Metabolism and Uptake of L-Proline by Human Kidney Cortex

Home , Glutamic acid, Hyperprolinemia, Proline, Proline (data page)

Pediat. Res. 7: 818-825 (1973) ' Amino acids kidney hydroxyproline proline iminoglycinuria Metabolism and Uptake of L-Proline by Human Kidney Cortex

P. HOLTZAPPLE, [38] M. GENEL, C. REA, AND S. SEGAL

Departments of Pediatrics and Medicine, University of Pennsylvania School of Medicine, and the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Extract L-Proline transport and metabolism have been investigated in normal adult human kidney cortical slices. The uptake of proline from buffers containing both high (10 HIM) and low (0.23 HIM) 14G-proline concentrations is intimately related to the extent of intracellular proline metabolism. The accumulation of radioactivity from both the 10 and 0.23 HIM proline media was against high radioactivity gradients, with distribution ratios of 18 and 3, respectively. Recovered proline from the intracellular fluid (IGF) accounted for less than 12% of the intracellular radioactivity in the tissue extracts. Glutamic acid accounted for greater than 75% of the intracellular form of the soluble radioactivity in tissue extracts. Total oxidation to 14CC>2 from both proline concentrations accounted for more than 50% of the total proline taken up by the slices. Kinetic analysis of the entry process suggested the existence of two saturable systems : one operative at low or physiologic proline concentrations (apparent Km 2 HIM) x 1 with a low capacity (Fmax 40 juM-ml ICF~ -30 min" ) and shared with neutral amino acids, and a second with an affinity to 10-fold less (apparent Km 17 HIM) with a high 1 1 capacity (Fraax 160 /uM-ml ICF~ -30 min" ) and unshared by the neutral amino acids. Separation of the influence of metabolism from the analysis of the transport systems by the use of proline analogues showed that thioproline inhibited proline uptake by 80%, primarily by reducing the conversion of proline to glutamic acid, whereas 3,4- dehydroproline did not affect the conversion of proline to glutamic acid but principally appeared to alter the affinity of the transport system for proline.

Speculation These studies suggest that changes in the rate of proline metabolism in the human kidney may alter the rate of tubular reabsorption, thus controlling renal proline excretion. It is possible that renal iminoglycinuric syndromes may not only be secondary to membrane transport disorders but may result from localized metabolic defects in the tubule cells.

Introduction ent during the early postnatal period in humans and gradually diminishes by the 6th month of life as the In humans, excessive excretion of the iminoacids—pro- net tubular reabsorption of glycine and the iminoacids line and hydroxyproline—and glycine is found in cer- increases. Delayed postnatal maturation of the imi- tain clinical situations, either as an isolated benign noacid and glycine transport system in the renal tu- aminoaciduria or associated with other systemic find- bule appears to be responsible for this benign finding ings. Hyperexcretion of iminoacids and glycine is pres- [3, 25].

