Lavell M. Henderson

Absorption of d- and l-carnitine by the intestine and kidney tubule in the rat

The process by which L- and D-carnitine are absorbed was investigated using the live rat and the isolated vascularly perfused intestine. A lumenal dose of 2-6 nmol in the perfused intestine resulted in less than 5% transport of either isomer to the perfusate in 30 min. The L-isomer was taken up by the intestinal tissue about twice as rapidly as the D-isomer by both the perfused intestine (52.8% and 21.6%, respectively) and the live animal (80% and 50%, respectively) in 30 min. After 1 h 90% of the L-carnitine had accumulated in the intestinal tissue and was released to the circulation over the next several hours. Accumulation of D-carnitine reached a maximum of 80% in 2 h and release to the circulations was similar to that of L-carnitine. Uptake of both L-[14C]carnitine and acetyl-L-[14C]carnitine was more rapid in the upper jejunal segment than in other portions of the small intestine. Acetylation occurred in all segments, resulting in nearly 50% conversion to this derivative in 5 min. Increasing the dose of L-carnitine reduced the percent acetylation. The uptake of both isomers was a saturable process and high concentrations of D-carnitine, acetyl-L-carnitine and trimethylaminobutyrate inhibited L-carnitine uptake. In the live animal after 5 h, the distribution of isotope from L-[14C]carnitine and D-[3H]carnitine differed primarily in the muscle where 29.5% of the L-carnitine and 5.3% of the D-carnitine was found and in the urine where 2.9% of the L-carnitine and 7.1% of the D-carnitine was found. The renal threshold for L-carnitine was 80 microM and for D-carnitine 30 microM, in the isolated perfused kidney. Approx. 40% of the L-carnitine but none of the D-carnitine excreted in the urine was acetylated. L-Carnitine and D-carnitine competed for tubular reabsorption.

The metabolism of D- and L-lysine specifically labeled with 15N

Abstract 1. 1. An investigation of the metabolism by the rat of D - and L -lysine labeled with 14 C and 15 N in either the α- or ϵ-amino group has shown that pipecolate, previously identified as the L -isomer, was found from both D - and L -lysine following removal of the α-amino group. D -Lysine was a much better precursor of L -pipecolate than was L -lysine. 2. 2. The conversion of L -lysine to α-aminoadipate, as shown by 14 C recovery, was too limited to permit an accurate determination as to which nitrogen atom of lysine was retained in this conversion. 3. 3. The present data, together with previous observations which indicated that L -pipecolate was metabolically inert, suggest that the principal pathway of L -lysine metabolism does not include L -pipecolate as an intermediate.

Effect of ascorbic acid deficiency on the in vivo synthesis of carnitine

The effects of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and kidney carnitine levels were not affected by the deficiency, but scorbutic animals had 50% less carnitine in heart and skeletal muscle than control animals. Labeled carnitine precursors, 6-N-tri-methyl-L-lysine and 4-N-trimethylaminobutyrate, both of which require ascorbate for their enzymatic hydroxylation, were injected into the vena cava of control, pair-fed and scorbutic animals. The distribution of isotope in compounds present in the liver and kidney after 1 h was determined. The uptake of trimethyllysine by the liver was less than 2% in 1 h, while the kidney took up approx. 20% of the 14C. Control and pair-fed animals converted trimethyllysine to kidney trimethylaminobutyrate 8--10 times as well as did scorbutic animals. Trimethylaminobutyrate hydroxylase, present in the liver but almost absent from the kidney, converted nearly all of substrate taken up by the liver to carnitine in both the scorbutic and control animals.

The metabolism of D- and L-lysine in the intact rat, perfused liver and liver mitochondria.

