Hydrolysis of iodothyronine glucuronides by obligately anaerobic bacteria isolated from human faecal flora

Abstract 35 bacterial strains isolated from the human faecal flora were screened for hydrolysis of the glucuronides of 3,3',5-triiodothyronine and 3,3'-diiodothyronine. Two Gram-positive obligately anaerobic strains possessed glucuronidase activity. These strains probably belong to the genus Eubacterium , but ethanol was produced in high concentrations during glucose fermentation, which makes final classification difficult. Considering the number of bacteria in the intestinal flora (> 10⁸/ml) and the biliary excretion of iodothyronine conjugates, the strains must be able to hydrolyse a major part of the total daily intestinal supply of these iodothyronine metabolites. The study extends previous observations with faecal suspensions of human and rat origin [24]. The relevance of bacterial β-glucuronidase activity for a possible enterohepatic circulation of iodothyronines is discussed.     

Distribution of estrone sulfatase activity in the intestine of germfree and conventional rats

The intestinal content, the mucosa and the rest of the intestinal wall of germfree (GF) and conventional ( CVL ) rats were tested for in vitro hydrolysis of [3H]estrone sulfate. In homogenates from GF rat intestine some estrone sulfate hydrolysis was detected in those from the proximal small intestine (PSI) (4.2 +/- 0.1% hydrolyzed after 4 h), but not in those from the distal small intestine (DSI) and the caecum. Estrone sulfate was also hydrolyzed by the homogenates of the mucosa and the rest of the intestinal wall from each of the segments tested (PSI: 12.8 +/- 0.4% (mucosa) and 21.5 +/- 2.1 (wall); DSI: 8.2 +/- 0.9% (mucosa) and 17.3 +/- 1.7% (wall); caecum: 8.8 +/- 1.6% (mucosa) and 17.3 +/- 0.5% (wall) ). In the homogenates of CVL rat intestine, the estrone sulfatase activity in the rest of the intestinal wall did not differ considerably from the values for GF rats, when expressed per mg protein of the homogenate. The mucosa of the CVL rats, however, showed higher rates of hydrolysis than the mucosa of the GF rats. The microbial estrone sulfatase activity in the intestinal content of CVL rats, tested by anaerobic incubation, was high in the caecum (91.7 +/- 6.6% after 4 h), but very low in the PSI (2.2 +/- 0.7%) and DSI (1.3 +/- 0.5%). Serial dilutions of the caecal content also showed higher viable numbers of estrone sulfate hydrolyzing bacteria. These results add further weight to the suggestion that estrone sulfate may be absorbed from the small intestine, but has to be hydrolyzed in the caecum by the gut microflora prior to absorption.     

Influence of microbial bile salt desulfation upon the fecal excretion of bile salts in gnotobiotic rats

The fecal excretion of intraperitoneally injected 24-14C-labeled taurocholate (TCA), taurolithocholate (TLCA) and the respective 3-sulfate esters (TCA-3-S; TLCA-3-S), were compared in germfree (GF) rats, conventional (CV) rats, and in gnotobiotic rats associated with Clostridium Cl-8 or this same strain Cl-8 plus the bile desulfating Clostridium S1, respectively. TCA and TLCA were about two times more rapidly excreted by CV animals than by GF animals; the time required for 50% excretion of total label injected (t 1/2) was 6.6 days vs 14.9 for TCA, and 4.4 vs 8.9 for TLCA. In GF and in CV animals, TCA-3-S and TLCA-3-S were excreted more rapidly than their nonsulfated analogues; the t 1/2 values of TCA-3-S and TCA were 2.7 days vs 14.9 in GF rats, and 3.1 vs 6.6 days in CV animals. The t 1/2 values of TLCA-3-S and TLCA were 2.7 days vs 8.9 in GF rats, and 1.5 vs 4.4 days in CV rats. In gnotobiotic rats associated with Clostridium strains S1 + Cl-8, fecal bile salts were nearly 100% deconjugated and desulfated and the 50% excretion times of TCA-3-S and TLCA-3-S approximated to those of TCA and TLCA in GF animals. T 1/2 of TCA-3-S in gnotobiotic S1 + Cl-8 animals was 12.2 days vs 14.9 for TCA in GF animals. In gnotobiotic S1 + Cl-8 animals the t 1/2 of TLCA and TLCA-3-S was 12.5 and 11.0 days, respectively. These results illustrate clearly the important effect the intestinal microflora has upon the metabolic half-life of bile salts. Moreover, they demonstrate that desulfation of bile salts by the intestinal microflora takes place in intestinal segments from where a certain degree of reabsorption is still possible, and thus point to the fact that microbial desulfation is an important variable in the overall elimination of bile salts.     

