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The Renal Clearance of Amino Acids in

J. C. Crawhall, … , C. J. Thompson, R. W. E. Watts

J Clin Invest. 1967;46(7):1162-1171. https://doi.org/10.1172/JCI105609.

Research Article

The renal clearance of , lysine, , arginine, and glycine has been compared with the simultaneously determined glomerular filtration rate in nine cystinuric patients. Five were studied before and after stabilization on penicillamine therapy, two were studied only while taking penicillamine, and two were studied only in the absence of penicillamine administration. The renal clearances of -penicillamine and of penicillamine were also determined in the patients who were taking the drug.

Amino acids were determined by quantitative ion exchange chromatography, and the reliability of the method has been evaluated in terms of its reproducibility and of the recovery of known amounts of amino acids added to plasma and to . The plasma levels of cystine and of the basic amino acids were less than normal in all the patients. Cysteine- penicillamine and penicillamine disulfide were cleared by the at rates similar to that of cystine. Two of the patients had glycine clearances that were considerably above the normal value. The renal clearance of cystine exceeded the glomerular filtration rate in six of the nine patients. The results form a continuum from values approximately equal to the glomerular filtration rate to values about twice this amount. The renal clearances of cystine and of the basic amino acids vary independently of one another in the disease.

The significance of these results […]

Find the latest version: https://jci.me/105609/pdf Journal of Clinical Investigation Vol. 46, No. 7, 1967

The Renal Clearance of Amino Acids in Cystinuria *

J. C. CRAWHALL, E. F. SCOWEN, C. J. THOMPSON, AND R. W. E. WATTS t (From the Medical Professorial Unit, [Dunn Laboratories], St. Bartholomew's Hospital, London, E.C.1., England)

Summary. The renal clearance of cystine, lysine, ornithine, arginine, and glycine has been compared with the simultaneously determined glomerular filtration rate in nine cystinuric patients. Five were studied before and after stabilization on penicillamine therapy, two were studied only while taking penicillamine, and two were studied only in the absence of penicillamine ad- ministration. The renal clearances of cysteine-penicillamine and of penicil- lamine disulfide were also determined in the patients who were taking the drug. Amino acids were determined by quantitative ion exchange chromatog- raphy, and the reliability of the method has been evaluated in terms of its reproducibility and of the recovery of known amounts of amino acids added to plasma and to urine. The plasma levels of cystine and of the basic amino acids were less than normal in all the patients. Cysteine-penicillamine and penicillamine disulfide were cleared by the kidney at rates similar to that of cystine. Two of the patients had glycine clearances that were considerably above the normal value. The renal clearance of cystine exceeded the glomeru- lar filtration rate in six of the nine patients. The results form a continuum from values approximately equal to the glomerular filtration rate to values about twice this amount. The renal clearances of cystine and of the basic amino acids vary independently of one another in the disease. The significance of these results is discussed in terms of the nature of the renal tubular transport defect that underlies the disorder.

Introduction Frimpter and co-workers (5) each have also re- During our investigations of the action of D(-) - ported single patients in whom the clearance of penicillamine 1 in the treatment of cystinuria (1, 2) cystine was significantly greater than the glomeru- we have measured plasma and urinary amino acids lar filtration rate. Glycine has also been before and during therapy with this drug. Dent, reported to be increased in cystinuria (5). Senior, and Walshe (3) reported that the renal In this paper we present cystine, lysine, orni- clearance of cystine was close to the glomerular thine, arginine, and glycine clearance data of nine filtration rate (GFR) in the disease; this was patients with cystinuria, in six of whom the renal confirmed in three out of four patients studied by clearance of cystine was greater than GFR. The Arrow and Westall (4) and in three of the four effect of penicillamine therapy on the blood and patients studied by Frimpter and associates (5). urinary concentrations and on the renal clearances Dent and Senior (6), Arrow and Westall (4), and of these amino acids and of cysteine-penicillamine and penicillamine disulfide is also reported. Some * Submitted for publication October 17, 1966; accepted of these results have been the subject of a prelimi- March 21, 1967. t Address requests for reprints to Dr. R. W. E. Watts, nary communication (7). Dunn Laboratories, St. Bartholomew's Hospital, London, E.C.1., England. Methods 1 D(-)-penicillamine will be subsequently referred to Determination of the renal clearances of the amino as penicillamine. acids and simultaneous measurement of GFR. The amino 1162 RENAL CLEARANCE OF AMINO ACIDS IN CYSTINURIA 1163