818 L-Proline metabolism and uptake 819

Renal iminoglycinuria with normal proline metabo- a boiling water bath for 6 min. Aliquots (0.2 ml) of the lism and normal proline plasma concentrations which tissue extract and the media were added to scintilla- persists beyond the immediate postnatal period is as- tion vials with 10 ml toluene-ethanol (72/28) which cribed to selective genetically determined iminoglycine contained 3% Liquifluor [26] and counted at 90% transport defects in the renal tubule [13, 22]. In vivo efficiency in a Packard model 3003 Tri-Carb scintilla- proline infusion studies in man suggest distinct mem- tion spectrometer [28]. brane transport systems for proline and glycine with Tissue water, expressed as the percentage of the differing degrees of mutual interaction and specificity total tissue weight, was determined for each surgical [16, 17]. According to these clinical studies, the mem- specimen by the difference in weight of 3 slices after brane system with a high affinity for proline at physio- blotting and after drying in vacuo at 105° for 24 hr. logic concentrations has a low transport capacity, ex- Extracellular fluid space (ECF) estimation for each sur- cludes glycine, and is saturated at normal plasma gical specimen was determined by incubation with 0.5 levels of proline. The shared glycine-iminoacid system lid inulin-14C-carboxyl, specific activity 3.23 mCi/g has a low affinity for proline but a high capacity and is [12, 26]. 14 the deleted system in homozygotes with benign imino- In selected experiments CO2 production was deter- glycinuria. mined by trapping the evolved carbon dioxide in the In contrast to these membrane transport defects, a flasks fitted with center wells. Slices were weighed be- familial hyperprolinemia syndrome which may be asso- fore incubation. Injection of 0.3 ml 6 N sulfuric acid ciated with mental retardation and renal malforma- through the serum stopper flask cap ended the incuba- 14 tions has been described [5, 15, 19, 20]. Deficiency of tion. The liberated CO2 was trapped in the center either proline oxidase or A'-pyrroline-5-carboxylic acid well after injecting 1.0 ml 1 M Hyamine into the well. dehydrogenase results in hyperprolinemia. The imi- After equilibration for 45 min by shaking at room noglycinuria is ascribed to the excessive filtered load of temperature, the well was emptied into a counting- proline and competition with glycine and hydroxypro- vial, rinsed three times with scintillator, and counted line for the mutually shared iminoglycine transport [28]. system. In other experiments the radioactive metabolites in Inasmuch as the only data of proline handling by the tissue extract and media were assayed by descend- the human kidney have come from in vivo tubular ing paper chromatography. Aliquots of the extracts reabsorption studies [16, 17], we have investigated pro- containing at least 1,200 cpm of radioactivity were ap- line transport and metabolism in human renal cortical plied on Whatman no. 1 paper [29]; 90-95% recovery slices in a series of in vitro experiments. The results of of the applied radioactivity was achieved. Twenty mi- these studies form the basis of this report. crograms of proline, ornithine, glutamic acid, and y- aminobutyric acid were run as standards [27]. After de- Methods velopment, 1-cm segments of the papers were cut, added to scintillation counting vials, wetted with 3 ml Kidneys were obtained from nine adult patients un- scintillator, and counted [28]. dergoing nephrectomy for either localized intrarenal Intracellular proline concentrations were deter- or extrarenal pathology. Disease-free cortical areas (as mined in three surgical specimens by homogenization determined by histologic evaluation) of the kidneys of at least 100 mg tissues in 5 volumes 3% sulfosali- were used for preparation of the slices. The techniques cylic acid followed by ion exchange chromatography for determining amino acid uptake in human kidney [30]. Trapped extracellular proline was accounted for cortical slices were similar to those described by Fox et by assuming an extracellular proline concentration of al. [7]. Slices weighing 4-8 mg were prepared with a 0.23 HIM, similar to human plasma levels [4, 14, 21]. Stadie-Riggs microtome. Each slice was incubated in 2 ml Krebs-Ringer bicarbonate buffer (pH 7.4) at 37° in Calculations a metabolic shaker under an atmosphere of 95% 14 O2-5% CO2 with fid L-U- C-proline, specific activity The uptake of proline is expressed as the radioactive 219 mCi/mM [26], and the appropriate amounts of distribution ratio, i.e., the ratio of counts per minute carrier L-proline [27]. At the end of the incubation the per milliliter of ICF to counts per milliliter of media, slices were rinsed rapidly in physiologic saline, blotted calculated from the soluble radioactivity in the tissue on filter paper, weighed, and the soluble radioactivity extract [7]. A chemical distribution ratio for proline in the slice was extracted into 1 ml water by heating in was calculated from the percentage of proline in the 820 HOTZAPPLE ET AL.

ECF + volume of ICF X distribution ratio)] [18]. In the substrate concentration studies, total entry into the slices was determined from the radioactivity in the tissue extract at steady state conditions [23]. The ra- 14 dioactivity which evolved as CO2 was added to the soluble radioactivity remaining in the slice as described by Baerlocher et al. [1].

