Intestinal Transport of β-Lactam Antibiotics: Analysis of the Affinity at the H+/Peptide Symporter (PEPT1), the Uptake into Caco-2 Cell Monolayers and the Transepithelial Flux

Purpose. This study on the intestinal transport of -lactam antibiotics was undertaken to investigate the correlation between cellular transport parameters and the bioavailability. Methods. Transport of 23 -lactam antibiotics was characterized by measuring their ability to inhibit the uptake of glycylsarcosine into Caco-2 cells, their uptake into the cells and their total flux across the cell monolayers. Results. Ceftibuten and cyclacillin were recognized by PEPT1 with affinity constants comparable to those of natural dipeptides (K_i = 0.3 and 0.5 mM, respectively). Cefadroxil, cefamandole, cephradine, cefaclor, cefuroxime-axetil, cefixime, cephalotin, cephalexin and ampicillin also interacted with PEPT1 (K_i = 7-14 mM). In contrast, cefapirin, cefodizime, cefuroxime, cefmetazole, ceftazidime, benzyl-penicillin, ceftriaxone, cefpirome, cefotaxime, cefepime, cephaloridine and cefsulodin displayed no affinity to the transport system (K_i > 20 mM). The uptake into the cells and the transepithelial flux was highest for those -lactam antibiotics, which showed the strongest inhibition of [¹⁴C]Gly-Sar transport (p < 0.0001). Exceptions were cefuroxim-axetil and cephalotin. Conclusions. The probability of oral bioavailability for -lactam antibiotics is mainly determined by their affinity to PEPT1. A threshold K_i value of 14 mM with respect to Gly-Sar uptake is required.     

Transepithelial flux of early and advanced glycation compounds across Caco-2 cell monolayers and their interaction with intestinal amino acid and peptide transport systems

Maillard products arise from condensation reactions between amino acids or proteins with reducing sugars during food processing. As ubiquitous components of human food, these early or advanced glycation products may be subject to intestinal absorption. The present study was performed to investigate the intestinal uptake of Maillard products and to determine whether they are substrates for peptide and amino acid transporters expressed at the apical membrane of Caco-2 cells. At a concentration of 10 mM, N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine and N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine inhibited the [(14)C]glycylsarcosine uptake mediated by the H(+)-peptide co-transporter PEPT1 by 13 to 45%. For N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine, an inhibitory constant of 8.7 mM was determined, reflecting a low affinity to PEPT1 in comparison with natural dipeptides. Uptake of L-[(3)H]lysine was weakly affected by N(epsilon)-(carboxymethyl)-L-lysine, N(alpha)-hippuryl-L-lysine and N(alpha)-hippuryl-N(epsilon)-(carboxymethyl)-L-lysine but strongly inhibited by N(epsilon)-(1-deoxy-D-fructosyl)-L-lysine (81%). None of the Maillard products was able to inhibit the uptake of L-[(3)H]leucine by more than 15%. We also studied the transepithelial flux of Maillard products across Caco-2 cell monolayers cultured on permeable filters. The flux rates of Maillard products ranged from 0.01 to 0.3%/cm(2) per h and were shown to be much lower than those of carrier substrates such as glycylsarcosine, L-proline and the space marker [(14)C]mannitol. We conclude that the Maillard products investigated in the present study are neither transported by PEPT1 nor by carriers for neutral amino acids. The low transepithelial flux measured for these compounds most probably occurs by simple diffusion.     

Vitamin D<Subscript>3</Subscript> supplementation does not modify cardiovascular risk profile of adults with inadequate vitamin D status

Purpose

The Nutrition Societies in Germany, Austria, and Switzerland recommend a daily intake of 20 µg vitamin D₃ for adults when endogenous synthesis is absent. The current study aimed to elucidate whether this vitamin D₃ dose impacts cardiovascular risk markers of adults during the winter months.