TABLE I Reproducibility of the determination*

Reproducibility (n comparisons) Aqueous solution Urinet Plasmaj Amino acid Mean SD n Mean SD n Mean SD n Cystine 100.0 2.72 12 102.2 6.69 6 101.9 8.11 21 Ornithine 100.4 2.56 12 98.6 3.69 6 104.3 8.58 21 Lysine 99.6 1.79 12 102.0 6.80 6 100.5 6.94 21 Arginine 101.3 1.81 12 101.7 6.09 6 99.9 7.66 21 Glycine 104.1 6.50 11 101.4 4.16 6 102.6 6.66 21 * In the case of aqueous amino acid solutions, a volume (1.0 ml) of the standard amino acid solution, which contained 0.1 pmole of each amino acid, was analyzed and the mean value obtained expressed as a percentage of this. In the case of urine and plasma, each sample was analyzed twice, the value of 100% was arbitrarily assigned to the result of the first determination, the result of the second analysis was expressed as a percentage of this, and the standard deviation of the (n) individual comparisons for each amino acid about the corresponding mean was calculated. t Cystinuric. t Normal. acid clearances and GFR were measured simultaneously, inulin (45 mg per kg body weight dissolved in 200 ml the renal clearance of inulin being used to measure GFR 0.9%o NaCl) was infused over the course of approxi- in all but one study (Patient 1, study before penicilla- mately 20 minutes and was followed by a maintenance in- mine treatment), when the endogenous creatinine clear- fusion (25 mg inulin per minute per 100 ml approximate ance was employed. The patients were encouraged to GFR, which had been previously determined on the drink water freely during the 9 hours before the test; basis of the 24-hour endogenous creatinine clearance). they were otherwise fasting, and penicillamine was with- The urine collection periods were timed to the end of held for 12 hours before the investigation. The amounts micturition and were begun not less than 30 minutes of inulin infused and the timing of the blood and urine after the maintenance infusion was begun. Each urine collections were the following: 1) (Patients 1, 4, and 5). collection period lasted approximately 15 minutes. Blood The loading infusion of inulin (4 g dissolved in 500 ml samples were obtained at recorded times immediately be- 0.9% NaCl solution) was given over the course of 30 fore the study, at the beginning of the first clearance pe- minutes and followed by a maintenance infusion (2 g inu- riod, in the middle of the second and third clearance pe- lin per 500 ml 0.9%b NaCl solution) at a rate of 2 ml riods, and immediately after the third clearance period. per minute for the rest of the study. Timed 30-minute The plasma inulin concentrations were plotted against urine collection periods (without catheterization) were time, and the concentration at the midpoint of each begun 30 minutes after the start of the maintenance in- clearance period was determined by interpolation. We fusion. Blood samples were obtained immediately be- introduced this modification of the clearance technique fore the study and at the midpoint of each clearance to improve the design of the procedure, not because the period. first method gave unsatisfactory clearance data. 2) (Patients 2, 3, 6, 7, 8, and 9). The loading dose of Preparation of blood samples for analysis. Blood was drawn from an antecubital vein with a heparinized sy- TABLE II ringe and immediately centrifuged at approximately 50 C The recovery of amino acids added to urine and plasma* to separate the erythrocytes. Plasma (2.5 ml) was re- moved, a known amount of (0.1 /Amole, 0.1 ml Recovery solution) was added as an internal standard, and the Plasmat protein was precipitated with salicylsulfonic acid (2.5 Urinet ml). The precipitate was removed by centrifugation and washed. The combined supernatants were evaporated to Glycine 99.5 (10.5)§ 96.3 (15.6)§ dryness in a rotary evaporator at 400 C. Cystine 96.1 ( 4.1) 100.6 ( 9.8) Ornithine 107.0 ( 4.9) 98.7 (11.0) Analytical methods. Amino acids were determined Lysine 99.5 (10.1) 111.7 (21.1) as described by Crawhall, Thompson, and Bradley (8) Arginine 99.3 ( 5.3) 100.3 (11.4) with a Technicon amino acid analyzer. The standard amino acid mixtures for calibrating the instrument were * A known amount (0.1 Mmole) of each amino acid also obtained from Technicon Co.2 Cysteine-penicillamine (1.0 ml) of the standard mixture of amino acids in 0.1 N HCl was added to each specimen of urine and plas ma. and penicillamine disulfide were prepared as described An equal volume of 0.1 N HCI was added to the control previously (2). DL-Ornithine and DL-norleucine were samples. t Cystinuric, 16 determinations. I Normal, 28 determinations. 2Technicon Instruments Co., Ltd., Hanworth Lane, § Standard deviation. Chertsey, Surrey, England. 1164 CRAWHALL, SCOWEN, THOMPSON, AND WATTS

