the current study, we sought to establish the experimental conditions required to conduct the SBT in piglets and noninvasively determine total intestinal sucrase activity at 3 time points after weaning.     

Assessment of the 14C-Glycocholic Acid Breath Test

The 1-(¹⁴C)-glycine-glycocholic-acid breath test has been performed on 104 subjects and a normal range established. Abnormal results due to bacterial deconjugation of bile salts were found not only in patients with the “contaminated bowel” syndrome and in those with ileal resection but also in a third group, patients with cholangitis. Abnormal results were also found in patients with gastrocolic fistula and staphylococcal enterocolitis, while mildly abnormal results were also found in some patients with liver disease.     

Molecular Characterization of a Variant of Bacillus anthracis-Specific Phage AP50 with Improved Bacteriolytic Activity

The genome sequence of a Bacillus anthracis-specific clear plaque mutant phage, AP50c, contains 31 open reading frames spanning 14,398 bp, has two mutations compared to wild-type AP50t, and has a colinear genome architecture highly similar to that of gram-positive Tectiviridae phages. Spontaneous AP50c-resistant B. anthracis mutants exhibit a mucoid colony phenotype.     

The Shape Trail Test: Application of a New Variant of the Trail Making Test

Objective

The Trail making test (TMT) is culture-loaded because of reliance on the Latin alphabet, limiting its application in Eastern populations. The Shape Trail Test (STT) has been developed as a new variant. This study is to examine the applicability of the STT in a senile Chinese population and to evaluate its potential advantages and disadvantages.

Method

A total of 2470 participants were recruited, including 1151 cognitively normal control (NC), 898 amnestic mild cognitive impairment (aMCI), and 421 mild Alzheimer disease (AD) patients. Besides the STT, the Mini mental state examination and a comprehensive neuropsychological battery involving memory, language, attention, executive function and visuospatial ability were administered to all the participants. In a subgroup of 100 NC and 50 AD patients, both the STT and the Color Trail Test (CTT) were performed.

Results

In NC, the time consumed for Part A and B (STT-A and STT-B) significantly correlated with age and negatively correlated with education (p<0.01). STT-A and B significantly differed among the AD, aMCI and NC. The number that successfully connected within one minute in Part B (STT-B-1 min) correlated well with STT-B (r = 0.71, p<0.01) and distinguished well among NC, aMCI and AD. In the receiver operating characteristic curve analysis, the AUCs (area under the curve) for STT-A, STT-B, and STT-B-1min in identifying AD were 0.698, 0.694 and 0.709, respectively. The STT correlated with the CTT, but the time for completion was longer.

Conclusion

The TMT is a sensitive test of visual search and sequencing. The STT is a meaningful attempt to develop a “culture-fair” variant of the TMT in addition to the CTT.     

Twenty-Minute Fasting Version of the US 13C-Urea Breath Test for the Diagnosis of H. pylori Infection

Background. In large-scale multi-center clinical trials, the US ¹³C-urea breath test (UBT) has proven to have a sensitivity and specificity of approximately 95%. Ingestion of a meal to delay gastric emptying has advantages of increasing the level of signal as well as prolonging the duration of significantly increased ¹³C excretion, at the expense of requiring 40 to 60 minutes to complete the test. Our aim was to explore the utility of the ¹³C-UBT with a total duration of 30 minutes or less.
Methods. After a baseline breath sample was obtained, 125 mg of ¹³C-urea was given in 100 ml of water, and additional breath samples were taken after 20 and 30 minutes. The results of the UBT were compared to histological assessment, culture, and the rapid urease test. ¹³C-UBTs were carried out on normal volunteers who underwent gastroscopy during which six mucosal biopsies were taken. Three biopsies were for histological evaluation (Genta stain), two for culture, and one was for agar gel rapid urease testing. The UBT was conducted 2 to 3 days either before or after the endoscopic procedure.
Results. The cutoff value for a positive UBT was enrichment of 2.4Δ%₀ (delta over baseline). Of the 66 tests, 51%₀ were Helicobacter pylori -positive. There were no false positive UBTs and only two false negative UBTs at 20 minutes (sensitivity, 96%; specificity, 100%). At 30 minutes, one other UBT was false negative (gray zone of 2.36%₀.) (sensitivity, 94%; specificity, 100%).
Conclusion. These results suggest that omission of the meal and shortening the duration of the US ¹³C-UBT to 20 minutes still may maintain excellent specificity and sensitivity of the test.     

Breath [13CO2] recovery from an oral glucose load during exercise: comparison between [U-13C] and [1,2-13C]glucose.