pH-dependency of iodothyronine metabolism in isolated perfused rat liver.

1. Isolated livers from fed male rats were perfused for 2 h with T4 (L-thyroxine), T3 (L-3,3',5-tri-iodothyronine) or rT3 (L-3,3',5'-tri-iodothyronine) at different pH values (7.1--7.6) in a fully synthetic medium, whereby normal metabolic functions were maintained without addition of rat blood constituents or albumin. 2. T3 output into the medium and net T3 production reached a maximum at a pH of the medium of 7.2 and significantly decreased with alteration of the pH when livers were perfused with T4 as a substrate. 3. However, the net T4 and T3 uptake by the liver, as well as the hepatic T4 and T3 content after perfusion, were not dependent on the pH of the perfusion when livers were offered T4 or T3 as substrates respectively. 4. Determination of intracellular pH by the analysis of the distribution of the weak acid dimethyloxazolidinedione allows the conclusion that the pH optimum of iodothyronine 5'-deiodinase in the intact perfused liver corresponds to the maximum determined in vitro for the membrane-bound enzyme localized in the endoplasmic reticulum. 5. The rapid 5'-deiodination of rT3 to 3,3'-T2 (L-3,3'-di-iodothyronine), the fast disappearance of 3,3'-T2, and the fact that no net rT3 production from T4 could be detected, supports the hypothesis that in rat liver iodothyronine 5'-deiodinase activity seems to predominate over iodothyronine 5-deiodinase activity. 6. Thus the rat liver can be considered in normal physiological situations as an organ forming T3 from T4 and deiodinating rT3 originating from extrahepatic tissues, whereby the cellular iodothyronine 5'-deiodination rate is controlled by the intracellular pH.     

Hydrolysis of iodothyronine conjugates by intestinal bacteria

Glucuronide and sulfate conjugation are important pathways in the peripheral metabolism of thyroid hormone. These reactions occur predominantly in the liver, and especially the glucuronides are excreted in the bile. Although an enterohepatic circulation after intestinal hydrolysis of iodothyronine conjugates is suggested by several authors, substantial proof has not been presented so far. In the present paper experimental work from our group is reviewed. The studies showed that fecal suspensions of human or rat origin hydrolysed iodothyronine conjugates, whereas oral administration of antibiotics to rats strongly reduced this capacity. Obligately anaerobic intestinal bacteria were found to be responsible for the hydrolysis and several species belonging to the major residents of the intestinal flora of man and rat could be isolated and identified. Recent studies with conventional and decontaminated rats produced strong support for the existence of an enterohepatic circulation of thyroid hormone. Our findings are discussed in connection with other relevant studies on this subject.     

Hydrolysis of iodothyronine conjugates by intestinal bacteria

Abstract Glucuronide and sulfate conjugation are important pathways in the peripheral metabolism of thyroid hormone. These reactions occur predominantly in the liver, and especially the glucuronides are excreted in the bile. Although an enterohepatic circulation after intestinal hydrolysis of iodothyronine conjugates is suggested by several authors, substantial proof has not been presented so far.
In the present paper experimental work from our group is reviewed. The studies showed that fecal suspensions of human or rat origin hydrolysed iodothyronine conjugates, whereas oral administration of antibiotics to rats strongly reduced this capacity. Obligately anaerobic intestinal bacteria were found to be responsible for the hydrolysis and several species belonging to the major residents of the intestinal flora of man and rat could be isolated and identified.
Recent studies with conventional and decontaminated rats produced strong support for the existence of an enterohepatic circulation of thyroid hormone. Our findings are discussed in connection with other relevant studies on this subject.     

