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.

Purification and characterization of N-acetylmuramyl-L-alanine amidase from human plasma using monoclonal antibodies.

N-Acetylmuramyl-L-alanine amidase (EC 3.5.1.28) cleaves the amide bond between N-acetyl muramic acid and L-alanine in the peptide side chain of different peptidoglycan products. The enzyme was purified from human plasma using a three-step column chromatography procedure. Monoclonal antibodies were produced against the purified human enzyme. By coupling of a high affinity monoclonal antibody to sepharose beads an immunoadsorbent column was prepared. Using this second purification method it was possible to purify large amounts of the amidase from human plasma in a single step. SDS-PAGE showed one single band of 70 kDa and two-dimensional electrophoresis showed the presence of multiple isomeric forms of the protein with pI between 6.5 and 7.9. Two different methods were used for determination of substrate specificity, a HPLC method separating peptidoglycan monomers from the reaction products after incubation with amidase and a colorimetric method when high molecular weight peptidoglycan was used as a substrate for amidase. It is shown that the disaccharide tetra peptide, disaccharide penta peptide and the anhydro disaccharide tetrapeptide are good substrates for the amidase and that muramyl dipeptide and disaccharide dipeptide are not a substrate for the amidase. Using one of the monoclonal antibodies against the amidase it was shown in FACScan analysis that N-acetylmuramyl-L-alanine amidase is present in granulocytes but not in monocytes from unstimulated peripheral blood of a healthy donor. The presence of N-acetylmuramyl-L-alanine amidase in granulocytes is a novel finding and perhaps important for the inactivation of biologically active peptidoglycan products still present after hydrolysis by lysozyme.

Effects of inhibition of type I iodothyronine deiodinase and phenol sulfotransferase on the biliary clearance of triiodothyronine in rats.

Recent studies using isolated rat hepatocytes have indicated that the bioactive form of thyroid hormone, T3, is metabolized in liver predominantly by conjugation with glucuronic acid or sulfate. In contrast to T3 itself and the stable glucuronide, T3 sulfate is rapidly degraded by successive deiodination of the tyrosyl and phenolic rings. In the present study we have investigated the biliary excretion of T3 metabolites in male Wistar rats under pentobarbital anesthesia. The animals were injected iv with 1) saline, 2) the deiodinase inhibitor propylthiouracil (PTU; 1 mg/100 g BW), 3) the phenol sulfotransferase inhibitor dichloronitrophenol (2.6 mumol/100 g BW), or 4) a combination of both drugs. After 15 min, 10 muCi [125I]T3 were administered iv, and bile was collected for 30-min periods until 4 h after tracer injection. Secretory products were analyzed by HPLC. In control animals, 22.4% of the dose was excreted in bile mainly in the form of T3 glucuronide. In PTU-treated rats biliary excretion was increased to 36.0% of the dose (P less than .001) due to a dramatic increase in the sulfates of T3 and 3,3'-diiodothyronine. Dichloronitrophenol by itself had no effect on the biliary clearance of T3, but greatly inhibited PTU-induced excretion of sulfates. These results strongly suggest that sulfation and subsequent deiodination is an important pathway of T3 metabolism in vivo.

Enterohepatic circulation of triiodothyronine (T3) in rats: importance of the microflora for the liberation and reabsorption of T3 from biliary T3 conjugates.

