Otakar Koldovský

Uptake, Activation, and Esterification of Fatty Acids in the Small Intestine of the Suckling Rat

Extract: The small intestinal mucosal phase of fatty acid absorption was studied in suckling and adult rats. Fatty acid binding protein (FABP) is present in the cytosol of jejunal mucosa of 6-day-old rats in amounts equivalent to that found in mucosal cytosol of adult rats (16.4% and 15.0%, respectively). The percentage of oleic acid binding to FABP is the same in 6-day-old and adult rats (13.9% and 10.2%, respectively). The specific activity of jejunal microsomal oleoyl-CoA synthetase is high in the fetus, falls abruptly after birth, but increased by the third day of life to remain constant thereafter into adult life. In contrast the specific activity of acyl-CoA:monoglyceride acyltransferase is low in the fetal jejunum, gradually increases, and is significantly higher in the 6- and 12-day-old rat than in the adult. Uptake of oleic acid by jejunal slices of 6-and 11-day-old animals is three- to fivefold higher than uptake by jejunal slices prepared from adults rats. The rate of esterification of oleic acid is higher in jejunal slices from 6- and 11-day-old rats, reflecting the enhanced uptake of oleic acid.Speculation: In the suckling rat, the increased intestinal mucosal epithelial cell capacity for fatty acid esterification coincides with a diminished lipolytic activity within the lumen. This paradox suggests that the fatty acid esterification process in the small intestine of the suckling rat may be involved with aspects of lipid metabolism other than that of fatty acid absorption. Extrapolating these observations on experimental animals to the human neonate, we suggest as working hypothesis that inefficient fat absorption should not be attributed to diminished mucosal epithelial cell function.

Epidermal Growth Factor Delays Gastric Emptying and Small Intestinal Transit in Suckling Rats

Suckling (12-d-old) rats that were fasted for 15 h received epidermal growth factor (EGF) s.c. (0.5 and 1.0 μg per rat, i.e. approximately 2 and 4 μg/100 g of body weight), together with motility markers 51Cr-EDTA or Poly R-478, and were killed 45 min later. Counts were measured separately in the stomach and the small intestine, which was divided into 12 segments. Administration of EGF delayed gastric emptying. In controls, the stomach contained 26.1 ± 1.6% (mean ± SEM); in EGF-treated rats the stomach contained 75.9 ± 10.2% and 75.7 ± 2.5% of the total 51Cr-EDTA counts given. EGF had the maximum effect(1.0 μg) when given simultaneously with 51Cr-EDTA. Significant, but lower, effects of EGF were seen with the administration of EGF preceded by 10 min or followed by 10 and 20 min with the administration of 51Cr-EDTA(65.8 ± 5.8%, 60.0 ± 6.4%, and 54.1 ± 4.2%, respectively). Small intestinal transit was also delayed. Administration of anti-EGF antiserum did not affect gastric emptying, but accelerated small intestinal transit as determined 30 min after administration of51 Cr-EDTA. These studies are the first to demonstrate the effect of EGF on gastrointestinal motility in vivo in suckling mammals.

Activity of β-galactosidase, β-glucuronidase and N-acetyl-β-glucosaminidase in human rectal mucosa

Abstract Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents. The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent Km values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM. The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.