Comparative lectin-histochemistry on the pre-epithelial mucus layer in the distal colon of conventional and germ-free rats

The mucin composition of the rat distal colonic pre-epithelial mucus layer (PML) was studied by lectin histochemistry in conventional (CV), and germ-free (GF) rats to define effects exerted by the gut flora. No peanut agglutinin (PNA) binding was observed in the PML of GF rats, while the PML of their CV counterparts showed a considerable PNA linkage, indicating terminal Gal-beta1,3-GalNAc residues. Soybean agglutinin (SBA) and Helix pomatia agglutinin (HPA) stained the PML mucins in CV and in GF rats, indicating terminal GalNAc moieties. A quantitative difference in the Limax flavus agglutinin (LFA) binding capacity was found between CV and GF rats, indicating terminal sialic acid moieties: the staining intensity of bound LFA/ FiTC was higher in CV rats than in GF rats. No linkage of Datura stramonium agglutinin (DSA) and of wheat germ agglutinin (WGA) was found in the PML of GF rats, indicating the absence of terminal GlcNAc, while in CV rats, a clearly marked border was visible next to the luminal content as a "nipple edge" when stained with DSA or WGA. Canavalia ensiformis agglutinin (ConA), indicative for branched mannose, stained PML mucins and goblet cell mucins of GF rat distal colon. In CV rats, both locations were free of ConA binding sites. These results suggest degrading effects, exerted by the gut flora on the rat colonic pre-epithelial mucus layer.     

Differences in development of lymphocyte subpopulations from gut-associated lymphatic tissue (GALT) of germfree and conventional rats: Effect of aging

The aim of the study was to compare the phenotype of lymphocyte subpopulations of the GALT (gut-associated lymphatic tissue) in germfree (GF) and conventionally (CV) reared rats,i.e. to analyze the effect of microbial colonization on the development of intestinal lymphocyte subsets. Surface marker characteristics were studied in cell suspensions isolated from Peyer’s patches, mesenteric lymph nodes, spleen and the intraepithelial lymphocyte compartment of 2- and 12-month old inbred AVN rats. The pattern of T lymphocyte phenotypes in Peyer’s patches, mesenteric lymph nodes and spleen determined by FACS analysis did not reveal differences between GF and CV rats. In contrast, a 2-month conventionalization of GF rats led to substantial changes in the composition of intestinal intraepithelial lymphocyte subsets (IELs): increase of CD4⁺, CD8α⁺, CD8β⁺, TcR α/β⁺ bearing lymphocytes was observed after colonization of rats with normal microflora. Surprisingly, the relative numbers of lymphocytes bearing TcR γ/δ⁺ did not change during conventionalization. The effect of aging was also studied and differences in IELs composition of aged (GF) and (CV) rats were found to be more pronounced: 6,6% and 30% of lymphocytes bearing TcR α/β were present among IELs in two-month old GF and CV rats, respectively. 30% of IELs in 2-month old GF rats, 80% of IEL from 12-month old CV rats were found to bear TcR α/β. This finding demonstrates that during conventionalization and aging the TcR α/β bearing population of IELs substantially expands. It suggests that mainly this lymphocyte subset responds to microflora stimuli and is probably involved in the protection of the epithelial cell layer of intestinal mucosa.     

Glucose tolerance, insulin and catecholamine levels in germfree rats.

Glucose was administered intravenously to 50- and 100-day-old GF and CV rats. Fasting blood glucose levels in GF and CV rats were found to be comparable. Glucose tolerance tests showed that GF and CV rats clear glucose from the blood at a similar rate. Although insulin concentrations in 100-day-old GF rats tended to be somewhat lower than in CV rats, the percentage increase during the 30-min period after glucose administration was similar, and matched the increase in blood glucose. Levels of plasma catecholamines were analyzed fluorometrically and were found to be comparable in 100-day-old GF and CV rats. It was concluded that insulin insufficiency plays no role in the syndrome of metabolic anomalies demonstrated by the germfree rat.     