Results

Cellular Uptake of Proline Intracellular proline concentrations in cortical tissue from three patients were determined before incubation. Proline concentrations of 0.86, 0.74, and 0.18 mM were found. By assuming a mean plasma concentration of 0.23 mM [4, 14, 21], an intracellular to extracellular gradient for proline exists in the human kidney. Subse- cc quently, experiments were performed with 10 and 0.23 mM proline concentrations. The uptake of proline with time into kidney slices is depicted in Figure 1. Rapid uptake of proline at the 15 30 60 120 low concentration occurred; maximal radioactivity Minutes gradients of 19 at steady state were obtained after a Fig. 1. Time curve o£ isotopic proline uptake in human kidney 90-min incubation. Steady state conditions at the high slices from 10 mM and 0.23 mM proline concentrations. The sym- media concentration of proline were reached by 30 bols represent the mean uptake in five different specimens in- min of incubation with radioactivity gradients of 4. In cubated in duplicate flasks. The brackets represent the SE O£ the mean uptake. ICF: intracellular fluid. pH dependence studies maximal uptake from both media concentrations was seen at a pH of 7.4. Table 1. Intracellular I4G-containing compounds after incubation of human kidney cortex slices with l4G-proline Intracellular Metabolites of z-Proline

Specimen1 Distribution Proline, Glutamic acid, The intracellular form of the accumulated radioac- ration % tivity was ascertained in slices from 4 different kidneys T 11.92 12 77 (Table I). After a 30-min incubation with 0.23 mM G 7.92 0 86 proline little or none of the intracellular radioactivity H 12.11 2 98 M 8.02 0 94 was recovered as chemical proline. The major soluble metabolite in the intracellular space was glutamic 1 Cortical slices were incubated with 0.23 mM proline for 30 min acid, comprising 77, 86, 94, and 98%, respectively. The as described in Methods. remainder of the recovered radioactivity on the chro- 2 The distribution ratio is the ratio of counts per minutes per milliliter of intracellular fluid per counts per minute per milli- matography paper migrated to an area which corre- liter of media. sponded to the standards for ornithine and y-aminobutyric acid. The combined total of these latter two me- tissue extract as determined from the chromatography. tabolites was always less than 10% of total intracellu- This ratio has been corrected for the radioactive pro- lar forms. line trapped in the ECF [18]. The calculation is as At the higher media concentrations there was an follows: the expected proline radioactivity in ECF increase in the proportion of the intracellular radioac- equals the total counts per minute on the chromatog- tivity that was represented by proline. In two speci- raphy paper X the fraction of the total tissue radioac- mens incubated for 30 min with 10 mM proline, the tivity in the ECF. The fraction of the total tissue ra- intracellular proline was 50 and 23%, respectively, cor- dioactivity in ECF = [(volume of ECF)/(volume of respondingly, there was a proportional reduction in L-Proline metabolism and uptake 821 the percentage of the radioactivity migrating as glu- 0.23 mM L-PROLINE tamic acid, accounting for 48 and 66%, respectively. Chromatography of an aliquot of media after a 30- min incubation showed that more than 98% of the l4 radioactivity could be recovered as proline, which indi- • C02 cated that there was little or no efflux of proline metab- OTHER olites from the slices. GLUTAMIC ACID PROLINE 4.0% Oxidation of Proline by Human Cortex and Estima- tion of Total Proline Uptake 24 - 5.5% In addition to the tissue metabolities of the trans- 14 20 - ported proline, oxidation of proline to CO2 was also a significant factor in evaluation of total uptake of proline by kidney slices. Oxidation of proline by slices ^ I6h from one surgical specimen with respect to time is 3.3% shown in Figure 2. At both the 0.23 mM and 10 mM 12 - media concentrations there is an initial lag phase during which a slow progressive increase in carbon dioxide production is noted. After 30 min an accelerated phase is evident; production levels off after 90 min of 6.9% incubation. 9.4% In Figures 3 and 4 the estimate of total cellular 21.3% uptake of proline in terms of both the amount oxidized and the amount of intracellular metabolites in 5' 15' 30' 60' 90' 120' one specimen is depicted. From the 0.23 mM media INCUBATION TIME concentration, chemical proline at 5 min represents Fig. 3. The metabolic fate of proline taken up by human kidney slices from 0.23 mM proline after varying incubation times. The data are expressed as micromolar concentration of proline taken up per each milliliter of intracellular fluid (ICF). The numbers 36 - • 10 mM Proline at the top of the bars represent the percentage of the total intra- • 0.23 mM Proline cellular radioactivity which is proline. ^ 30 r only 21% of the total amount of transported proline. / As the oxidative rate increases after the first 30 min, r 24 - "D chemical proline represents less than 5% after 30 min M - of incubation. At this media concentration, a true •1 18 / chemical gradient for proline could not be achieved. / / With uptake of proline from the 10 mM media concen- / o> tration, a true chemical gradient of approximately 2.0 .jE 12 — 7 / was achieved after a 60-min incubation, but still over o ol 60% of the transported proline had been metabolized __ 6 by 60 min.