In normal rats, T3 glucuronide (T3G) is the major biliary T3 metabolite, but excretion of T3 sulfate (T3S) is greatly increased after inhibition of type I deiodinase, e.g. with 6-propyl-2-thiouracil (PTU). In this study, the fate of the T3 conjugates excreted with bile was studied to assess the significance of a putative enterohepatic circulation of T3 in rats. Conventional (CV) or intestine-decontaminated (ID) rats received iv [125I]T3G or [125I]T3S, the latter usually after pretreatment with PTU (1 mg/100 g BW). Radioactivity in plasma and bile or feces was analyzed by Sephadex LH-20 chromatography and HPLC. Within 1 h, 88% of injected T3G was excreted in bile of CV or ID rats, independent of PTU. About 75% of the injected T3S was excreted within 4 h in PTU-treated rats, in contrast to only 20% in controls. Up to 13 h after iv administration of T3G or T3S (+PTU) to intact ID and CV rats, fecal radioactivity consisted of more than 90% T3 in all CV rats, 95% of T3S in T3S-injected ID rats, and 30% T3 and 67% T3G in T3G-injected ID rats. In overnight-fasted CV rats injected with T3G, total plasma radioactivity rapidly declined until a nadir of 0.10% dose/ml at about 2.5 h, but radioactivity reappeared with a broad maximum of 0.12% dose/ml between 5.5-10 h. In the latter phase, plasma radioactivity consisted of predominantly I- and T3 in a ratio of 2:1. Reabsorption was diminished in fed CV rats and prevented in ID rats. Plasma T3 4-10 h after iv T3G injection to overnight-fasted CV rats was 12, 2, and 3 times higher than that in bile-diverted rats, fed CV rats, and ID rats, respectively, and similar to that 4 h after the injection of T3 itself. Total plasma radioactivity as well as plasma T3 6-13 h after iv administration T3S in PTU-treated rats were significantly increased in CV vs. ID rats, e.g. T3 0.016% vs. 0.005% dose/ml. These results demonstrate a significant enterohepatic circulation of T3 in rats in which bacterial hydrolysis of T3 conjugates excreted with bile plays an important role.

Humoral immune response against Campylobacter jejuni lipopolysaccharides in Guillain-Barré and Miller Fisher syndrome.

In this study we characterized the IgG antibodies against lipopolysaccharides (LPS) of Campylobacter jejuni in serum from patients with Guillain-Barré syndrome (GBS), Miller Fisher syndrome (MFS), C. jejuni enteritis and normal controls. In patients with GBS and MFS long-lasting titers of IgG1 and IgG3 antibodies against LPS from GBS and MFS associated C. jejuni were found. The subclass and course of these antibodies were highly associated with those of antibodies against GM1 and GQ1b in GBS and MFS patients. However, in C. jejuni enteritis and normal controls anti-LPS antibodies were predominantly IgG2. Antibody binding with LPS was reduced after treatment with choleratoxin and sialidases, suggesting that the ganglioside-like epitopes in LPS are immunodominant. These results further indicate that antecedent C. jejuni infections determine the specificity and isotype of anti-ganglioside antibodies in GBS and MFS patients.

In vitro and in vivo studies of substance P receptor expression in rats with the new analog [indium-111-DTPA-Arg1]substance P.

UNLABELLED: We evaluated the potential usefulness of a new radiolabeled substance P (SP) analog, [111In-DTPA-Arg1]SP, as a radiopharmaceutical for the in vivo detection of SP receptor-positive (SPR+) immunologic disorders (i.e., inflammatory bowel disease and arthritis) and tumors (i.e., carcinoid). METHODS: Substance P, [DTPA-Arg1]SP and [3-(p-hydroxyphenyl)propionyl-Arg1]SP (Bolton-Hunter-SP, [BH-SP]) were tested as competitors for 125I-BH-SP to SPR in rat brain cortex membranes. An autoradiographic displacement study of the submandibular gland of the rat with the 125I-BH-SP as radioligand and [DTPA-Arg1]SP as competitor was performed. Tissue distribution and ex vivo autoradiography were studied in rats, with and without pretreatment with the selective nonpeptide antagonist CP96,345 to quantify specific binding. In vivo metabolism of [111In-DTPA-Arg1]SP was performed in control rats. Gamma-camera scintigraphic studies were carried out with control rats to visualize the SPR+ salivary glands in rats bearing the SPR+ transplantable pancreatic tumor CA20948 and in rats with SPR+ adjuvant arthritic joints, which was induced after injection of a homogenate of Mycobacterium tuberculosis. RESULTS: Substance P, [DTPA-Arg1]SP and BH-SP dose-dependently inhibited binding of 125I-BH-SP to SPR in rat brain cortex membranes with IC50 values of 0.2, 4 and 2 nM, respectively. In an autoradiographic displacement study of the submandibular gland with 125I-BH-SP as radioligand, an IC50 of 2.7 nM was found for [DTPA-Arg1]SP. In vivo metabolism of the radiopharmaceutical in the rat revealed a renal clearance rate of 50% of the injected radioactive dose in 30 min and a rapid enzymatic degradation of the radiopharmaceutical, resulting in an effective half-life of the intact radiopharmaceutical in blood of approximately 3 min. Tissue distribution and ex vivo autoradiographic studies in rats showed uptake and specific binding of radioactivity in isolated tumors and submandibular and parotid glands. Optimum SPR+ target-to-background ratios were found 24 hr after injection of [111In-DTPA-Arg1]SP. Visualization of normal SPR+ tissues, such as the salivary glands by gamma camera scintigraphy, after administration of [111In-DTPA-Arg1]SP was demonstrated in untreated rats. Pathological SPR+ processes were visualized both in rats bearing the transplantable pancreatic tumor CA20948 and in those with adjuvant mycobacteria tuberculosis-induced arthritic joints. CONCLUSION: [Indium-111-DTPA-Arg1]SP can be used successfully to visualize SPR+ processes in vivo by gamma camera scintigraphy.