Thyroid status in the obese syndrome of rats

The thyroid function was explored by comparing serum total and free iodothyronine levels in young male genetically obese Zucker rats and in their lean littermates, aged from 6 to 8 weeks old. Total and free thyroxine (T4) and 3,5,3'triiodothyronine (T3) levels were significantly decreased in obese rat serum while total 3,3',5'-triiodothyronine (rT3) remained constant. Radioactive T4 half life is slower in the plasma of obese rats. Peripheral synthesis of T3 from deiodination of T4 is also decreased in obese rat liver homogenate. These modifications produce changes in liver mitochondria oxidative phosphorylation and in marker enzyme activity, which are usually associated with hypothyroidism and hypothalamic disturbances. Genetic obesity probably involves activation of peripheral deiodination of T4 to rT3 which induces biochemical and metabolic changes.     

Hydrolysis of guanosine and adenosine 3',5'-monophosphates by rat blood.

Cyclic nucleotide phosphodiesterase activity was measured in whole blood, plasma, and suspensions of platelets and erythrocytes from rats. In fresh whole blood, apparent phosphodiesterase activity was low, but it rose strikingly during the hour after blood withdrawal. The apparent phosphodiesterase activity in platelet-free plasma showed no such increase, but that in platelet-enriched plasma increased in parallel with that in whole blood. The apparent phosphodiesterase activity of blood or of platelet-enriched plasma also was increased markedly by sonication. The increase in rat blood phosphodiesterase activity with aging thus appeared to be due to damage of platelets. Most of the phosphodiesterase activity in rat erythrocytes and platelets was located in the soluble fraction of sonicated preparations, but the total enzyme activities from the two sources exhibited marked differences in substrate specificity. With erythrocyte preparations, the rate of hydrolysis of muM concentrations of cyclic AMP was approx. 50 times that of cyclic GMP, while with platelet preparations, cyclic GMP was hydrolyzed about 20 times faster than cyclic AMP at muM levels. The activity of phosphodiesterase in platelets was much greater than that in erythrocytes at all concentrations of both substrates.     

Iodothyronine sulfate-hydrolyzing anaerobic bacteria isolated from human fecal flora

Abstract 23 Bacterial strains isolated from human fecal flora were screened for hydrolysis of iodothyronine sulfates. Three obligate anaerobic bacterial strains possessed sulfatase activity. Two strains were identified as Peptostreptococcus productus , the other strain probably belonged to the genus Eubacterium or Lachnospira . In anaerobic incubations with growing bacteria up to 78% of the iodothyronine sulfates were deconjugated in 24 h. Hydrolysis was dependent on the bacterial strains and on the iodothyronine sulfates tested. This study extents previous observations of similar iodothyronine sulfatase activities associated with bacteria from rat intestinal microflora [5]. By analogy, hydrolysis of iodothyronine sulfates by anaerobic bacteria from human intestinal microflora probably represents an exo-enzymatic process.     

哺乳动物硒蛋白的研究进展

硒是哺乳动物和人必需的微是元素。硒的生物学功能主要是以硒蛋白的形式表现的。到目前为止,已经克隆并测定ｃＤＮＡ顺序的哺乳动物硒蛋白有９种停,它们是细胞内谷胱甘肽过氧化物酶、细胞外谷胱甘肽过氧化物酶、磷脂氢谷胱甘肽过氧化物酶、胃肠谷胱甘肽过氧化物酶、Ｉ型碘化甲状腺原氨酸５′脱碘酶、Ⅱ型碘化甲状腺原氨酸５′脱磺酶、Ⅲ型碘化甲状腺原氨酸５′脱碘酶、硒蛋白Ｐ和硒蛋白Ｗ。这些硒蛋白中硒参入到蛋白分子是通过硒半     

Iodothyronine 5'-deiodinase activity in rat brown adipose tissue during development