i • i i i , i Substrate Concentration Studies 15 30 60 90 120 The uptake of proline by the human cortex cells Minutes appears to be concentration dependent. Analysis of the Fig. 2. Oxidation of radioactive proline to "CO2 from media con- process suggests two modes of uptake as shown in Fig- centrations of 0.23 mM and 10 mM with time. Oxidation is expressed as micromolar concentration of proline oxidized per milli- ure 5 which is an Eadie-Hofstee plot of a study in one liter of intracellular fluid (ICF) by assuming complete oxidation human kidney (specimen K). The derived Km and Fmax of the proline molecule. from the data shown in the plot and from a similar 822 HOTZAPPLE ET AL.

10.0 mM L-PROLINE Effects of Inhibitors and Sodium or Oxygen Depriva- tion The effects of other amino acids and deprivation of • l4C0 sodium and oxygen on proline uptake and metabolism 90 24.5% OTHER are shown in Table II. Proline uptake from 0.065 mM GLUTAMIC ACID media concentration is inhibited by a-aminoisobutyric PROLINE 24.6% acid. Both a-aminoisobutyric acid and valine produce a 50% reduction in the accumulation of radioactivity. 70 - 40.1% 90 "HIGH SYSTEM" "LOW SYSTEM

80 KIDNEY Km(mM/L) Vmax(/iM/ml ICF/30') Km Vmax

K 23 53 0.7 7.8 70 50 T 10 75 09 13

+ OXIDATION 60 * \ K 17 105 2.0 25 50 1 T 12 160 1.5 40 42.7% 45.3% 40 30

30 O -C02

• +C02 20 \ V 60.9% r 10 10 i , ^ t i I I I 2 4 6 8 10 12 14 16 18 20 22 V [S] 5' 15' 30' 60' 90' 120 Fig. 5. Eadie-Hofstee plot of concentration-dependent proline up- INCUBATION TIME take in human kidney cortex (specimen K). Each point is the Fig. 4. The metabolic fate of proline taken up by human kidney mean value obtained from 5-mg slices incubated for 30 min in triplicate. The data is expressed without (Q) and with (•) the slices from 10 mM proline after variation of incubation times. amount of oxidation at each substrate concentration. The ap- The data are expressed as micromolar concentration of proline parent K values and F, values are shown in the inset. taken up per each milliliter of intracellular fluid (ICF). The m nax numbers at the top of the bars represent the percentage of the 1 total intracellular radioactivity which is proline. Table II. Inhibition of proline uptake