Methods

The study was conducted in Halle (Saale), Germany (51^o northern latitude) as a placebo-controlled, double-blinded, randomised trial (from January to April). A total of 105 apparently healthy subjects (male and female, 20–71 years old) were included. Subjects were randomly allocated to two groups. One group received a daily 20-µg vitamin D₃ dose (n = 54), and the other group received a placebo (n = 51) for 12 weeks. Outcome measures included blood pressure, heart rate, concentrations of renin, aldosterone, serum lipids and vascular calcification markers, and haematologic variables such as pro-inflammatory monocytes.

Results

Blood pressure and systemic cardiovascular risk markers remained unchanged by vitamin D₃ supplementation, although serum 25-hydroxyvitamin D₃ increased from 38 ± 14 to 73 ± 16 nmol/L at week 12. The placebo and vitamin D groups did not differ in their final cardiovascular risk profile.

Conclusion

Daily supplementation of 20 µg vitamin D₃ during winter is unlikely to change cardiovascular risk profile.

     

Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form

Maltosine, a 3-hydroxy-4-pyridinone derivative of lysine formed in the course of the advanced Maillard reaction, is an effective metal chelating agent. It therefore represents an interesting compound for the treatment of metal ion storage diseases. We synthesized 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-l-norleucine (free maltosine) and its dipeptide derivatives alanylmaltosine (Ala-Mal) and maltosinylalanine (Mal-Ala) and examined the transepithelial flux of these compounds across Caco-2 cells and their interaction with membrane transporters. Transepithelial flux of maltosine was significantly higher when added as Ala-Mal and Mal-Ala than in free form. Assays at Caco-2 cells and at HeLa cells expressing the human peptide transporter (hPEPT)1 revealed that Ala-Mal and Mal-Ala show medium to high affinity to the system. Only free but not peptide-bound maltosine inhibited the uptake of l-[³H]lysine in Caco-2 and OK cells. Maltosine dipeptides were transported by hPEPT1 across cell membranes and accumulated in hPEPT1-transfected HeLa cells. In electrophysiological measurements at hPEPT1-expressing Xenopus laevis oocytes, Ala-Mal and Mal-Ala induced significant inward directed currents. We conclude that Ala-Mal and Mal-Ala are transported by hPEPT1 into intestinal cells and then hydrolyzed to free maltosine and alanine. The results suggest that the oral bioavailability of maltosine can be increased significantly by applying this drug candidate in peptide-bound form.     

Transport of angiotensin-converting enzyme inhibitors by H+/peptide transporters revisited

Angiotensin-converting enzyme (ACE) inhibitors are often regarded as substrates for the H+/peptide transporters (PEPT)1 and PEPT2. Even though the conclusions drawn from published data are quite inconsistent, in most review articles PEPT1 is claimed to mediate the intestinal absorption of ACE inhibitors and thus to determine their oral availability. We systematically investigated the interaction of a series of ACE inhibitors with PEPT1 and PEPT2. First, we studied the effect of 14 ACE inhibitors including new drugs on the uptake of the dipeptide [14C]glycylsarcosine into human intestinal Caco-2 cells constitutively expressing PEPT1 and rat renal SKPT cells expressing PEPT2. In a second approach, the interaction of ACE inhibitors with heterologously expressed human PEPT1 and PEPT2 was determined. In both assay systems, zofenopril and fosinopril were found to have very high affinity for binding to peptide transporters. Medium to low affinity for transporter interaction was found for benazepril, quinapril, trandolapril, spirapril, cilazapril, ramipril, moexipril, quinaprilat, and perindopril. For enalapril, lisinopril, and captopril, very weak affinity or lack of interaction was found. Transport currents of PEPT1 and PEPT2 expressed in Xenopus laevis oocytes were recorded by the two-electrode voltage-clamp technique. Statistically significant, but very low currents were only observed for lisinopril, enalapril, quinapril, and benazepril at PEPT1 and for spirapril at PEPT2. For the other ACE inhibitors, electrogenic transport activity was extremely low or not measurable at all. The present results suggest that peptide transporters do not control intestinal absorption and renal reabsorption of ACE inhibitors.     