Somatostatin receptor subtype expression in cells of the rat immune system during adjuvant arthritis.

Somatostatin is a neuropeptide that is widely distributed throughout the body. It acts as a neurohormone and a neurotransmitter and may also have an immunomodulatory role. The genes for five subtypes of somatostatin receptors (sst) have been cloned, suggesting that the diverse effects of the peptide might be mediated by different receptors. We are interested in studying the role of sst ininflammation, using an animal model. Because of the up-regulation of sst expression in inflamed joints in human rheumatoid arthritis, we chose rat adjuvant arthritis as an experimental model. In order to determine which of the sst subtypes might be important in immune modulation, subtype expression in leukocytes isolated from different lymphoid tissues of the rat was studied. Also, the expression levels of the most abundantly expressed sst mRNAs in leukocytes from spleen and blood were compared in rats with adjuvantarthritis and controls, using a semi-quantitative approach. Furthermore, the effect of systemic administration of a long-acting somatostatin analogue, octreotide, which binds selectively to sst subtypes 2 and 5 (sst2 and sst5), on the incidence and the severity of rat adjuvant arthritis, was studied. The main sst expressed in cells of the rat immune system, both resting and activated, were found to be sst3 and sst4. This contrasts with the human and murine situations, in which sst2 appears to be the main subtype expressed in the immune system. No quantitative differences in sst subtype mRNA levels in leukocytes from spleen and blood were found between rats with adjuvant arthritis and controls. Finally, no effect of systemic administration of octreotide on either the incidence or severity of adjuvant arthritis in Lewis rats was found. As octreotide binds selectively to sst2 and sst5, the absence of an immunomodulatory effect of this analogue in rat adjuvant arthritis corroborates our finding that these sst subtypes are not expressed in cells of the rat immune system. In conclusion, cells of the rat immune system appear to express a spectrum of sst (sst3 and sst4) different from that found in human granulomatous and autoimmune disease (mainly sst2). Therefore, the rat adjuvant arthritis model appears to be suitable only for studying the immunomodulatory effects of somatostatin analogues which have a high affinity for sst3 and sst4, but not for studying the immunomodulatory effects of octreotide, which has a high affinity only for sst2 and sst5.

Background immunoglobulin-secreting cells specific for intestinal peptidoglycan-polysaccharides in mice.

Natural antibodies to soluble peptidoglycan-polysaccharide complexes (PPC) from the human intestinal flora have been found in mammalian sera. In this study the occurrence and frequency of PPC-specific immunoglobulin-secreting cells (anti-PPC Ig-SC) were determined in lymphoid organs of normal (C57BL x CBA)F1 mice. One out of 100 IgM-SC in the spleen and Peyer's patches was found to be specific for PPC. In the small intestine a small number of anti-PPC IgA-SC were present probably responsible for IgA secretion in the gut lumen since very low serum concentrations anti-PPC IgA were found. Anti-PPC IgG-SC were not detected, although some anti-PPC IgG was found in the serum. It is concluded that the spleen is the major lymphoid organ responsible for the production of natural antibodies to PPC.