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1- RENAL CLEARANCE OF AMINO ACIDS IN CYSTINURIA 1165

obtained commercially.3 The reproducibility of this t- 00 (1 '- en rawt- \C \0 \0 method was tested for aqueous amino acid solutions, .. * . * . . . ." *! .~ C1 e- e4 --I _ e m eno urine, and plasma (Table I). The recoveries of known t- 00 m \-4 e- r;\ 6O'40eC~ -4 l amounts of the amino acids for which we report ana- lytical data are shown in Table II. en_t X) 0%uie'me mc en0 00 Creatinine and inulin were estimated by the methods of Edwards and Whyte (9) and Schreiner (10), re- spectively.

00 " o6w om o e5o m-ooi Patients. The clinical states of the patients are sum- . . . cli e13 6 .~ .:: .4 t. .6 marized in the Appendix. They were hospitalized for these 10 'xICVo '4+4utLn CN-4 ,t . 5 cd investigations. Patients 2, 3, 6, 7, and 9 ate a repetitive 0 'In diet for at least 6 days before the studies; Patients 1, 4, .E and 8 ate an unrestricted diet. 4) r0- 40 Id X~of o6 0 - -_4 ---s - Results I-00 -000 00 00 to 000A Ci1;- lC-i e's!~ The concentrations of glycine, cystine, lysine,

a4- ornithine, and arginine in plasma and urine of the 0% . m1 z VQ*= nine patients during the clearance periods are pre- -4 - U)41).,J A %0-4--el- sented in Table III. Clearance measurements were not made on Patients 3 and 7 before penicil-