Thio- 3,4-De- Specimen Con trol AIB^ Valine hydro- Na+- Anaero- study in another specimen (specimen T) is seen in the proline proline Frees biosis inset of the figure. On the basis of cellular content of Media, proline and its soluble metabolites one mode of entry 0.065 has a high affinity (apparent Km values of 0.7 and 0.9 mM rnM/liter) but a low capacity for proline transport G 11. 624 4.02 5 .25 2.41 2.86 4.15 (maximal velocity of 7.8 and 13 ^M/ml/30 min). The S 13. 71 6.07 6 .73 2.24 1.89 other mode has a low affinity (apparent K values of H 12..11 7 .03 1.42 m M 8. 02 1.83 2.61 23 and 30 niM/liter) but a large capacity for proline Media, 10 entry (Vnwx of 53 and 75 ^M/ml/30 min). In account- mM ing for all of the transport in proline by adding the G 4. 24 3.22 2 .62 0.63 2.68 1.99 14 S 4. 61 2.83 3 .28 2.23 3.05 amount of proline oxidized to CO2 at the various 14 substrate concentrations to the C remaining in the 1 The inhibitor concentrations were 25 mM for 0.065 HIM proline tissue extract, the apparent affinity of proline for the and 50 mM for the 10 niM proline concentration. saturable sites does not change; however, the capacity 2 AIB : a-aminoisobutyric acid. as indicated by the maximal velocity of uptake in- 3 Tris was substituted for sodium in the preparation of the buffer. creases 2-3-fold in both the "high" and "low" Km sys- 4 All values represent the radioactive distribution ratios at 30 tems. min of incubation. L-Proline metabolism and uptake 823