Natural antibodies to 65 kD mycobacterial heat shock protein in rats do not correlate with susceptibility for Mycobacterium tuberculosis induced adjuvant arthritis.

Natural antibodies to 65 kD heat shock protein (hsp65) of Mycobacterium bovis were found in the sera of Lewis rats. The levels of these natural hsp65 antibodies differed substantially between the individual rats. Each rat was subsequently tested for its susceptibility to develop arthritis following injection of M. tuberculosis in incomplete Freund adjuvant. It was found that the incidence and severity of the induced arthritis did not differ between groups of Lewis rats with relatively high and relatively low natural antibody levels to hsp65. Inoculation of rats without natural antibodies to hsp65 with intestinal contents did not induce hsp65 antibodies, although the rats were able to respond to the antigen.

Are intestinal bacteria involved in the etiology of rheumatoid arthritis? Review article.

Observations in bowel-related joint diseases give support to this hypothesis. In Crohn's disease and ulcerative colitis, the bowel wall inflammation is complicated in about 20% of the patients by joint inflammation. Bowel infection by Salmonella, Shigella and Yersinia can provoke joint inflammation and supports an etiological link between bowel bacteria and arthritis. The arthropathic properties of the most abundant group of intestinal bacteria, i.e. the obligate anaerobic bacteria, were studied in an animal model. Cell wall fragments (CWF), with peptidoglycan as the major component, from some Eubacterium and Bifidobacterium species induced a severe chronic polyarthritis in Lewis rats after a single intraperitoneal injection. Eubacterium was found in numbers of 10(8)-10(9) per gram in stools of healthy subjects and rheumatoid arthritis (RA) patients. CWF of isolated strains of E. aerofaciens were arthropathic. Soluble peptidoglycan polysaccharide complexes (PG-PS) originating from the obligate anaerobic flora were purified from human intestinal contents. PG-PS from ileostomy fluid that proved to be less processed by intestinal enzymes induced chronic arthritis in rats after a single administration in oil in the base of the tail. It was concluded that the human intestinal bowel contains soluble bacterial cell wall products that are arthropathic in an animal model. Peptidoglycan (PG) or its subunits was reported to be present in mammalian tissues. Immunohistochemical studies from our group showed the presence of intestinal PG-PS in sections of normal rat spleen. Bacterial cell wall or PG-induced joint inflammation in rats is proven to be absolutely dependent on functional T cells. T-cell lines were isolated from the lymph nodes of rats with an E. aerofaciens CWF arthritis. A helper T-cell line B13 was in vivo arthritogenic in knee or ankle joints upon intravenous injection in rats and proliferated in vitro on syngeneic spleen cells alone, but was additionally stimulated by intestinal PG-PS and E. aerofaciens CWF. It was postulated that the arthritogenic T cells that seem to be autoreactive are, in fact, recognizing bacterial PG-PS on antigen-presenting cells (APC). It is generally accepted that RA is a T-cell-dependent process and that therefore the reaction is directed at small peptides bound by the major histocompatibility complex of APC. The only peptides present in arthritis inducing intestinal PG-PS and in CWF are PG peptides interlinking the sugar chains. We feel that the immunoreaction against PG peptides plays a pivotal role in experimental and human arthritis of an unknown etiology.

Inflammatory properties of peptidoglycan are decreased after degradation by human N-acetylmuramyl-L-alanine amidase.

Human N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28) degrades peptidoglycan, a major component of bacterial cell walls with potent pro-inflammatory cytokine-inducing properties. We postulate that degradation of peptidoglycan by N-acetylmuramyl-L-alanine amidase is important for the inactivation of inflammatory peptidoglycan products in human tissues. The inflammatory activities of peptidoglycan digested by lysozyme and/or amidase were investigated using two properties of peptidoglycan: its capacity to induce the release of the inflammatory cytokines IL-1, IL-6 and TNF-alpha in vivo and in vitro and its capacity to induce arthritis in Lewis rats. The results show that after subsequent treatment with both lysozyme and amidase, the peptidoglycan products were unable to induce arthritis in Lewis rats. The production of pro-inflammatory cytokines in mice after intravenous injection of cell wall fragments was lower after in vitro degradation of the cell wall fragments by amidase. These in vivo results were confirmed with whole blood assays in which the production of pro-inflammatory cytokines was measured after stimulation with lysozyme- and amidase-treated peptidoglycan. The results show that human N-acetylmuramyl-L-alanine amidase possesses an enzymatic activity capable of inactivating inflammatory peptidoglycan by lowering its cytokine-inducing properties.