Abstract 1. 1. Isolated rat liver or kidney mitochondria readily form saccharopine from l -lysine when incubated anaerobically. Sacchropine is metabolized to ⇐inoadipic and glutamic acids under aerobic conditions. α-Aminoadipic and pipecolic acids, but not saccharopine, were isolated from the urine of a rat injected intraperitoneally with radioactive l -lysine. 2. 2. d -Lysine was metabolized very slowly by isolated liver mitochondria and by the isolated perfusedd liver. However, the intact rat converted a considerable amount of d -lysine to pipecolic acid. 3. 3. The pipecolic acid formed by the intact rat from either d - or l -lysine was shown to the l -isomer by its failure to be oxidized by d -amino acid oxidase (EC 1.4.3.3). This l -pipecolic acid was not metabolized by isolated liver or kidney mitochondria or by isolated perfused rat liver. 4. 4. The relative contrtibutions of d - and l -lysine to formation of l -pipecolic acid are such that approx. 96% of the pipecolic acid isolated after administration of dl -lysine would arise from the d -lysine. The initial reaction of d -lysine metabolism in the rat probably involves α-deamination. The initial reaction of l -lysine metabolism is not firmly established.

Transport and metabolism of carnitine precursors in various organs of the rat

Isolated, vascularly perfused small intestine, liver, and kidney were used to investigate their interdependence in the absorption and metabolism of carnitine precursors in the rat. During 30 min of recirculating perfusion, the small intestine absorbed trimethyllysine, hydroxytrimethyllysine, and trimethylaminobutyrate fairly well when they were administered via the lumen or the perfusate. Trimethylaminobutyrate was synthesized from either trimethyllysine or hydroxytrimethyllysine by the small intestine, but further hydroxylation of trimethylaminobutyrate to carnitine did not occur. Trimethyllysine and hydroxytrimethyllysine were not readily absorbed by the liver. In contrast, trimethylaminobutyrate and trimethylaminobutyraldehyde were rapidly absorbed from the perfusate and readily incorporated into carnitine by the liver. Trimethyllysine and hydroxytrimethyllysine were taken up slowly by the kiodney and partially converted to trimethylaminobutyrate during 3409 min of perfusion. Trimethylaminobutyrate was neither absorbed readily by the kidney nor was it hydroxylated to carnitine. These results were compared to whole animal studies performed over an equivalent time period. The data suggest that the isolted small intestine absorbs trimethyllysine well, but it probably plays a minor role in metabolizing physiological quantities of this compound in the whole animal where other organs are competing for the same substrate. In both the isolated organ and in the whole animal, the kidney absorbs and metabolizes trimethyllysine more readily than the liver; whereas the liver absorbs trimethylaminobutyrate more rapidly than either the kidney or the small intestine and, unlike these organs, converts it to carnitine.

The formation of oxalic acid from the side chain of aromatic amino acids in the rat.

1. 1. 14C from DL-[I-14C]tryptophan and DL[2-14C]tryptophan was incorporated into urinary oxalate of intact rats about the same extent, but DL-[3-14C]tryptophan did not label urinary oxalate. The incorporation of 14C from DL[2-14C]alanine into oxalate was only about 150 of that from DL-[2-14C]tryptophan. 2. 2. When sodium benzoate was given to rats together with either DL-[2-14C]-tryptophan or DL-[3-14C]tryptophan, the specific activities of the hippurate isolated from the urine were about equal, indicating that glyoxylate is not an intermediate in the major pathway by which oxalate arises from the side chain of tryptophan. 3. 3. The two terminal carbon atoms, but not the methylene carbon, of the side chain of [14C]tryptophan labeled oxalate when the amino acid was incubated with snake venom L-amino-acid oxidase [EC 1.4.3.2]. However, decarboxylation by auto-oxidation of the indolepyruvate thus formed was much more rapid than oxalate formation. 4. 4. DL-[2-14C]tyrosine and DL-[2-14C]phenylalanine also labeled oxalate when given to intact rats or after incubation with L-amino acid oxidase. 5. 5. The quantity of oxalate derived from the side chain moiety of the three aromatic amino acids tested was estimated to be less than 3% of the total oxalic acid excreted by the normal rat.

