Gottfried Krampitz

Decarboxylation of pyruvic acid in aqueous solution by thermal proteinoids

Abstract Thermal proteinoids accelerate the conversion of pyruvic acid to carbon dioxide and acetic acid. The reaction follows Michaelis-Menten kinetics. Co-poly(glutamic acid-threonine) and co-poly (glutamic acid-leucine) were found to be the most active of simple co-polymers and even more active than proteinoid. The activity of proteinoid is remarkably reduced when aspartyl-imide bonds are opened by treatment with alkali or by heating in buffered solution. Proteinoid is gradually inactivated by progressive acid hydrolysis.

OUABAIN-LIKE FACTORS IN HUMAN URINE: IDENTIFICATION OF A Na-K-ATPase INHIBITOR AS VANADIUM- DIASCORBATE ADDUCT

We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of>30g NaCl/day 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Sephadex G-10, one of threc OLFs eluted in the early subfractions FIV/1–2 close to 3H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with Mraround 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF-1 (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 cross-reacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-K-ATPase with IC50of around 1.5 × 10-5M and 1.5 × 10-4M, respectively. Identification of OLF-2 was first attem...

Endogenous sodium pump inhibitors in human urine further identification of inhibitors of Na-K-ATPase

We investigated the presence of endogenous Na-K-ATPase inhibitor(s), ie, ouabain-like factors (OLFs), in the urine of salt-loaded healthy subjects. For this purpose 24-h urine was collected on days 3, 4, and 5 of high sodium intake (> 30 g NaCl/day). The samples then were lyophilized. Redissolved urine concentrates were acidified (pH 3.5) and subjected to gelchromatography on a Sephadex G-25 column where the OLFs eluted in the post-salt fraction IV. When lyophilized fraction IV was rechromatographed on Sephadex G-10, OLFs with molecular mass (M(r) of approximately 400 eluted in a late fraction IV/8 separate from added ouabain, ouabagenin (or digoxin), which eluted shortly after void volume. With the subsequent reverse-phase HPLC of fraction IV/8 a polar OLF-1 eluted in fraction IV/8a after the void volume in the water phase and a more apolar OLF-2 eluted at 20% acetonitrile in fraction IV/8d. Only the more apolar OLF-2 cross-reacted with a digoxin antibody. By preparative thin-layer chromatography OLF-1 and OLF-2 were purified as single compounds with potent dose-dependent Na-K-ATPase inhibition and Ki-values approximating 1.5 x 10(-5) mol/L and 1.5 x 10(-4) mol/L, respectively. Mass-spectroscopy (MS) showed M(r) of 391 and 1H-NMR characterized the endogenous urinary apolar OLF-2 as a compound that is structurally totally unrelated to ouabain; infrared (IR) spectroscopy of OLF-1 and OLF-2 also revealed no similarity with ouabain.(ABSTRACT TRUNCATED AT 250 WORDS)