On the enterohepatic cycle of triiodothyronine in rats; importance of the intestinal microflora.

Until 70 h after a single iv injection of 10 uCi [125I]triiodothyronine (T3), normal rats excreted 15.8 +/- 2.8% of the radioactivity with the feces and 17.5 +/- 2.7% with the urine, while in intestine-decontaminated rats fecal and urinary excretion over this period amounted to 25.1 +/- 7.2% and 23.6 +/- 4.0% of administered radioactivity, respectively (mean +/- SD, n = 4). In fecal extracts of decontaminated rats 11.5 +/- 6.8% of the excreted radioactivity consisted of T3 glucuronide (T3G) and 10.9 +/- 2.8% of T3 sulfate (T3S), whereas no conjugates were detected in feces from normal rats. Until 26 h after ig administration of 10 uCi [125I]T3, integrated radioactivity in blood of decontaminated rats was 1.5 times higher than that in normal rats. However, after ig administration of 10 uCi [125I]T3G or [125I]T3S, radioactivity in blood of decontaminated rats was 4.9- and 2.8-fold lower, respectively, than in normal rats. The radioactivity in the serum of control animals was composed of T3 and iodide in proportions independent of the tracer injected, while T3 conjugates represented less than 10% of serum radioactivity. These results suggest an important role of the intestinal microflora in the enterohepatic circulation of T3 in rats.

Assay for N-acetylmuramyl-L-alanine amidase in serum by determination of muramic acid released from the peptidoglycan of Brevibacterium divaricatum.

A method is reported for the determination of N-acetylmuramyl-L-alanine amidase in serum. Muramic acid, released from the interpeptide bridges of Brevibacterium divaricatum peptidoglycan, is measured by a modified colorimetric method. Using this procedure, it was possible to determine N-acetylmuramyl-L-alanine amidase in aliquots of less than 10 microliters human serum with an incubation time of 10 min. Amidase activity was found in all the sera tested (n = 11). The relevance of this simple and fast assay is discussed.

Selective decontamination and the anaerobic faecal flora.

Selective decontamination with non-absorbable antibiotics of the gastrointestinal tract of patients with impaired host defense is increasingly applied to prevent infections with gram-negative facultatively anaerobic rods. In vivo experiments on the effect of antibiotics on the human intestinal flora were performed with (ex-germ-free) mice. The experiments have shown that the human obligately anaerobic flora did not change during selective decontamination with polymyxin B, but Enterobacteriaceae were eliminated. The magnitude of differences between the sensitivity to antibiotics of the human obligately anaerobic flora and the Enterobacteriaceae in vitro may be used as an index for the usefulness of the antibiotic for selective decontamination. Binding of the antibiotics to intestinal contents has been found to be important for the estimation of the daily dose. The release of the bound antibiotic could contribute to the maintenance of equable concentrations in the intestine.

Effect of a soluble bacterial carbohydrate fraction on the viscosity of intestinal contents in healthy subjects and patients with Crohn's disease.

Supernatants of faecal suspensions from patients with Crohn's disease (CD) showed much lower viscosity than those from healthy subjects. Material responsible for the viscosity could be precipitated with ethanol. Gel filtration indicated that the viscosity was not due to the glycoprotein fraction but to a fraction with higher molecular weight and relatively high contents of muramic acid suggesting a bacterial origin. The concentration and viscosity of this fraction are less in faeces from patients with CD than in that of healthy subjects.

The obligate anaerobic faecal flora of patients with Crohn's disease and their first-degree relatives.