v) e lamine therapy was begun or on Patients 8 and 9 = .. -i CtNs a,0 )O00o- UZuy 'I od...0 4441) u) of ou0 while they were on penicillamine treatment. Pa- ax --- - - _q ci tient 4 was studied twice before and twice while t-Zo U)6M- CO- _ Min taking penicillamine. The fasting plasma cystine _l4 _4 C C4 C4 C1 C1 Cq C4 e C14 N concentrations agree with the values obtained in this disease by previous investigators (4, 5, 11), .-4) d4me 00r-%0 It 01%0 0X00 be r L. ._ and penicillamine treatment reduced it by more than half in all cases. The concentrations of cys- --o Uote NU n teine-penicillamine sometimes exceeded the con- %0%0 0 -4 "tCi00t-o O%4-00 _ _ _ centrations of cystine, and the concentrations of -- --- _ e e _.eI 14-___I 4-_ -4 V_4_1 J. penicillamine disulfide were considerably lower cd r 0 than those of the other . The precision o - ui to E Cu%%0o .14 r-merge000%0oow 0 tpoC)OoQn 4) " ci ci' "'iC' . SC'iC'i 4 de ed of the penicillamine disulfide analysis is, therefore, 1oX'"' less than that of the other amino acids. The con- 4) :k o0 U) 00 en d centrations of lysine and arginine in the- plasma agree with the data of Frimpter (5, 12), being rather lower than earlier reported values in nor- a- 00 m c- mal subjects (11, 12). The concentration of orni- thine is more variable among different patients 0404 and within the normal range (2.95 to 10.6 jMmole 0pro Odes~3t QCd 44S.° u0000 per 100 ml) (13) in some of the studies. Penicil- lamine administration did not alter the plasma con- C500 C;Cs1 C;L _s 1-4 11. rag lo c centrations of the basic amino acids. S~~~~~~~~~~L oo~~m i~% -4 0%'-44- 00 r-t-.. r3.0% 1-l1)%0 00 4 O 4 .6 4 X The amino acid clearances and the simultane- * - -4 -- -- _-4 ously determined GFR are shown in Table IV.

-4C1 v- Cl M C, M .d C4 M The ratio of cystine clearance (Ccystine) to GFR U was unaltered by penicillamine administration (Pa- cio04 .~4 0 0o 0C tients 1, 2, 4, 5, and 6), and varies between about U) NS1 0 bn one and two. In six of the nine patients the cys-

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00 Ch RENAL CLEARANCE OF AMINO ACIDS IN CYSTINURIA 1167 and in protein hydrolyzates is well documented, 1.3 [] but there have been few detailed investigations of its validity when applied to biological fluids, 1.1 which often present a more complicated analytical 1.01 problem. Preliminary experiments confirmed 0.91 that the reproducibility of the present method 0.8 0 (Table I) when applied to aqueous solutions was p0.7 0 PATIE0* satisfactory, the standard deviations of the indi- 0.6 vidual values about their mean (expressed as 0.5