Iodothyronine 5'-deiodinase activity in rat brown adipose tissue has a characteristic pattern of developmental changes that is completely different from that of the liver. Fetal brown fat exhibits an extremely high iodothyronine 5'-deiodinase activity that is approx. 10-fold that in adult rats. Even though brown fat iodothyronine 5'-deiodinase activity falls suddenly at birth, there is a new peak in the activity around days 5-7 of life, whereas it remains very low afterwards. Just after birth, brown adipose tissue iodothyronine 5'-deiodinase activity is already capable of stimulation by noradrenaline. The postnatal peak in brown fat iodothyronine 5'-deiodinase correlates with the known increase in the thermogenic activity of the tissue in the neonatal rat, thus reinforcing the suggestion that local 3',3,5-triiodothyronine generation could be an important event related to thermogenesis in brown adipose tissue. However, the high fetal activity was only slightly related to the thermogenic activity of brown fat. Moreover, the increased iodothyronine 5'-deiodinase activity of brown adipose tissue during fetal and neonatal life suggests a substantial contribution by brown fat in the overall extrathyroidal 3',3,5-triiodothyronine production in these physiological periods.     

Influence of intestinal bacterial desulfation on the enterohepatic circulation of dehydroepiandrosterone sulfate

Selective association of germ-free (GF) rats with dehydroepiandrosterone sulfate (DHEAS) desulfating bacteria allowed us to assess the exact impact of intestinal bacterial desulfation on the excretion and enterohepatic circulation of orally administered DHEAS. Germ-free rats selectively associated with the DHEAS-desulfating strain Peptococcus niger H4 (H4 rats) excreted 50% of the total label recovered within 17 h vs 21 h in GF rats and 13 h 23 min in conventional (CV) rats. Germ-free rats excreted 30% of the total label recovered via their urine. However, association of GF rats with the desulfating microorganism increased urinary excretion to 46%, comparable to the 45.5% found in CV rats. Fractionation of fecal label yielded 70% sulfoconjugated DHEAS and 2% unconjugated dehydroepiandrosterone in GF rats vs 5 and 77% in CV rats, and 55 and 14% in H4 rats, respectively. Our results demonstrate that the intestinal bacterial desulfation of DHEAS stimulated the enterohepatic circulation of DHEAS. This in turn increased the urinary excretion of label resulting in an accelerated elimination of labeled DHEAS from the body.     

Acid Hydrolysis of Wheat Gluten Induces Formation of New Epitopes but Does Not Enhance Sensitizing Capacity by the Oral Route: A Study in “Gluten Free” Brown Norway Rats

Background

Acid hydrolyzed wheat proteins (HWPs) are used in the food and cosmetic industry as emulsifiers. Cases of severe food allergic reactions caused by HWPs have been reported. Recent data suggest that these reactions are caused by HWPs produced by acid hydrolysis.

Objectives

To examine the sensitizing capacity of gluten proteins per se when altered by acid or enzymatic hydrolysis relative to unmodified gluten in rats naïve to gluten.

Methods

High IgE-responder Brown Norway (BN) rats bred on a gluten-free diet were sensitized without the use of adjuvant to three different gluten products (unmodified, acid hydrolyzed and enzymatic hydrolyzed). Rats were sensitized by intraperitoneal (i.p.) immunization three times with 200 µg gluten protein/rat or by oral dosing for 35 days with 0.2, 2 or 20 mg gluten protein/rat/day. Sera were analyzed for specific IgG and IgE and IgG-binding capacity by ELISA. IgE functionality was measured by rat basophilic leukemia (RBL) assay.

Results

Regardless of the route of dosing, all products had sensitizing capacity. When sensitized i.p., all three gluten products induced a strong IgG1 response in all animals. Acid hydrolyzed gluten induced the highest level of specific IgE but with a low functionality. Orally all three gluten products induced specific IgG1 and IgE but with different dose-response relations. Sensitizing rats i.p. or orally with unmodified or enzymatic hydrolyzed gluten induced specific IgG1 responses with similar binding capacity which was different from that of acid hydrolyzed gluten indicating that acid hydrolysis of gluten proteins induces formation of ‘new’ epitopes.

