Gertrud Rehner

Comparative studies on the high-performance liquid chromatographic determination of thiamine and its phosphate esters with chloroethylthiamine as an internal standard using pre- and post-column derivatization procedures

Abstract Two improved reversed-phase high-performance liquid chromatographic procedures for the rapid separation and sensitive fluorimetric quantification of thiamine and its phosphate esters are presented using pre-column and post-column derivatiziation. Further, for the first time chloroethylthiamine has been introduced into thiamine determination as an internal standard which allows the analytical procedure to be controlled. Complete separation and sensitive detection can be achieved by both methods within 15 min. The pre-column derivatization technique is easier to perform but is sometimes accompanied by technical problems caused by the derivatization reagent. In the post-column derivatization procedure the chromatographic system was not attacked but a chemically inert derivatization pump was essential. Analysis of rat intestinal tissue by both methods including a simplified extraction scheme yielded the same results, indicating that the techniques are interchangeable.

High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide

This paper reports a high-performance liquid chromatographic (HPLC) technique to determine biotin in biological samples. Biotin and the internal standard dethiobiotin are converted into fluorescent derivatives by using panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] as a fluorescence label. Biotin is extracted from biological tissue with trichloroacetic acid and the extract is purified by a combination of solid-phase extraction on C18 cartridges, ion-exchange chromatography on DOWEX formate resin, and thin-layer chromatography. The purified sample extract is derivatized in the presence of a crown ether. The resulting panacyl esters can be separated on reversed-phase as well as on normal-phase HPLC. Normal phase HPLC is preferable because it provides higher sensitivity and demands less sample pretreatment. Analysis of rat intestinal tissue revealed that only about 13% of the biotin is present in free form whereas 87% is bound in proteins from which it can be released by hydrolysis. Biotin values determined by this method are comparable to those obtained by other techniques.

Optimized high-performance liquid chromatographic procedure for the separation and quantification of the main folacins and some derivatives: I. Chromatographic system

Abstract An improved reversed-phase high-performance liquid chromatographic (HPLC) procedure for the rapid separation and sensitive fluorimetric quantification of the main folacins and some degradation products is presented. The system is the first to include two substances that can be used as internal standards. It allows the complete separation of all substances within 26 min. The fluorescence of reduced folates is measured directly after the column whereas other compounds are converted oxidatively to fluorescent products by post-column derivatization. Among several oxidants tested, potassium peroxodisulphate was found to be the most suitable.

Wirkung von Maillard-Produkten und Lysinoalanin auf die Bioverfügbarkeit von Eisen, Kupfer und Zink

ZusammenfassungEs wurde die Bioverfügbarkeit von Eisen, Kupfer und Zink untersucht, wenn diese essentiellen Spurenelemente 1. mit einigen isolierten Maillard-Produkten bzw. LAL, oder 2. mit thermisch unterschiedlich belasteten Testnahrungen einmalig an Säuglingsratten verabreicht wurden. Durch die isolierten Testsubstanzen ließen sich bei allen drei Elementen Effekte auf der präresorptiven und/oder postresorptiven Ebene erzielen. Die Testnahrungen beeinflußten — auch wenn sie fünf Wochen lang verfüttert wurden — nur die Bioverfügbarkeit des Kupfers.SummaryBioavailability of iron, copper, and zinc was investigated in suckling rats. The essential trace elements were given once either with several isolated Maillard products and with LAL, respectively, or with differently heat-treated formula diets. The isolated substances revealed effects on bioavailability of all the elements tested, either on the preresorptive or on the postresorptive level. The formula diets affected only the bioavailability of copper — even when fed for several weeks.

High-performance liquid chromatographic determination of nicotinic acid and nicotinamide in biological samples applying post-column derivatization resulting in bathmochrome absorption shifts

An ion-pair reversed-phase high-performance liquid chromatographic procedure for the rapid separation and sensitive quantitation of nicotinic acid (NA) and nicotinamide (NAM) in biological samples was developed. The vitamers were separated within 10 min on an octadecylsilica column applying a linear gradient of tetrabutylammonium phosphate and methanol. NA and NAM were converted to highly absorbing derivatives by a modified Königs reaction using a double post-column derivatization arrangement consisting of two pumps and two knitted tubular reactors. The proposed method is highly sensitive and specific and applicable to biological materials as was shown by the analysis of rat intestinal tissue.