The purpose of the present experiment was to compare 13CO2 recovery at the mouth, and the corresponding exogenous glucose oxidation computed, during a 100-min exercise at 63 +/- 3% maximal O2 uptake with ingestion of glucose (1.75 g/kg) in six active male subjects, by use of [U-13C] and [1,2-13C]glucose. We hypothesized that 13C recovery and exogenous glucose oxidation could be lower with [1,2-13C] than [U-13C]glucose because both tracers provide [13C]acetate, with possible loss of 13C in the tricarboxylic acid (TCA) cycle, but decarboxylation of pyruvate from [U-13C]glucose also provides 13CO2, which is entirely recovered at the mouth during exercise. The recovery of 13C (25.8 +/- 2.3 and 27.4 +/- 1.2% over the exercise period) and the amounts of exogenous glucose oxidized computed were not significantly different with [1,2-13C] and [U-13C]glucose (28.9 +/- 2.6 and 30.7 +/- 1.3 g, between minutes 40 and 100), suggesting that no significant loss of 13C occurred in the TCA cycle. This stems from the fact that, during exercise, the rate of exogenous glucose oxidation is probably much larger than the flux of the metabolic pathways fueled from TCA cycle intermediates. It is thus unlikely that a significant portion of the 13C entering the TCA cycle could be diverted to these pathways. From a methodological standpoint, this result indicates that when a large amount of [13C]glucose is ingested and oxidized during exercise, 13CO2 production at the mouth accurately reflects the rate of glucose entry in the TCA cycle and that no correction factor is needed to compute the oxidative flux of exogenous glucose.     

Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient

The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.     

The Role of the Carbohydrate Recognition Domain of Placental Protein 13 (PP13) in Pregnancy Evaluated with Recombinant PP13 and the DelT221 PP13 Variant

Introduction

Placental protein 13 (PP13), a placenta specific protein, is reduced in the first trimester of pregnancy in women who subsequently develop preeclampsia. A naturally occurring PP13 deletion of thymidine at position 221 (DelT₂₂₁ or truncated variant) is associated with increased frequency of severe preeclampsia. In this study we compared the full length (wildtype) PP13 and the truncated variant.

Methods

Full length PP13 or its DelT₂₂₁ variant were cloned, expressed and purified from E-Coli. Both variants were administrated into pregnant rats at day 8 of pregnancy for slow release (>5 days) through osmotic pumps and rat blood pressure was measured. Animals were sacrificed at day 15 or day 21 and their utero-placental vasculature was examined.

Results

The DelT₂₂₁ variant (11 kDA) lacked exon 4 and a part of exon 3, and is short of 2 amino acids involved in the carbohydrate (CRD) binding of the wildtype (18 kDA). Unlike the wildtype PP13, purification of DelT₂₂₁ variant required special refolding. PP13 specific poly- clonal antibodies recognized both PP13 and DelT₂₂₁ but PP13 specific monoclonal antibodies recognized only the wildtype, indicating the loss of major epitopes. Wildtype PP13 mRNA and its respective proteins were both lower in PE patients compared to normal pregnancies. The DelT₂₂₁ mutant was not found in a large Caucasian cohort. Pregnant rats exposed to wildtype or DelT₂₂₁ PP13 variants had significantly lower blood pressure compared to control. The wildtype but not the DelT₂₂₁ mutant caused extensive vein expansion.

Conclusion

This study revealed the importance of PP13 in regulating blood pressure and expanding the utero-placental vasculature in pregnant rats. PP13 mutant lacking amino acids of the PP13 CRD domain fails to cause vein expansion but did reduce blood pressure. The study provides a basis for replenishing patients at risk for preeclampsia by the full length but not the truncated PP13.     

Detoxification of an aliphatic amine by N-acetylation: experimental and clinical studies.

Incubation of serum serpin (alpha-1-antitrypsin) with 5 mM 1,6-diaminohexane causes significant loss of heterozygotic inhibitor activity. While serpin genes have several alleles, the enzyme complex that acetylates the amine to render it suitable for excretion has primarily two phenotype populations, i.e. slow and fast N-acetylators. This study shows that diacetyl-1,6-diaminohexane does not cause in vitro loss of serpin activity, and that all regional cases (eleven in all) referred to us during one year with a diagnosis of occupational organic diisocyanate asthma were slow acetylators except one who presented a marginally fast reaction type.     