Conclusions

In rats not tolerant to gluten acid hydrolysis of gluten enhances the sensitizing capacity by the i.p. but not by the oral route. In addition, acid hydrolysis induces formation of new epitopes. This is in contrast to the enzymatic hydrolyzed gluten having an epitope pattern similar to unmodified gluten.     

Iodothyronine 5′-deiodinase activity in rat brown adipose tissue during development

Iosothyronine 5′-deiodinase activity in rat brown adipose tissue has a characteristic pattern of developmental changes that is completely different from that of the liver. Fetal brown fat exhibits an extremely high iodothyronine 5′-deiodinase activity that is approx. 10-fold that in adult rats. Even though brown fat iodothyronine 5′-deiodinase activity falls suddenly at birth, there is a new peak in the activity around days 5–7 of life, whereas it remaines very low afterwards. Just after birth, brown adipose tissue iodothyronine 5′-deiodinase activity is already capable of stimulation by noradrenaline. The postnatal peak in brown fat iodothyronine 5′-deiodinase correlates with the known increase in the thermogenic activity of the tissue in the neonatal rat, thus reinforcing the suggestion that local 3′,3,5-triiodothyronine generation could be an important event related to thermogeneis in brown adispose tissue. However, the high fetal activity was only slightly related to the thermogenic activity of brown fat. Moreover, the increased iodothyronine 5′-deiodinase activity of brown adipose tissue during fetal and neonatal life suggests a substantial contribution by brown fat in the overall extrahydroidal 3′,3,5-triiodothyronine production in these physiological periods.     

Growth rate of male germfree Wistar rats fed ad libitum or restricted natural ingredient diet

Germfree life and feed restriction initiated at an early age are known to extend life span. We have examined growth rate and life expectancy in germfree (GF) and conventional (CV) male Lobund Wistar rats, fullfed (F) or restricted (R) to 12 grams/day of natural ingredient diet L-485. GF-F and CV-F rats show comparable growth rates during the first 6 months of life. Thereafter, the GF-F rat falls behind, with its body weight stabilizing at 85% of the CV rat's weight at 2 years of age (510 g vs 435 g). In contrast, GF-R rats become slightly, but significantly, heavier than CV-R rats after an initial 6 months of comparable growth. At 2 years of age GF-R rats weigh 12% more than the CV-R rats (340 g vs 300 g). Physiological parameters were examined in each treatment group in animals that had to be sacrificed because of contamination. These gnotobiotic (GN) rats (see text) and their CV counterparts were grouped as adult (7 to 11 months) and old (18 to 28 months) rats. The most significant findings were: GN-F rats have smaller hearts than CV-F rats, both on an absolute and relative basis; restriction did not affect absolute testes size but elevated serum testosterone levels; serum T4 was reduced by restriction only in CV rats, and declined with age in all groups; and serum T3 was higher in adult GN-F and GN-R rats, but fell to CV levels in old age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intestinal mucosal morphometry and ileal epithelial renewal in conventional and germ-free rats fed an amylomaize starch diet.

Intestinal mucosal morphometry and ileal epithelial renewal were studied in conventional (CV) and germ-free (GF) rats fed either poorly digestible amylomaize or normal maize starch diets. Intestinal morphometry and position of labelled enterocytes were studied at various times after tritiated thymidine injection. With amylomaize starch diet, no difference was observed in the size of crypts (C), villi (V) and C + V between duodenum and jejunum both in CV and GF rats. In the ileum, however, values were significantly lower than those in the duodenum and jejunum. Furthermore, the presence of the microbial flora led to higher values when compared with GF values. Despite the morphological modifications in the ileum, no significant difference was detected in the labelled cell positions and epithelial renewal time between CV and GF values. This suggests that the resistant part of amylomaize starch was responsible for the modification in mucosal morphometry and the longer ileal epithelium renewal time in CV rats which then becomes similar to that in GF rats.