Optimized high-performance liquid chromatographic procedure for the separation and determination of the main folacins and some derivatives : II. Extraction method and application to rat liver

Different methods for the extraction of folacins from biological materials and the hydrolysis of pteroylpolyglutamates prior to high-performance liquid chromatography were investigated. Acetone precipitation of proteins led to higher extraction rates of folates from biological materials as examined by using an endogenous labelling technique. It also caused less destruction of some folates but could not be combined with subsequent hydrolysis of pteroylpolyglutamates. Pteroylpolyglutamates were hydrolysed by a partially purified suspension of pteroylpolyglutamates hydrolase (PPH). Comparative studies on the folate content of rat liver revealed that complete hydrolysis of polyglutamates could also be achieved by incubating the homogenized tissue at 37 degrees C to allow endogenous PPH to act. This procedure causes interconversions of folates and is therefore not suitable for the analysis of biological materials.

Effect of proteins on availability of zinc I. Gastrointestinal transit time of casein and whey protein and zinc absorption in weaned rats

SummaryThe availability of zinc from isolated casein (CasD) was compared with that from whey protein (WpD) in 23–25 day old rats. The study was designed to show the course of the gastrointestinal transit time of either chyme (radiolabeled by141Ce as a non-absorbable marker) or zinc (as65Zn) in groups of 9 to 12 animals each. Animals were killed either 1/4, 1/2, 1, 2, 6, 12 or 24 hours after intragastric intubation of the protein suspensions. Immediately afterwards, intragastric pH was measured and the determination of zinc retention in intestinal tissues and liver as well as in the carcass was performed.30 and 60 minutes after intubation the intragastric pH of the CasD group was significantly lower than that of the WpD group. The precipitation behaviour of the two protein fractions — compact curd formation by the CasD versus flocculent structure of the WpD — was determining for the pattern of gastric emptying. With the WpD the chyme was emptied according to an exponential function; while the CasD precipitate left the stomach in three distinct phases. With either protein suspension zinc left the stomach earlier than the bulk of the chyme, indicating a partial disintegration of the zinc-protein-complexes. With the WpD, zinc was emptied exponentially, whereas with the CasD a biphasic emptying pattern was found. Ileum was found to be the main zinc absorbing segment, mainly due to the long time of contact with zinc. After 2 and 12 hours zinc retention from the CasD was significantly higher than that from the WpD, however, after 24 hours retention was significantly better from the WpD.From the present study it can be concluded that, for comparison of zinc availability from diets containing different proteins, short-term experiments are not appropriate. Furthermore, it can be supposed that preabsorptive processes in the stomach are crucial for the availability of zinc.ZusammenfassungZiel der an 23 bis 25 Tage alten Ratten durchgeführten Untersuchungen war es, die Verfügbarkeit von Zink aus intragastral verabreichten Casein-(CasD-) bzw. Molkenprotein-(WpD-)Suspensionen zu vergleichen. Um den zeitabhängigen Verlauf des gastrointestinalen Durchgangs des Chymus (mit nichtresorbierbarem141Ce markiert) und des Zinks (als65Zn) zu ermitteln, wurden je 9 bis 12 Tiere 1/4, 1/2, 1, 2, 6, 12 und 24 Stunden nach dem Sonden getötet. Unmittelbar danach wurde der intragastrale pH-Wert sowie die Retention von Zink im Darmgewebe, Leber und Restkörper gemessen.30 Minuten nach Intubation der Proteine war der pH-Wert im Magen der CasD-Gruppe signifikant niedriger als der der WpD-Gruppe. Die unterschiedliche intragastrale Präzipitation der beiden Proteine — kompaktes Präzipitat bei Casein, feinflockiges bei Molkenprotein — dürfte ausschlaggebend sein für das Entleerungsmuster aus dem Magen: der Chymus aus WpD wurde einer Exponentialfunktion entsprechend entleert, während der Chymus aus CasD den Magen in drei Phasen mit unterschiedlicher Geschwindigkeit verließ. Das Zink aus beiden Proteinsuspensionen wurde schneller aus dem Magen entleert als die Masse des Chymus, woraus auf eine zumindest partielle Loslösung des Zinks aus der Proteinbindung geschlossen werden kann. Das Zink aus der WpD wurde exponentiell, das Zink aus der CasD nach einem biphasischen Muster entleert. Als Folge der besonders langen Verweildauer des Zinks im Ileum erwies sich dieser Darmabschnitt als Hauptresorptionsort. Die Zink-Retention war 2 und 12 Stunden nach Verabreichung der Nahrungen signifikant höher aus der CasD als aus der WpD, nach 24 Stunden jedoch war aus der WpD signifikant mehr Zink retiniert worden als aus der CasD. Aus dieser Untersuchung ist zu folgern, daß zur Beurteilung der Zink-Verfügbarkeit aus unterschiedlichen Proteinen Kurzzeitversuche ungeeignet sind. Weiterhin muß angenommen werden, daß die präresorptiven Prozesse im Magen für die Zinkverfügbarkeit von wesentlicher Bedeutung sind.