CARBOXY-TERMINAL PROCESSING OF MOUSE EPIDERMAL GROWTH FACTOR IN MOUSE GASTROINTESTINAL TRACT IN VIVO

Breast-fed mammals consume epidermal growth factor (EGF) in milk. We characterized processing of mouse EGF (mEGF) in gastrointestinal (GI) tract of 14-day-old auckling (Su) anesthetized mice using reversed phase-HPLC. 125I-mEGF (4-10 ng/mouse) in 50 μl of 0.05 M phosphate buffer + 0.1% BSA, pH 7.4 (intestine) or pH 5.5 (stomach) was introduced into ligated stomach, duodenum, jejunum, midjejunum or ileum. After 10 or 30 min, radioactivity in luminal flushings (LF) and wall of GI segments was extracted and analyzed by RP-HPLC. Standards ( 125I-mEGF-1: 1 amino acid less at C-terminus; 125I-mEGF-5: 5 amino acids less at C-terminus; and 125I-mEGF-6: 6 amino acids less at C-terminus) were prepared by controlled proteolysis (Endocrinology 118:875). Formation by the tissues of C-terminally processed 125I-mEGF was defined by coelution with standards. Isolated peaks were characterized by binding to anti EGF antibody and EGF-speciflc receptors. 125I-mEGF was found to be intact in LF of stomach; in LF of duodenum it was partially degraded to 125I-mEGF-1. In LF and wall of jejunum, midjejunum and ileum, 125I-mEGF-1, 125I-mEGF-5 and 125I-mEGF-6 were detected. All peaks isolated from GI tissues were fully immunoreactive. Receptor binding 125I-mEGF-I was significantly greater than that of intact 125I-mECF, whereas receptor bindings of 125I-mEGF-5 and 125I-mEGF-6 were significantly diminished. Thus luminally administered EGF undergoes C-terminal processing in the GI tract; type of biologically active fragments produced varies with segment.

Effects of Milk-Borne Physiological Concentrations of Insulin-Like Growth Factors-I or -II (IGF-I, -II) Upon Growth in the Artificially Fed (AR) Suckling Rat

Insulin-Like Growth Factors-I and -II are potent mitogenic peptides that are synthesized by many cell types in mammals (Sara and Hall, 1990, Styne, 1998). These hormones appear to be important in growth regulation of the organism, including during perinatal life (Cohick, et al., 1993,Philipps, et al., 1988). This presumption is strengthened by recent observations of mice bearing nonfunctional genes for IGF-I, -II (Baker, et al., 1993,Lau, et al., 1994), since these have significant growth restriction as well as a high perinatal mortality rate. Attention has also recently focused upon the presence of IGF’s in milk of many species in biologically relevant concentrations. Studies from our laboratories (Philipps, et al., 1991) as well as others (Donovan, et al., 1990) have shown that IGF-I and -II are present in the milk of at least several species. IGF’s are present in most biological fluids in close association with binding proteins and it has been suggested that these binding proteins play a significant role in modulation of IGF-receptor interaction and as a potential reservoir for IGF in the circulation. The serum concentration of IGFBP, particularly BP2 in the suckling, may be responsive to levels of nutritional sufficiency.

INTRAVENOUSLY ADMINISTERED 125I-EGF IS SECRETED INTO BILE AND LUMEN OF SUCKLING RAT STOMACH AND INTESTINE

Parenterally administered epidermal growth factor (EGF) produces trophic effects on various organs of suckling mammals. Present studies investigated distribution of intravenously administered rat EGF (rECF) in liver and GI tract of 12-day-old suckling rats anesthetized with fentanyl (6 μl/rat). The bile duct was cannulated, and 125I-rEGF (2-4 ng/rat) was administered via femoral vein. After 5, 30, 60 and 120 min, animals were killed and extracted radioactivity (RA) from tissue was characterized by immunoaffinity chromatography. The highest levels of RA were found in liver (46.1±3.0% of total); and 98% was immunoreactive (IR) at 5 min. Both total and IR-RA in liver decreased with time. Large amounts of administered RA were detected in bile at 60 min (2.2%) and 120 min (5.1%); more than 99% of the RA was IR. Concentrations of RA in bile were 15 X greater than that in blood. RA in wall and luminal flushings of stomach and small intestine increased with time. More than 95% of the RA extracted from wall and luminal flushings of small intestine was found to be IR. Cannulation of the bile duct caused a significant reduction in the levels of RA in luminal flushing of the small intestine; however, the percentage of IR was unaffected. IR in the wall and flushings of stomach was more than 60% at 30 min, decreasing to 7-9% at 60 and 120 min. These results suggest that IR EGF is secreted into the lumen via bile, as well as across the wall of the GI tract in suckling rats.