High-affinity interaction of sartans with H+/peptide transporters

Sartans are very effective drugs for treatment of hypertension, heart failure, and other cardiovascular disorders. They antagonize the effects of angiotensin II at the AT(1) receptor and display p.o. bioavailability rates of 13 to 80%. Because some sartans sterically resemble dipeptide derivatives, we investigated whether they are transported by peptide transporters. We first assessed the effects of sartans on [(14)C]glycylsarcosine uptake into Caco-2 cells expressing H(+)/peptide transporter (PEPT) 1 and into SKPT cells expressing PEPT2. Losartan, irbesartan, valsartan, and eprosartan inhibited [glycine-1-(14)C]glycylsarcosine ([(14)C]Gly-Sar) uptake into Caco-2 cells in a competitive manner with K(i) values of 24, 230, 390, and >1000 microM. Losartan and valsartan also strongly inhibited the total transepithelial flux of [(14)C]Gly-Sar across Caco-2 cell monolayers. In SKPT cells, [(14)C]Gly-Sar uptake was inhibited with K(i) values of 2.2 microM (losartan), 65 microM (irbesartan), 260 microM (valsartan), and 490 microM (eprosartan). We determined by the two-electrode voltage-clamp technique whether the compounds elicited transport currents by PEPT1 or PEPT2 when expressed in Xenopus laevis oocytes. No currents were observed for any of the sartans, but the compounds strongly and reversibly inhibited peptide-induced currents. Uptake of valsartan, losartan, and cefadroxil was quantified in HeLa cells after heterologous expression of human PEPT1 (hPEPT1). In contrast to cefadroxil, no PEPT1-specific uptake of valsartan and losartan was found. We conclude that the sartans tested in this study display high-affinity interaction with PEPTs but are not transported themselves. However, they strongly inhibit hPEPT1-mediated uptake of dipeptides and cefadroxil.     

Influence of a proton gradient on the transport kinetics of the H+/amino acid cotransporter PAT1 in Caco-2 cells.

The recently cloned proton-coupled amino acid transporter 1 (PAT1) not only accepts several amino acids as substrates but also pharmaceutically relevant L-proline or GABA derivatives such as cis-4-hydroxy-L-proline, L-azetidine-2-carboxylic acid (LACA), 3-amino-1-propanesulfonic acid, nipecotic acid, and the antituberculotic agent D-cycloserine. Because human intestine expresses hPAT1 at the brush border membrane, the transporter may serve as a new oral drug delivery route. Using the human intestinal cell line Caco-2, we have investigated the influence of an inwardly directed proton gradient on the kinetic parameters of L-proline uptake. H+ altered only the apparent affinity of L-proline transport and not the maximal transport velocity. Similarly, treatment of the cells with diethylpyrocarbonate (DEPC), known to chemically modify histidyl residues and block their function, affected only the Kt value of L-proline transport. Both increasing pH and DEPC treatment strongly increased the inhibition constants (Ki) of several drugs at hPAT1. It is concluded that H+ stimulates hPAT1 primarily by increasing the substrate affinity with no detectable influence on the maximal transport velocity of the transporter.     

Experimental investigations on the effect of etilefrinhydrochloride (Effortil) on heart and circulation of animals with partial ischaemic damage of the myocardium (author's transl)

The effect of Etilefrin on the blood flow of normal and partial ischaemic myocardium with heat conductance probes as well as on the ventricular dynamics was investigated on 11 dogs under propiomazine-pentobarbital narcosis. Etilefrin was applied in doses of 0.04 mg/kg bw. and 0.2 mg/kg bw. At low doses, the blood flow was only elevated in normal myocardium, likewise the systolic aortic pressure, the diastolic aortic pressure decreased, the heart frequency was elevated, whereas the systolic pressure in the left ventricle was only slightly increased for a short time, the enddiastolic pressure unchanged, dp/dtmax rose significantly, but the heart minute volume only slightly. At higher doses, elevation of blood flow also appeared in partial ischaemic myocardium, the aortic pressure, systolic and diastolic, decreased considerably at first and then 20 min. p.i. rose to above the control value. The heart frequency was more elevated than at lower doses, the pressures in the left ventricle showed no obvious changes, dp/dtmax and heart minute volume were likewise considerably elevated. Radiological measurements of the ejection fraction and the mean circumferential fiber shortening velocity at higher Etilefrin doses showed also a positive inotropic effect. In consideration of this fact, therapy with Etilefrin appears useful in disturbances of cardiac function and at least shows no contraindication by means of myocardial blood flow.     