The obligate anaerobic faecal floras of patients with Crohn's disease, their first-degree relatives, and healthy control subjects were compared. The flora of Crohn's patients contained more anaerobic gram-positive coccoid rods and gram-negative rods than that of healthy subjects; on this basis patients and healthy subjects formed two clusters with minor overlap. Nine of 26 children of Crohn's patients were also included within the Crohn's disease cluster. During 5 to 7 years of follow-up study 3 of them presented with remitting abdominal pain, diarrhoea, or weight loss, and in 1 of them Crohn's disease was diagnosed; none of the 17 children with a normal flora showed symptoms possibly due to Crohn's disease. It is concluded that the abnormal flora may be indigenous to subjects predisposed to Crohn's disease, suggesting a direct or indirect relationship between the abnormal faecal flora and Crohn's disease.

Cell wall fragments from major residents of the human intestinal flora induce chronic arthritis in rats.

To investigate the involvement of human intestinal flora in joint inflammation, cell wall fragments of 9 anaerobic gram positive bacteria of the human fecal flora were prepared and tested for arthropathic properties in the rat. A single intraperitoneal injection of cell wall fragments from Eubacterium aerofaciens or Bifidobacterium species induced persistent chronic arthritis, in contrast to those from Eubacterium rectale, Clostridium species and Lactobacillus leichmanii. The results show that cell wall fragments of major residents from the human fecal flora can induce chronic arthritis in the rat and support the hypothesis that normal human intestinal flora plays a role in the induction of arthritis in man.

Regulation of the bacterial microflora of the vagina in cyclic female rats.

The bacterial microflora was examined in the vagina of cyclic female rats kept under normal laboratory conditions. Large variations occurred during the cycle with high numbers of bacteria (10(5)-10(8) per vagina) during proestrus and estrus and low numbers (10(1)-10(4) per vagina) during the diestrus period. Histological analysis of in situ vaginal tissue and transplanted vaginal tissue revealed an association of high bacterial numbers with the presence of large amounts of cellular debris in the vaginal lumen during the period of epithelial keratinization. Absence of phagocytosis in leucocytes at mestestrus suggested that leucocytes did not play an active role in reduction of bacterial numbers between estrus and metestrus. Accurate measurement of the pH in the vaginal lumen failed to reveal differences which could explain the reduction in bacterial numbers between estrus and metestrus. The cyclic changes in the bacterial population-consisting of species which are normally present in the intestinal flora-- seem to be controlled by cyclic changes in the amounts of cellular debris in the vaginal lumen.

Immunohistology of joint inflammation induced in rats by cell wall fragments of Eubacterium aerofaciens.

After a single intraperitoneal injection of cell wall fragments of Eubacterium aerofaciens, a main resident from the human intestinal flora, an acute arthritis develops within 2 days which is followed by a chronic arthritis that lasts at least 90 days. In an earlier report the histological appearance of the joint inflammation during this period has been described. In this study we investigated in more detail the cell types that are involved in the development of arthritis by using cell-type-specific monoclonal antibodies in an immunohistological assay. In the acute phase of arthritis, T-helper cells appeared in the synovial tissue together with ED1-positive (ED1+) and ED3-positive (ED3+) macrophages. After a temporary decline at day 12 all macrophage subsets, as well as T-helper cells, reappeared or increased again at day 33. Later, in the chronic phase (days 47-90), an increased number of ED1-positive (ED1+) cells in the synovial tissue and a decreased number of ED2-positive (ED2+) cells in the synovial lining was the most prominent finding when compared with control rats. These results indicate that, apart from T lymphocytes, macrophages also play an important role in the development and continuation of chronic arthritis in this model.

Binding to and antibacterial effect of aztreonam, temocillin, gentamicin and tobramycin on human faeces.

Aztreonam, temocillin, gentamicin and tobramycin were studied for their effect on the human faecal flora in vitro and for their usefulness for selective decontamination (SD) of the gastrointestinal tract. The sensitivities of the obligately anaerobic flora and the Gram-negative facultatively anaerobic bacteria were determined and the ratio was expressed as SD factor. The high SD factor of the flora from most subjects for aztreonam and tobramycin indicates that the drugs are useful for SD in contrast to temocillin and gentamicin. Binding to and subsequent release of tobramycin from faeces are presumed to facilitate the maintenance of adequate concentrations in the intestine despite the discontinuous intake.