0.4 100%o) being 2.72% or less for cystine and the

0.3 O-PATIENT 9 basic amino acids. Similar values are reported by

0.2 Spackman, Stein, and Moore (14) and Dickin-

0.1 son, Rosenblum, and Hamilton (13). We have no explanation for the larger standard deviation 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 CARGININE G.F.R. that we observed for glycine. The results obtained with urine and plasma showed a wider spread of FIG. 1. RELATIONSHIP BETWEEN THE RATIOS Clyuine/ GFR AND Carginlne/GFR. The regression line was cal- values about the corresponding means, lysine and culated by the method of averages (the value enclosed ornithine having the largest standard deviations in in brackets was omitted from the calculation). The star urine and plasma, respectively. This could have indicates two identical values. C =clearance; GFR= been due to small amounts of interfering sub- glomerular filtration rate. stances that behaved inconsistently on the ion exchange column. Average recoveries within 5% tine clearance is greater than the glomerular of the theoretical results were obtained for glycine, filtration rate. In general, the clearance of cys- cystine, lysine, and arginine in urine and for gly- teine-penicillamine tends to parallel the cystine cine, cystine, ornithine, and arginine in plasma. clearance, but the clearances of the basic amino The recovery of ornithine from urine and of. ly- acids do not necessarily parallel the clearances of sine from plasma was usually high, suggesting in cystine. The present values for the ratio Carginine/ this case also that there was interference by an- GFR range from 0.33 to 1.13 except for Patient 9, other Ninhydrin-reacting compound, which was in whom this value was 0.051. The ranges of the not being separated from the amino acid con- ratios Clysine/GFR and Cornithine/GFR are 0.44 to cerned; this was tryptophan in the case of plasma 0.78 and 0.36 to 0.59, respectively. Patient 9 is of lysine. Urine contains no tryptophan, so that this special interest because her high cystine clearance effect is not observed when it is analyzed. The (Ccystine/GFR = 1.47) is associated with smaller range of values for the recovery data is wider for increases in the basic amino acid clearances than plasma than for urine, possibly due to the forma- in the other patients, this being particularly tion of small and variable amounts of low molecu- marked for arginine (Carginsne/GFR = 0.05, Clysine/ lar weight substances during the deproteinization GFR = 0.30, Cornithine/GFR = 0.14). procedure, loss of free amino acids by absorption When the clearance ratios Cargiiine/GFR and onto the denatured protein precipitate, or both. Cline/GFR are compared graphically (Figure 1), Cystine is known to be absorbed onto plasma pro- the results for Patient 9 lie on the line describing teins before precipitation (13), but loss due to the results for the other patients. this can be minimized by precipitating the plasma proteins within 15 minutes of drawing the blood, Discussion as was done in the present studies. Salicylsulfonic Accuracy of the method of amino acid analysis. acid is a convenient protein precipitant because it Ion exchange chromatography with automated does not have to be removed before the protein- methods of determining the amino acids in the col- free supernatant is applied to the column of ion umn effluent has been widely applied to the analy- exchange resin. Dickinson and associates (13) sis of blood and urine. Its reliability for the concluded that it was at least as satisfactory as any determination of amino acids in aqueous solutions of the other methods that they studied; however, 1168 CRAWHALL, SCOWEN, THOMPSON, AND WATTS Gerritsen, Rehberg, and Waisman (15) think because some of the patients (1, 2, 6, 7, and 8) did that it to significant loss of some amino acids not have radiologically demonstrable calculi. as compared with ultrafiltration. The cystine Frimpter and co-workers (5) have discussed determinations on both urine and plasma were the significance of bidirectional flow of amino acids among the most satisfactory from the point of view across the tubule cell as a possible mechanism to of recovery and reproducibility (Tables I and II); account for cystine clearances greater than GFR the possible discrepancies that we have enumerated in cystinuria. Transport from the lumen of the would not explain the relatively large differences renal tubule involves an influx pathway (IRT) between, for example, cystine clearance and glo- from the renal tubule into the cell and an efflux merular filtration rate that we report. pathway (Epo) from the cell into the peritubular Significance of Ccy8tjne/Cjwui,& greater than 1. capillaries. Some reverse flow can also be envi- While investigating the renal amino acid clear- saged along the pathway (ERT) from the cell into ance of nine patients with cystinuria, we have en- the lumen of the tubule and along an influx path- countered six patients in whom the renal clear- way (Ipc) from the peritubular capillaries into ance of cystine was greater than the GFR. Only the cell. In this system, the experimentally de- one of these was of a magnitude comparable to the termined ratio Ccyttine/GFR depends upon the ac- patient reported by Frimpter and associates (5), tivities of IRT and ERT, and the occurrence of ra- and the others appear to represent a continuous tios greater than unity could be accounted for by range from clearances equal to the GFR to clear- a partial or complete defect that slows the rate of ances twice the GFR. No attempt was made in transport along IRT without affecting