Oxidized fat reduces milk triacylglycerol concentrations by inhibiting gene expression of lipoprotein lipase and fatty acid transporters in the mammary gland of rats

Feeding oxidized fats to lactating rats causes a strong reduction of triacylglycerol concentration in the milk. The reason for this, however, has not yet been elucidated. Pregnant Sprague-Dawley rats were assigned to 2 groups of 11 rats each and fed diets containing either fresh fat (FF group) or an oxidized fat (OF group) from d 1 to d 20 of lactation. Concentrations of triacylglycerols and long-chain fatty acids in the milk and weight gain of suckling pups were lower in the OF group than in the FF group (P < 0.05). Concentrations of medium-chain fatty acids in the milk and messenger RNA (mRNA) abundance of lipogenic enzymes in the mammary gland did not differ between the 2 groups of rats. However, the OF group had a lower concentration of triacylglycerols and nonesterified fatty acids (NEFA) in plasma and lower mRNA concentrations of lipoprotein lipase and fatty acid transporters in the mammary gland than the FF group (P < 0.05). Moreover, the OF group had higher mRNA concentrations of hepatic lipase, fatty acid transporters, and several genes involved in fatty acid oxidation in the liver than the FF group (P < 0.05). The present findings suggest that a dietary oxidized fat lowers the concentration of triacylglycerols in the milk by a reduced uptake of fatty acids from triacylglycerol rich-lipoproteins and NEFA into the mammary gland. The study, moreover, indicates that an oxidized fat impairs normal metabolic adaptations during lactation, which promote the utilization of metabolic substrates by the mammary gland for the synthesis of milk.     

Feeding oxidized fat during pregnancy up-regulates expression of PPARα-responsive genes in the liver of rat fetuses

Background

Feeding oxidized fats causes activation of peroxisome proliferator-activated receptor α (PPARα) in the liver of rats. However, whether feeding oxidized fat during pregnancy also results in activation of PPARα in fetal liver is unknown. Thus, this study aimed to explore whether feeding oxidized fat during pregnancy causes a PPARα response in fetal liver. Two experiments with pregnant rats which were administered three different diets (control; oxidized fat; clofibrate as positive control) in a controlled feeding regimen during either late pregnancy (first experiment) or whole pregnancy (second experiment) were performed.

Results

In both experiments pregnant rats treated with oxidized fat or clofibrate had higher relative mRNA concentrations of the PPARα-responsive genes acyl-CoA oxidase (ACO), cytochrome P₄₅₀ 4A1 (CYP4A1), L-type carnitin-palmitoyl transferase I (L-CPT I), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) in the liver than control rats (P < 0.05). In addition, in both experiments fetuses of the oxidized fat group and the clofibrate group also had markedly higher relative mRNA concentrations of ACO, CYP4A1, CPT I, MCAD, and LCAD in the liver than those of the control group (P < 0.05), whereas the relative mRNA concentrations of PPARα, SREBP-1c, and FAS did not differ between treatment groups. In the second experiment treatment with oxidized fat also reduced triacylglycerol concentrations in the livers of pregnant rats and fetuses (P < 0.05).

Conclusion

The present study demonstrates for the first time that components of oxidized fat with PPARα activating potential are able to induce a PPARα response in the liver of fetuses. Moreover, the present study shows that feeding oxidized fat during whole pregnancy, but not during late pregnancy, lowers triacylglycerol concentrations in fetal livers.