The proportions, however, of the intracellular meta- lower media concentration, the effect of the sodium- bolities did not change. Glutamic acid accounted for free media not only reduced proline uptake by 80% more than 90% and proline only 5% of the intracellu- but also reduced the level of intracellular proline from lar accumulation in two specimens in which chroma- 19% in the control slices to 0%. Under conditions of tography of the tissue extract was accomplished. The anaerobiosis uptake was impaired to the same extent neutral amino acids has less, if any, effect on proline as seen with sodium-free conditions; however, chemical uptake from the 10 HIM media concentration. proline in the extract accounted for 50% of the accu- The inhibition of proline uptake by thioproline [31] mulated intracellular radioactivity at 30 min of incu- {thiozolidine-4-carboxylic acid), a metabolizable pro- bation. The inhibition of proline uptake from 10 mM line analogue, and 3,4-dehydroproline [32] was greater media concentration was less for both sodium-free and than that of the neutral amino acids (Table II). Al- anaerobic conditions. However, under both conditions though the inhibitory effect of these two agents on the proportion of intracellular proline increased from proline uptake is similar, the intracellular forms of the control uptake levels. radioactivity are different. In two specimens (specimens H and M), chemical proline accounted for more Discussion than 60% of the intracellular radioactivity in slices incubated with 25 mM thioproline. A component Proline, a nonessential amino acid, is metabolized by which migrated in the •y-aminobutyric acid area of the mammalian tissues to glutamic acid in a two-step se- chromatogram accounted for 22 and 30% of the re- quence. It is first oxidized by a mitochondrial enzyme, maining intracellular radioactivity. In contrast, chro- proline oxidase, to A'-pyrroline-5-carboxylic acid [9]. matography of the extract of specimen M incubated Spontaneous conversion of A'-pyrroline-5-carboxylic with 25 mM 3,4-dehydroproline was not different from acid to glutamic-y-semialdehyde can occur. The A'-pyr- that of noninhibited control slices. Chemical proline roline-5-carboxylic acid can be dehydrogenated by A'- and glutamic acid accounted for 15 and 84% of the pyrroline-5-carboxylic acid dehydrogenase also present intracellular radioactivity, respectively. Thus, it ap- in mitochondria. Both of these enzymes have been pears that the mode of inhibition of proline uptake by found in rat and bovine liver and in human liver, these two proline analogues is different; thioproline kidney, and brain [5]. The resultant glutamic acid is has an effect on the intracellular metabolism of proline further metabolized by several reactions, the most im- as well as on uptake, while the 3,4-dehydroproline effect portant of which is transamination to a-ketoglutarate. appears to be inhibitory to proline uptake at the Decarboxylation to y-aminobutyric acid by glutamic plasma level only. acid dehydrogenase, an enzyme whose relative activity Both complete replacement of the media sodium by in the rat kidney is greater than that in the brain, has equimolar Tris or anaerobic incubation cause an in- recently been described in the human kidney [24]. hibition of proline uptake (Tables II and III). At the The results of our studies showing extensive proline metabolism in human renal cortical slices support the Table III. Effect of sodium and oxygen deprivation on concept that the human kidney may be an important 1 intracellular forms site for the regulation of proline homeostasis. This was Glutamic first suggested by Felig et al. [6] in studies demonstrat- Conditions D.R.2 Proline, acid, % % ing consistent renal extraction of proline in the post- absorptive and fasting conditions. In both states renal Media, 0.065 HIM arterial-venous differences were significantly greater Control 11.62 19 69 Sodium free 2.86 0 88 than differences across the hepatic bed [6]. Although Anaerobiosis 4.15 50 32 our data with the kidney slice do not allow estimations Media, 10 mM of the relative importance of the various pathways of Control 3.24 24 58 glutamic acid degradation, complete oxidation of pro- Sodium free 2.68 31 42 line appears to be the major fate of the transported pro- Anaerobiosis 1.99 40 27 line. We assume that the tubule cells which constitute 1 A 5-mg slice was incubated in 2 ml Krebs-Ringer-bicarbonate the majority of the cell types in a cortical slice are 14 buffer (pH 7.1) for 30 min with 0.2 ,uCi C-proline. Sodium-free responsible for this metabolism of proline. However, buffer was prepared with equimolar Tris. Anaerobic flasks were MacKenzie and Scriver [10] found that isolated rat gassed with N2/CO2 (95/5) for 45 sec and sealed. 