Epitope Characterization of an Aromatase Monoclonal Antibody Suitable for the Assessment of Intratumoral Aromatase Activity

Immunohistochemistry is one of the most suitable methods for the detection of intratumoral aromatase in order to identify patients who may respond to aromatase inhibitor therapy in hormone-dependent breast cancer. Previous studies showed statistically significant correlation between results of immnuohistochemistry and biochemical analysis in carcinoma components stained by aromatase monoclonal antibody 677. In this study, determination of the antigenic peptides recognized by aromatase antibodies through epitope mapping, combined with the new knowledge on aromatase-reductase interaction, provide insights for understanding various immunostaining patterns using different aromatase antibodies. Our studies on aromatase-reductase interaction also provided critical information on how aromatase and reductase interact with each other on the endoplasmic reticulum membrane, and identified key residues, including K108 of aromatase, that are involved in the interaction with reductase. Through epitope mapping and taking into consideration the interference with aromatase immunohistochemical staining by NADPH-cytochrome P450 reductase, we demonstrated that monoclonal antibody 677 is a suitable antibody for an assessment of intratumoral aromatase activity in breast cancer patients for making clinical management decisions. These results also provide valuable information to identify new aromatase antibodies for immunohistochemical diagnosis of hormone-dependent breast cancer in future.     

Hypothalamic Regulation of the Daily Rhythm in Hepatic Tyrosine Transaminase Activity

THERE is a daily rhythm in the activity of tyrosine transaminase in rat liver¹ which is characterized by a three-fold rise in enzyme activity from low daytime values to a peak several hours after dark. The oscillation persists in the absence of the pituitary or adrenal glands^2–4 and during fasting^5,6. Noradrenaline seems to play a role in the regulation of the enzyme and thereby contributes to the generation of the daily rhythm of activity⁷, but elevation of tissue noradrenaline in vivo suppresses the circadian enzyme rhythm at basal levels⁸. Noradrenaline inhibits tyrosine transaminase activity in vitro by competing with enzyme for binding with the pyridoxal-5′-phosphate co-factor⁷ and recent observations suggest that noradrenaline regulates tyrosine transaminase turnover in vivo by the same mechanism.     

Glutathione-Dependent Conversion of N-Ethylmaleimide to the Maleamic Acid by Escherichia coli: an Intracellular Detoxification Process

The electrophile N-ethylmaleimide (NEM) elicits rapid K⁺ efflux from Escherichia coli cells consequent upon reaction with cytoplasmic glutathione to form an adduct, N-ethylsuccinimido-S-glutathione (ESG) that is a strong activator of the KefB and KefC glutathione-gated K⁺ efflux systems. The fate of the ESG has not previously been investigated. In this report we demonstrate that NEM and N-phenylmaleimide (NPM) are rapidly detoxified by E. coli. The detoxification occurs through the formation of the glutathione adduct of NEM or NPM, followed by the hydrolysis of the imide bond after which N-substituted maleamic acids are released. N-Ethylmaleamic acid is not toxic to E. coli cells even at high concentrations. The glutathione adducts are not released from cells, and this allows glutathione to be recycled in the cytoplasm. The detoxification is independent of new protein synthesis and NAD⁺-dependent dehydrogenase activity and entirely dependent upon glutathione. The time course of the detoxification of low concentrations of NEM parallels the transient activation of the KefB and KefC glutathione-gated K⁺ efflux systems.     

The PHA Test as an Indicator of Phagocytic Activity in a Passerine Bird

Several techniques in ecological immunology have been used to assess bird immunocompetence thus providing useful information to understand the contribution of the immunological system in life-history decisions. The phytohaemagglutinin (PHA)-skin test has been the most widely employed technique being interpreted as the sole result of T lymphocytes proliferation and hence used to evaluate acquired immunological capacity. However, the presence of high numbers of phagocytic cells in the swelling point has cast some doubt about such an assumption. To address this issue, we collected blood from 14 days-old nestlings of spotless starling (Sturnus unicolor), administered subcutaneous PHA immediately after and then measured the swelling response 24 hours later. Differential counts of white blood cells suggested that an intense development of acquired immunological defences was taking place. The phagocytic activity of both heterophiles and monocytes was also very intense as it was the swelling response. Moreover, our results show, for the first time in birds, a positive relationship between the phagocytic activity of both kinds of cells and the swelling response. This broadens the significance of the PHA test from reflecting T lymphocytes proliferation -as previously proposed but still undetermined in vivo- to evaluate phagocytosis as well. In other words, our data suggest that the PHA swelling response may not be considered as the only consequence of processes of specific and induced immunity –T lymphocytes proliferation- but also of constitutive and nonspecific immunity –heterophiles and monocytes phagocytosis. We propose the extensive use of PHA-skin test as an optimal technique to assess immunocompetence.