Endogene Natriuretische und Ouabain-ähnliche Faktoren

SummaryThe existence of an endogenous natriuretic hormone and ouabain-like factors (OLF) has been postulated for many years. This postulate was based on our original observation that a small M.W. fraction in the serum after acute expansion of the extracellular fluid volume (ECFV) not only exhibited natriuretic activity but also inhibited the Na-K-ATPase enzyme in vitro similar to ouabain. Since then, numerous studies confirmed the presence of OLFs in serum, urine, cerebrospinal fluid, and various organs including the heart and hypothalamus. Some of these OLFs are well-known endogenous compounds, such as free unsaturated fatty acids, which inhibit in vitro transmembranous sodium transport, Na-K-ATPase and3H-ouabain binding to its membrane receptor or crossreact with digoxin antibodies. Chemically yet undefined OLFs of potentially hypothalamic origin were detected in various models of experimental and clinical hypertension and are suggested to play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs isolated from the urine of salt-loaded healthy subjects strongly enhance basal and vasopressin-stimulated release of calcium in vascular smooth muscle cells and platelets similar to the effects we had observed with endothelin. This urine fraction also exhibits natriuretic activity which increases in parallel with sodium intake. Further chromatographic separation and amino acid analysis confirmed the peptidic nature (M.W.<1000) of the natriuretic factor(s). However, the two biological activities, namely natriuretic and ouabain-like activities, reside in distinct and chemically different compounds. In face of the previous discovery of the atrial natriuretic peptides (ANP) it is of special interest that very recent observations strongly suggest a natriuretic factor of non-cardiac origin to play an important role in the natriuresis that follows ECFV expansion. In addition, numerous experimental data point to an interaction between the ANP and OLF systems. They should stimulate once again the final identification of these yet unknown endogenous natriuretic and ouabain-like factors.The existence of an endogenous natriuretic hormone and ouabain-like factors (OLF) has been postulated for many years. This postulate was based on our original observation that a small M.W. fraction in the serum after acute expansion of the extracellular fluid volume (ECFV) not only exhibited natriuretic activity but also inhibited the Na-K-ATPase enzyme in vitro similar to ouabain. Since then, numerous studies confirmed the presence of OLFs in serum, urine, cerebrospinal fluid, and various organs including the heart and hypothalamus. Some of these OLFs are well-known endogenous compounds, such as free unsaturated fatty acids, which inhibit in vitro transmembranous sodium transport, Na-K-ATPase and 3H-ouabain binding to its membrane receptor or cross-react with digoxin antibodies. Chemically yet undefined OLFs of potentially hypothalamic origin were detected in various models of experimental and clinical hypertension and are suggested to play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs isolated from the urine of salt-loaded healthy subjects strongly enhance basal and vasopressin-stimulated release of calcium in vascular smooth muscle cells and platelets similar to the effects we had observed with endothelin. This urine fraction also exhibits natriuretic activity which increases in parallel with sodium intake. Further chromatographic separation and amino acid analysis confirmed the peptidic nature (M.W. less than 1000) of the natriuretic factor(s). However, the two biological activities, namely natriuretic and ouabain-like activities, reside in distinct and chemically different compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

F013: Ouabain-like factors in human urine: Identification of a Na-K-ATPase inhibitor as vanadium-diascorbate adduct

We investigated endogenous Na-K-ATPase inhibitors, i.e. ouabain-like factors(OLFs), in the urine of salt-loaded healthy subjects. During an intake of > 30g NaCl/day 24h-urines were collected, lyophilized, redissolved and acidified to pH 3.5. With gelchromatography the inhibitory activity eluted in a post-salt fraction FIV from Sephadex G-25. When this fraction was again passed through Sephadex G-10, one of three OLFs eluted in the early subfractions FIV/1-2 close to H-ouabain and cross-reacted strongly with a ouabain antibody (NEN). Two additional OLFs with Mr around 400 eluted in a late subfraction FIV/8 which resolved after reverse-phase HPLC into a more polar OLF- (water phase) and a more apolar OLF-2 (20% acetonitrile). Only the more apolar OLF-2 cross-reacted with digoxin and ouabain antibodies. OLF-1 and OLF-2 purified to single compounds by preparative thin layer chromatography inhibited Na-K-ATPase with IC50 of around 1.5 x 10(-5) M and 1.5 x 10(-4) M, respectively. Identification of OLF-2 was first attempted because most material was available for further processing. Data from mass-spectroscopy, nuclear magnetic resonance (1H-NMR) and infrared spectroscopy characterized OLF-2 as structurally unrelated to ouabain but resembling ascorbic acid derivatives, i.e. vanadium (V) diascorbates (Mr 403) with similar elution times from RP-HPLC as OLF-2. They inhibited the enzyme in its E2-configuration with IC50 of 9 x 10(-5) M and 2 x 10(-6) M for V(IV)- and V(V)-diascorbate, respectively. OLF-1, OLF-2 and V-diascorbate raise intracellular free calcium in inner medullary collecting duct and vascular smooth muscle cells which also contract in vitro. V-diascorbate was also natriuretic in a bioassay. We suggest that V-diascorbates represent one of several OLFs excreted in human urine.