Renal tubular transport of proline, hydroxyproline, and glycine. II. Hydroxy-l-proline as substrate and as inhibitor in vivo.

Abstract

Renal tubular absorption of the free amino acids L-proline, hydroxy-L-proline, and glycine in man is accomplished by a transport system exhibiting preference for this group of amino acids (1, 2). The characteristics of saturability (for L-proline transport) and selective inhibition between the constituent substrates have been demonstrated for this system in man (1). Studies in vivo in the rat (3, 4) also reveal a transport system in that animal with preference for the same three amino acids. Studies in vitro with rat kidney cortex slices (4) and other tissues (5, 6) confirm this finding and also indicate that, within the system showing group preference, there may be discrete catalytic transport sites for the individual substrates (4, 6). The apparent order of substrate affinities for the transport system or systems is highest for L-proline and lowest for glycine (1, 4).A maximal rate for renal tubular absorption of L-proline (Tm Pro) has been shown in man (1). In the present work the behavior of free hydroxy-L-proline as substrate and its effect as an inhibitor within the system were studied. A Tm for hydroxyproline (Tm Hypro) was demonstrated; the imino acid was also shown to be a poor inhibitor of L-proline transport but more effective against glycine.* Submitted for publication November 8, 1965; accepted May 20, 1966. Supported by grant AM-05117-04 from the Division Methods SubjectsFour healthy adult Caucasian males between 30 and 39 years of age were studied. Two subjects (2 and 3) were given two infusions; the remaining two were given one infusion. Techniques Infusions were performed in the morning with the subjects in the resting state after an overnight fast. Sufficient water was given by mouth to ensure a diuresis exceeding 5 ml per minute. Urine was collected by spontaneous voiding while subjects were standing. The steady state infusion technique used in these experiments has been described previously (1). Hydroxy-L-proline was given in priming doses of 0.4 to 1.2 mmoles per kg (50 to 150 mg per kg) ; the sustaining dose was 3.5 /Amoles per kg per minute.Infusion of free hydroxy-L-proline is followed about 12 hours later by muscular ache, mild pyrexia, and nausea. These sensations disappear within 24 hours. This response is dose dependent, and the two subjects who were given two infusions felt greater discomfort with the larger infusion. Hydroxyprolinuria or persistence of hydroxyprolinemia were not observed at the time of symptoms. The origin of this reaction remains obscure. Materials Hydroxy-L-proline (chromatographically pure) was obtained commercially.1 Purity was confirmed by chromatographic analysis in n-butanol, acetic acid, water (12: 3: 5) followed by staining with Ninhydrin (0.2% in acetone) and sequential staining with isatin (0.2% in acetone) followed by Ehrlich's reagent (7). Preparation of samplesAll urine samples were preserved with thymol and stored immediately after collection at -20°C. Plasma was separated from heparinized whole venous blood by centrifugation within an hour after...

Renal tubular transport of proline, hydroxyproline, and glycine

“…infusions. Purity was confirmed by chromatographic methods and the solutions were prepared for infusion as described previously (1,2). L-proline, hydroxyproline-free (obtained from Nutritional Biochemical Corp., Cleveland, Ohio), which was used for the loading tests was given by mouth.…”

“…Amino acid content and endogenous clearance rates of amino acids were estimated in the overnight-fasted state in the manner previously described (1,2,8); random or 24-hr urine collections were not used. L-proline was infused i.v.…”

“…L-proline was infused i.v. into subj ects A,II,2, A,II,3, and B,I,4, and hydroxy-L-proline was infused separately into subjects A,II,2 and A,II,3, in order to achieve an estimate of their respective maximum rates of tubular absorption (Tm) (1,2). Intestinal absorption of L-proline was evaluated in subjects A,II,2 and B,II,7, and in two healthy adult males matched for weight and age.…”

“…; the concentration of proline was measured in these samples. All samples were prepared for analysis by procedures described elsewhere (1,2,8).…”

“…The mammalian kidney absorbs the imino acids, L-proline and 4-hydroxy-L-proline, and the amino acid glycine from the glomerular filtrate by at least one tubular transport agency, which is common to these particular solutes (1)(2)(3)(4). The specificity of the transport system is made apparent only when the concentrations in plasma are raised above their normal endogenous levels; the system is saturable and either imino acid will competitively inhibit the uptake of the other two substrates.…”