Carol J. Gross

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.

Comparative effects of exogenous lactase (β-galactosidase) preparations on in vivo lactose digestion

Microbial-derived β-galactosidase (β-gal) enzyme preparations improvein vivo lactose digestion and tolerance through enhanced gastrointestinal digestion of lactose. Three different β-gal preparations, Lactogest (soft gel capsule), Lactaid (caplet), and DairyEase (chewable tablet) and placebo were fed to lactose maldigesters with either 20 g or 50 g of lactose to compare the efficacy of these products and to further establish a dose-response relationship for use. All enzyme preparations dramatically reduced both the peak and total breath hydrogen production when fed with milk containing 20 g of lactose. Four capsules of Lactogest, two caplets of Lactaid, or two tablets of DairyEase (each treatment containing approx 6000 IU) reduced total hydrogen production significantly (P<0.05) below that observed with two capsules of Lactogest (containing approx 3000 IU) in a stoichiometric manner. Symptoms were significantly (P<0.05) less severe with all the β-gal products. In contrast, with 50 g of lactose in water, peak and total hydrogen production was modestly, but not significantly reduced by the enzyme treatment. Furthermore, symptom scores for bloating, cramping, nausea, pain, diarrhea, and flatus were not different between treatments and the control. The 50-g lactose dose appeared to overwhelm the ability of either 3000 or 6000 IU of β-gal to assist significantly with lactose digestion. Results from these studies demonstrate the relative equivalency of chewable, caplet, and soft-get β-gal products, based on IUs of enzyme fed.

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 development and nutritional state on the uptake, metabolism and release of free and acetyl-l-carnitine by the rodent small intestine

Intestinal carnitine levels and the incorporation and release of exogenous, [14C]carnitine were compared in intestine from adult rat and guinea pig. Total carnitine levels were 4-fold higher in rat as compared to guinea pig intestine. Retention of label was also 4-fold greater, 4 h after placing carnitine (7 nmol) in the lumen. Carnitine was detected in rat chow (64 nmol/g) but not in guinea pig chow. Intestinal carnitine was reduced 2-fold in rats fed a carnitine-free diet for 2 weeks, suggesting the importance of dietary carnitine in determining intestinal carnitine levels. Two conditions where fatty acid oxidation is increased (fasting and suckling) resulted in elevated carnitine levels and retention. In the 3-day fasted guinea pig, intestinal carnitine increased by 40% and retention of a lumenal dose of [14C]carnitine increased about 7-fold after 4 h. During suckling, carnitine levels peaked after 3 days (792 nmol/g) and decreased to near adult levels after 7 days (108 nmol/g). Retention of a lumenal dose of carnitine was greater after 4 h in 1-day old neonatal, than in adult intestine (82% vs. 7% of a 7 nmol dose, respectively). This reflects, in part, the larger intestinal carnitine pool on day 1 (352 nmol/g) than on day 29 (91 nmol/g). The calculated efflux of total intestinal carnitine after 4 h was similar for adults and neonates (72 vs. 58 nmol/g) suggesting that efflux relative to pool size was greater in the adult than in the neonate. Uptake of [14C]acetylcarnitine was similar to [14C]carnitine in 1-day old animals, but was retained to a lesser extent (36% vs. 82%, respectively) after 4 h. The calculated efflux of total intestinal carnitine when acetylcarnitine was the substrate was about 4-fold that when carnitine was the substrate. Incorporation of [14C]carnitine into enterocytes isolated from 3-day old animals was 4-fold greater than into enterocytes isolated from adults (152 vs. 36 pmol/mg protein after 60 min). Active transport of carnitine into enterocytes from neonates, but not from adults is suggested, since labeled free intracellular carnitine reached 4-fold the calculated equilibrium value in neonatal enterocytes, but did not exceed the equilibrium value in adult enterocytes.

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.

Genes and Enzymes of Azetidine-2-Carboxylate Metabolism: Detoxification and Assimilation of an Antibiotic

l-(-)-Azetidine-2-carboxylate (AC) is a toxic, natural product analog of l-proline. This study revealed the genes and biochemical strategy employed by Pseudomonas sp. strain A2C to detoxify and assimilate AC as its sole nitrogen source. The gene region from Pseudomonas sp. strain A2C required for detoxification was cloned into Escherichia coli and sequenced. The 7.0-kb region contained eight identifiable genes. Four encoded putative transporters or permeases for gamma-amino acids or drugs. Another gene encoded a homolog of 2-haloacid dehalogenase (HAD). The encoded protein, denoted l-azetidine-2-carboxylate hydrolase (AC hydrolase), was highly overexpressed by subcloning. The AC hydrolase was shown to catalyze azetidine ring opening with the production of 2-hydroxy-4-aminobutyrate. AC hydrolase was further demonstrated to be a new hydrolytic member of the HAD superfamily by showing loss of activity upon changing aspartate-12, the conserved active site nucleophile in this family, to an alanine residue. The presence of a gene encoding a potential export chaperone protein, CsaA, adjacent to the AC hydrolase gene suggested that AC hydrolase might be found inside the periplasm in the native Pseudomonas strain. Periplasmic and cytoplasmic cell fractions from Pseudomonas sp. strain A2C were prepared. A higher specific activity for AC hydrolysis was found in the periplasmic fraction. Protein mass spectrometry further identified AC hydrolase and known periplasmic marker proteins in the periplasmic fraction. A model was proposed in which AC is hydrolyzed in the periplasm and the product of that reaction is transported into and further metabolized in the cytoplasm.