Transport and metabolism of pyridoxine in rat liver

Evidence, obtained with in situ perfused rat liver, indicated that pyridoxine is taken up from the perfusate by a non-concentrative process, followed by metabolic trapping. These conclusions were reached on the basis of the fact that at low concentrations (0.125 microM), the 3H of [3H]pyridoxine accumulated against a concentration gradient, but high concentrations (333 microM) of pyridoxine or 4-deoxypyridoxine prevented this apparent concentrative uptake. Under no conditions did the tissue water:perfusate concentration ratio of [3H]pyridoxine exceed unity. The perfused liver rapidly converted the labeled pyridoxine to pyridoxine phosphate, pyridoxal phosphate and pyridoxamine phosphate and released a substantial amount of pyridoxal and some pyridoxal phosphate into the perfusate. Since muscle and erythrocytes failed to oxidize pyridoxine phosphate to pyridoxal phosphate, it is suggested that the liver plays a major role in oxidizing dietary pyridoxine and pyridoxamine as their phosphate esters to supply pyridoxal phosphate which then reaches to other organs chiefly as circulating pyridoxal.

Uptake of l-carnitine, d-carnitine and acetyl-l-carnitine by isolated guinea-pig enterocytes

Uptake and metabolism of L-carnitine, D-carnitine and acetyl-L-carnitine were studied utilizing isolated guinea-pig enterocytes. Uptake of the D- and L-isomers of carnitine was temperature dependent. Uptake of L-[14C]carnitine by jejunal cells was sodium dependent since replacement by lithium, potassium or choline greatly reduced uptake. L- and D-carnitine developed intracellular to extracellular concentration gradients for total carnitine (free plus acetylated) of 2.7 and 1.4, respectively. However, acetylation of L-carnitine accounted almost entirely for the difference between uptake of L- and D-carnitine. About 60% of the intracellular label was acetyl-L-carnitine after 30 min, and the remainder was free L-carnitine. No other products were observed. D-Carnitine was not metabolized. Acetyl-L-carnitine was deacetylated during or immediately after uptake into intestinal cells and a portion of this newly formed intracellular free carnitine was apparently reacetylated. L-Carnitine and D-carnitine transport (after adjustment for metabolism and diffusion) were evaluated over a concentration range of 2-1000 microM. Km values of 6-7 microM and 5 microM, were estimated for L- and D-carnitine, respectively. Ileal-cell uptake was about half that found for jejunal cells, but the labeled intracellular acetylcarnitine-to-carnitine ratios were similar for both cell populations. Carnitine transport by guinea-pig enterocytes demonstrate characteristics of a carrier-mediated process since it was inhibited by D-carnitine and trimethylaminobutyrate, as well as being temperature and concentration dependent. The process appears to be facilitated diffusion rather than active transport since L-carnitine did not develop a significant concentration gradient, and was unaffected by ouabain or actinomycin A.

Effect of vitamin C deficiency on hydroxylation of trimethylaminobutyrate to carnitine in the guinea pig.

The effect of ascorbate deficiency on carnitine biosynthesis was investigated in young male guinea pigs. Liver and skeletal muscle carnitine levels were reduced in scorbutic animals. Heart and kidney concentrations remained unchanged. 14C-labeled 4-N-trimethylaminobutyrate was administered to control, pair-fed and scorbutic animals and distribution of isotope in compound present in the liver after 30 min was determined. Control and pair-fed animals converted trimethylaminobutyrate to carnitine faster than scorbutic animals. Injection of ascorbate with the [14C]trimethylaminobutyrate reversed the decline in trimethylaminobutyrate hydroxylase (EC 1.14.11.1) activity in scorbutic animals.

Transport and metabolism of pantothenic acid by rat kidney

Transport of [14C]pantothenic acid was studied using brush-border membrane vesicles prepared from rat kidney. In the presence of a Na+ gradient an accumulation of pantothenic acid 3-fold above equilibrium was observed. The Km and Vmax found were 7.30 microM and 23.8 pmol/mg protein per min, respectively. Isolated perfused rat kidneys were employed to study excretion of pantothenic acid at various concentrations in the perfusate. At physiological plasma concentrations, the filtered pantothenic acid was largely reabsorbed by the active process observed in the vesicles. At higher concentrations, pantothenic acid was found to undergo tubular secretion. Penicillin inhibited this secretory process indicating that both compounds share a secretory mechanism. Live animal studies indicated that the only compound excreted after injection of [14C]pantothenic acid was free pantothenic acid. After 1 week only 38% of the administered dose was excreted in the urine, indicating that effective conservation was taking place in the whole animal.