2 D.R.: radioactive distribution ratio. glomeruli are able to convert proline to glutamic acid 824 HOTZAPPLE ET AL. and carbon dioxide. Therefore, some of the metabolic droproline did not affect the intracellular metabolism products of proline conversion could come from glo- of accumulated proline. The inhibitory effect was as merular metabolism in the cortical slice. great as that of thioproline on the radioactive accumu- In our studies and in the rat slice studies reported lation of proline. This suggests that the 3,4- by Baerlocher et al. [1, 2], the rapidity of the proline dehydroproline was active principally at the mem- degradation raises some questions as to the nature of brane carrier site for proline and exerted its inhibitory the intracellular proline gradients. Our data suggest effect on proline uptake by this interaction. that at the physiologic levels a true proline gradient The influence of metabolism on the transmembrane calculated from the percentage of intracellular proline movement of proline in the human kidney raises some present after incubation was only slightly more than questions concerning the etiology of prolinuria in both unity and could represent intracellular accumulation the familial hyperprolinemic syndromes with known by an equilibrating or facilitated diffusion system. enzymatic defects [5, 15, 19, 20] and the hyperprolinu- Because of the significant amount of proline metabo- ria renal tubule syndromes [13, 16, 17, 22]. The degree lism in the cortical slices, the characterization of the of prolinuria in the former syndromes may be related membrane transport system is difficult. Time- and con- not only to an increased filtered load of proline but centration-dependent studies suggest that a steady state also to defects in the tubular metabolism of proline, of entry is achieved after 30 min of incubation and resulting in impairment of tubular reabsorption of the that the transport system is saturable. Two modes of filtered proline load. The hyperiminoglycinuric phen- entry with differing affinities for a saturable membrane otypes, presumably disorders to specific membrane reactive site are apparently present. In these general transport carriers, may result from alterations at the characteristics the human transport process for proline renal level in the control of proline metabolism. A is similar to that which exists in the rat [11] and the local change in the kidney resulting in a reduction of rabbit tubule [8]. The affinity of the saturable system proline oxidation, usually rapid, could be responsible operative at the low proline concentrations in human for an increase in the excretion rate of proline. This kidney is similar to that estimated for both the rat and hypothesis remains to be evaluated. the rabbit transport system [8, 11]. In studying the effects of various inhibitors on the Summary transport system in human kidney, differences in the Studies of L-proline uptake by human kidney cortical physiologic system are apparent. The effects of inhibi- slices indicated the existence of two separate transport tors suggest that neutral amino acids as represented by systems with differing affinity and capacity for proline. a-aminoisobutyric acid reduce proline uptake propor- Oxidation and conversion to glutamic acid of the accu- tionally more from physiologic concentrations than mulated intracellular proline occurred so rapidly that from high media concentrations. The data from our a true gradient for proline at physiologic concentra- study and that of the isolated rabbit tubules system tions did not exist. Inhibition of proline metabolism delineate a component of the low K system for pro- m either by anaerobiosis or by thioproline resulted in a line which is shared with neutral amino acids [8]. In significant reduction in proline uptake by the slices. contrast, the same system in rat cortical slices does not The data suggest that changes in renal metabolism of interact with neutral amino acids as represented by proline may influence physiologic function of tubular either a-aminoisobutyric acid or alanine [11]. transport systems. In an attempt to separate proline metabolic effects from influencing the cellular entry process, proline an- References and Notes alogues were used. Thioproline is metabolized by rat liver mitochondria by the same enzyme, proline oxi- 1. BAERLOCHER, K. E., SCRIVER, C. R., AND MOHYUDDIN, F.: The ontogeny of amino acid transport in rat kidney. I. Effect of dase, which catalyzes the conversion of proline to glu- distribution ratios and intracellular metabolism of proline tamic-y-semialdehyde [9]. The fact that thioproline in- and glycine. Biochim. Biophys. Acta, 249: 353 (1971). hibited glutamic acid formation and caused an in- 2. BAERLOCHER, K., SCRIVER, C, AND MOHYUDDIN, F.: The on- crease in cellular radioactive proline is consistent with togeny of amino acid transport in rat kidney. II. Kinetics of this compound having a primary effect on proline oxi- uptake and effect of anoxia. Biochim. Biophys. Acta, 249: 364 dation. However, we cannot rule out the possibility (1971). 3. BRODEHL, J., AND GELLISSEN, K.: Endogenous renal transport that thioproline did not have an effect on the site of of free amino acids in infancy and childhood. Pediatrics, 42: proline transport in the cell membrane. DL-3,4-Dehy- 395 (1968). L-Proline metabolism and uptake 825