The effect of nutritional state and allopurinol on nucleotide formation in enterocytes from the guinea pig small intestine

The uptake of purine nucleosides (guanosine and hypoxanthine) and bases (guanine, hypoxanthine and adenine) and their incorporation into nucleotides were studied in enterocytes isolated from fed and 3-day fasted guinea pig jejunum. Both total uptake and synthesis of nucleotides were greater for these purines in the fasted, as compared to the fed state for the first 5 min, when the initial substrate concentration in the medium was 10 microM. Increased uptake did not result from a change in the relative distribution of synthesized nucleotides between the fed and fasted states. Reduced catabolism was observed in the medium by enterocytes from fasted as compared to fed animals after 1 min of incubation with both inosine and guanosine. Preincubation of enterocytes with allopurinol (a xanthine oxidase inhibitor) decreased total uptake but increased the formation of IMP from hypoxanthine. Xanthine oxidase activity measured in mucosa from fasted guinea pigs was lower than that from fed animals (6.29 vs. 9.30 nmol/min per mg protein, respectively). However, activities of the salvage enzymes adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase were not significantly different between the fed and fasted states. These data show that allopurinol treatment, and mucosal atrophy resulting from fasting, decrease xanthine oxidase activity and increase nucleotide synthesis from exogenous substrates in enterocytes from the guinea-pig small intestine, suggesting a regulatory function of mucosal xanthine oxidase in purine salvage by the small intestine.

Effect of nutritional state and allopourinol on purine metabolism in the rat small intestine

The effect of fasting and refeeding on the uptake and retention of purines by the small intestine of the rat was studied in vivo. Short-term uptake and incorporation into nucleotides of the purine bases adenine, guanine and hypoxanthine and the nucleoside inosine were evaluated in the proximal jejunum. After 5 min, more label was recovered in the intestinal contents in fasted rats, indicating that total absorption was reduced. However, intestinal retention of purines (50 nmol dose) was elevated with fasting (27.2 vs. 16.6 nmol/g for adenine, 5.7 vs. 3.0 nmol/g for guanine and 16.1 vs. 7.4 nmol/g for hypoxanthine, for fed vs. fasted, respectively). After 1 day of refeeding, retention remained elevated for adenine (27.4 nmol/g) and guanine (5.5 nmol/g). After 3 days of refeeding intestinal weight and retention of labeled purines returned to the unfasted levels. Nucleotide formation from all purine bases was greater in the intestinal tissue of fasted as compared to fed rats (25.4 vs. 11.4 nmol/g for adenine, 1.32 vs. 0.24 nmol/g for guanine, and 2.84 vs. 0.82 nmol/g for hypoxanthine). At a higher dose (3000 nmol) hypoxanthine and inosine were retained to a greater extent in the fasted than in the fed state. Pretreatment with allopurinol (a xanthine oxidase inhibitor) reduced the absorption of hypoxanthine, increased the retention of label in the tissue 4-fold or more, and elevated nucleotide formation 10-fold or more. Fasting and allopurinol treatment, both known affectors of xanthine oxidase activity, enhanced both the retention of dietary purine and nucleotide formation.

Pyridine nucleotide synthesis in normal and neoplastic human pituitary cells in culture

14C‐Nicotinic acid (NA) incorporation into nicotinamide adenine dinucleotide (NAD) was studied in cultures from 7 normal human pituitaries and 13 chromophobe adenomas. 14C‐NA (7.2 μM) was incubated with both normal and tumor cell cultures for periods up to 48 hours. Cells and culture media were examined separately for metabolites at 24 and 48 hours. NAD was the major labeled metabolite found in both normal and tumor cells, accounting for 65.4% for normal cells and 56.8% for tumor cells of the total cellular label at 48 hours. Nicotinamide (NAm), a product arising from NAD, was the only labeled metabolite found in the culture medium, aside from the 14C‐NA added initially. Total incorporation of 14C‐NA into NAD was estimated by adding the cellular 14C‐NAD and labeled products of NAD (NMN, NAm and NADP) to the 14C‐NAm found in the medium for each culture. Cultures derived from adenomas demonstrated greater than twice the rate of incorporation of 14C‐NA into NAD as did normal cell cultures (P < 0.01 at 24 hours and <0.001 at 48 hours). This difference did not appear to be related to the secretory status of the tumor, since both secreting and nonsecreting tumors demonstrated increased rates when compared to normal cells. This difference also persisted in two different culture media and with cells that had been maintained in culture for different lengths of time (6‐day dispersed cell cultures and 6‐ to 16‐week fragment cultures). Cancer 52:2100‐2106, 1983.