[30] High-performance liquid chromatographic determination of biotin in biological materials after crown ether-catalyzed fluorescence derivatization with panacyl bromide

This paper reports a high-performance liquid chromatographic (HPLC) technique to determine biotin in biological samples. Biotin and the internal standard dethiobiotin are converted into fluorescent derivatives by using panacyl bromide [p-(9-anthroyloxy)phenacyl bromide] as a fluorescence label. Biotin is extracted from biological tissue with trichloroacetic acid and the extract is purified by a combination of solid-phase extraction on C18 cartridges, ion-exchange chromatography on DOWEX formate resin, and thin-layer chromatography. The purified sample extract is derivatized in the presence of a crown ether. The resulting panacyl esters can be separated on reversed-phase as well as on normal-phase HPLC. Normal phase HPLC is preferable because it provides higher sensitivity and demands less sample pretreatment. Analysis of rat intestinal tissue revealed that only about 13% of the biotin is present in free form whereas 87% is bound in proteins from which it can be released by hydrolysis. Biotin values determined by this method are comparable to those obtained by other techniques.

Die Leber als multifunktionelles Organ

Als zentrales Organ des gesamten Metabolismus ubt die Leber sehr vielfaltige katabole und anabole Stoffwechselfunktionen aus. Die diskontinuierlich angefluteten Nahrstoffe, insbesondere die Kohlenhydrate, werden in der Leber zunachst gespeichert und nach und nach zur Distribution an den Blutkreislauf abgegeben. Durch diesen homoostatischen Regelmechanismus sorgt die Leber fur die Stabilitat des inneren Milieus. Hierzu ist sie durch ihre anatomische Lage pradestiniert: Die resorbierten Kohlenhydrate, Aminosauren, freie Fettsauren und das Glycerin werden fast quantitativ im Pfortaderblut gesammelt und unmittelbar der Leber zugefuhrt. Eine Ausnahme bilden lediglich die in der Darmschleimhaut bereits resynthetisierten Triglyceride, die uber den Ductus thoracicus dem Blut zugeleitet werden.

Transport of pteroylglutamic acid into brush border membrane vesicles from rat small intestine is a partially carrier-mediated process.

SummaryIntestinal transport of PteGlu was studied using BBMV from rat small intestine. Transport was neither coupled to a specific cation gradient nor was it influenced by variations of the membrane potential. In the presence of a transmembrane pH gradient (pHout < pHin) initial transport was significantly higher compared to studies without pH gradient. Under these conditions transport could be inhibited by pretreating the vesicles with DIDS, an inhibitor of anion exchange systems. Uptake of PteGlu could not be enhanced by preloading the BBMV with HPO42− and Cl− and was not sensitive to DIDS under these conditions. Uptake studies using different concentrations of PteGlu revealed dual transport kinetics in the presence of a pH gradient and linear uptake in its absence. It could be concluded that uptake is mediated by a PteGlu−/OH−-antiporter at low substrate concentrations and occurs by non-ionic diffusion at higher concentrations or in the absence of a pH gradient. In an additional series of experiments it could be shown that about one-third of the substrate is bound to the membrane and is not transported. The biological significance of this binding remains unclear.ZusammenfassungDer transmembranäre Transport von PteGlu wurde mittels BBMV aus Rattendünndarm untersucht. Der Transport war weder an einen spezifischen Kationengradienten gekoppelt noch durch Veränderungen des Membranpotentials zu beeinflussen. In Gegenwart eines transmembranären pH-Gradienten (pHout < pHin) waren die initialen Transportraten signifikant höher als in Versuchen ohne pH-Gradient. Unter diesen Bedingungen war der Transport zu inhibieren, wenn die BBMV mit DIDS, einem Hemmstoff von Anionenaustauschsystemen, vorbehandelt wurden. Die Aufnahme von PteGlu war nicht erhöht, wenn die BBMV mit HPO42− und Cl− vorbeladen wurden. Unter diesen Bedingungen hatte auch DIDS keinen hemmenden Effekt. Studien zur konzentrationsabhängigen Aufnahme ergaben eine duale Transportcharakteristik in Anwesenheit eines pH-Gradienten und eine lineare Aufnahme in Abwesenheit eines pH-Gradienten. Hieraus ist zu schließen, daß die Aufnahme von PteGlu bei niedrigen Substratkonzentrationen mittels eines PteGlu−/OH−-Antiporters vermittelt wird. Bei höheren Konzentrationen oder in Abwesenheit eines pH-Gradienten erfolgt die Aufnahme hingegen durch nichtionische Diffusion. In einer zusätzlichen Versuchsserie konnte gezeigt werden, daß ein Drittel des Substrates nicht transportiert, sondern an die BBM gebunden wird. Die biologische Bedeutung dieser Bindung bleibt unklar.