4. DICKINSON, J. C, ROSENBLUM, H., AND HAMILTON, P. B.: Ion 19. SELKOE, D. J.: Familial hyperprolinemia and mental retarda- exchange chromatography of the free amino acids in the tion. Neurology, 19: 494 (1969). plasma of the newborn infant. Pediatrics, 36: 2 (1965). 20. SIMILA, S.: Hyperprolinaemia type II. Ann. Clin. Res., 2: 143 5. EFRON, M. L.: Familial hyperprolinemia: Report of a second (1970). case, associated with congenital renal malformations, heredi- 21. SOUPART, P.: Free amino acids of blood and urine in the hu- tary hematuria and mild mental retardation with demonstra- man. In: J. T. Holden: Amino Acid Pools, p. 220 (Elsevier tion of an enzyme defect. New Engl. J. Med., 272: 1243 (1965). Publishing Company, Amsterdam, 1962). 6. FELIG, P., OWEN, O. E., WARREN, J., AND CAHILL, G. F., JR.: 22. TADO, K., MOVIKAWA, T., ANDO, T., YOSHIDA, T., AND MINA- Amino acid metabolism during prolonged starvation. J. Clin. GAWA, A.: Prolinuria. New renal tubular defect in transport Invest., 48: 584 (1969). of proline and glycine. Tohoka J. Exp. Med., 87: 133 (1965). 7. Fox, M., THIER, S., ROSENBERG, L., RISER, W., AND SEGAL, S.: 23. TENENHOUSE, A., AND QUASTEL, J. H.: Amino acid accumulation Evidence against a single renal transport defect in cystinuria. in Ehrlich ascites carcinoma cells. Can. J. Biochem. Physiol., New Engl. J. Med., 270: 556 (1964). 38: 1311 (1960). 8. HILLMAN, R. E., AND ROSENBERG, L. E.: Amino acid transport 24. WHELAN, D. T., SCRIVER, C. R., AND MOHYUDDIN, F.: Glutamic by isolated mammalian renal tubules. II. Transport systems acid decarboxylase and gamma-aminobutyric acid in mam- for L-proline. J. Biol. Chem., 244: 4494 (1969). malian kidney. Nature, 224: 916 (1969). 9. JOHNSON, A. B., AND STRECKER, H. J.: The interconversion of glutamic acid and proline. IV. The oxidation of proline by 25. WOOLF, L. I., AND NORMAN, A. P.: The urinary excretion of amino acids and sugars in early infancy. J. Pediat, 50: 271 rat liver mitochondria. J. Biol. Chem., 237: 1876 (1962). (1957). 10. MACKENZIE, S., AND SCRIVER, C. R.: Transport of L-proline and a-aminoisobutyric acid in the isolated rat kidney glo- 26. New England Nuclear, Boston, Mass. merulus. Biochim. Biophys. Acta, 241: 725 (1971). 27. Schwarz-Mann Research Company, New York, N. Y. 11. MOHYUDDIN, F., AND SCRIVER, C. R.: Amino acid transport in 28. Packard Instrument Company, Downers Grove, 111. mammalian kidney: Multiple systems for imino acids and 29. Fisher Scientific Company, King of Prussia, Penn. glycine in rat kidney. Amer. J. Physiol., 219: 1 (1970). 30. Beckman-Spinco amino acid analyzer model 120C, Beckman- 12. ROSENBERG, L. E., DOWNING, S. J., AND SEGAL, S.: Extracellular Spinco, Palo Alto, Calif. space estimation in rat kidney slices using C" saccharides and 31. Nutritional Biochemical Company, Cleveland, Ohio. phlorizin. Amer. J. Physiol., 202: 800 (1962). 32. Calbiochem Corporation, San Diego, Calif. 13. ROSENBERG, L. E., DURANT, J. L., AND ELSAS, L. J., II: Familial 33. The authors wish to express their gratitude to Drs. John iminoglycinuria: An inborn error of renal tubular transport. Murphy and Harry Schoenberg for supplying the surgical New Engl. J. Med., 278: 1407 (1968). specimens. 14. ROSENBERG, L. E., AND SCRIVER, C. R.: Amino acid metabolism. 34. This work was presented in part at the Society for Pediatric In: P. K. Bondy: Duncan's Diseases of Metabolism, Ed. 6 Research, Atlantic City, N. J., May 1970. (Saunders, Philadelphia, 1969). 35. Dr. P. Holtzapple is the recipient of United States Public 15. SCHAEFER, I. A., SCRIVER, C. R., AND EFRON, M. L.: Familial Service Career Development Award no. K9-HD-70026. hyperprolinemia, cerebral dysfunction and renal anomalies 36. The present address of Dr. M. Genel is: Department of Pe- occurring in a family with hereditary nephropathy and deaf- diatrics, Yale University School of Medicine, New Haven, ness. New Engl. J. Med., 267: 51 (1962). Conn. 16. SCRIVER, C. R.: Renal tubular transport of proline, hydroxy- 37. This work was supported by a grant from the John A. Hart- proline and glycine. III. Genetic basis for more than one mode ford Foundation and by Grant no. AM 10894 from the Na- of transport in human kidney. J. Clin. Invest., 47: 823 (1968). tional Institutes of Health. 17. SCRIVER, C. R., AND WILSON, O. H.: Amino acid transport: Evidence of genetic control of two types in human kidney. 38. Requests for reprints should be addressed to: P. HOLTZAPPLE, Science, 155: 1428 (1967). M.D., Children's Hospital of Philadelphia, 1740 Bainbridge 18. SEGAL, S., AND CRAWHALL, J. C: Transport of cysteine by hu- St., Philadelphia, Penn. man kidney cortex in vitro. Biochem. Med., 1: 141 (1967). 39. Accepted for publication May 22, 1973.