ERT. This the present work to fractionate cysteine from cys- concept of the disease is in accord with the in tine. However, the former amino acid cannot be vitro demonstration that kidney cortex slices from detected in urine, so that even if it is assumed that cystinuric patients seem capable of actively trans- all the plasma cysteine was oxidized to cystine dur- porting cystine (17), but it is not known if any ing the preparation and analysis of the plasma of the patients whose renal tissue was examined samples, the resulting error would reduce and not in this way (17) had cystine clearance that was increase the ratio Ccystine/GFR. greater than GFR. It has been suggested (4, 6) that the occurrence It has been shown that cystine does not exist of cystine clearances that are greater than the as such in the human renal cortex but is wholly glomerular filtration rate in cystinuria could be present in the reduced form (i.e., as cysteine) due to cystine being dissolved from calculi, and (18). Thus, it may be cysteine rather than cys- that this would be particularly likely to occur dur- tine that is transported along the pathways Ep0 ing a diuresis. Further studies of cystinuric stone- and ERT. The appearance of more cystine in the forming patients have shown that a consistent in- urine than can be accounted for by cystine filtered crease in the cystine excretion occurred only in one at the glomerulus could be due to increased de out of six patients during a diuresis of comparable novo synthesis of cystine in the renal tubular epi- magnitude to that achieved in the present studies thelium. Some support for this idea is provided (16). The rates of urine flow and the urinary by the observations of Frimpter (12) and of cystine concentrations vary during some of the Rosenberg, Durant, and Holland (19), who found clearance studies (e.g., study 2 on Patient 4, Ta- that the cystine concentrations in renal artery and ble III) without a corresponding change in the -renal venous blood were the same. Frimpter (12) cystine clearance or in the glomerular filtration also reported that the concentration of cysteine in rate (Table IV), and this supports the view that renal venous blood was less than that in the blood the occurrence of Ccytine/GFR ratios greater than that reached the kidney, producing a high ex- unity cannot be explained by particularly high traction of this amino acid. This observation was rates of urine flow during part of the investigation. not, however, confirmed by Rosenberg and as- In addition, we do not think that stone dissolution sociates (19). explains the cystine clearance values that exceeded The renal clearances of cysteine-penicillamine the glomerular filtration rate in the present work and penicillamine disulfide are of the same order RENAL CLEARANCE OF AMINO ACIDS IN CYSTINURIA 1169 of magnitude as the cystine clearance and do not The present observation that the magnitude of the depend on the dose of penicillamine under the con- transport defect varied among the different amino ditions of these studies. This suggests that the acids as well as among different patients raises cystinuric kidney may handle cystine and these the possibility that the control of the formation of "foreign" disulfides by the same, or similar, mecha- these mechanisms may depend upon the activity of nisms and that some of these disulfides appear in more than one gene. This situation could prevail the urine not only by glomerular filtration, but either because the individual amino acids have also by Ipc and ERT. It is also possible, by anal- separate carrier proteins or because there is a ogy with cysteine (18), that penicillamine disulfide modifying effect from other genes that influences and cysteine-penicillamine may be reduced to the activity of a single carrier protein in such a in the renal tubular cells. way as to alter its affinity for the individual Penicillamine administration lowered the plasma amino acids differentially. and urinary cystine concentrations, but the cor- Patient 9 is of particular interest because she responding values for the basic amino acids were shows an extreme degree of dissociation of the unaltered. The renal clearance values for the amino acid transport defects; thus, although the basic amino acids were, like the cystine clearance, ratio Ccystine/GFR was 1.49, the clearances of the unaltered by penicillamine administration. Thus, basic amino acids were relatively lower than there is no increased loss of basic amino acids, and those observed for any of the other patients. In the over-all excretion of cystine plus the cysteine particular, the excretion of arginine was extremely moeity of cysteine-penicillamine is decreased by low relative to that of cystine. Another patient penicillamine therapy (7). with similar clearance data has recently been en- Scriver, Efron, and Schafer (20) reported that countered in this laboratory (21), and patients the normal renal clearance of glycine was 4.7 (SD with high cystine but low arginine ex- 1.3) ml per minute per 1.73 M2. Some of the cretions have been reported by Crawhall, Saun- present group of patients had glycine clearances ders, and Thompson (22) and by Harris, Mitt- that were above this range, but only Patients 6 woch, Robson, and Warren (23). When the and 8 had glycine clearances that approached the clearance ratios Cargine/GFR and Cly8ine/GFR value (23 ml per minute per 1.73 m2) reported by are compared graphically (Figure 1), the results Frimpter and co-workers (5) for their patient for Patient 9 lie on an extension of the line that with a cystine clearance greater than the glomeru- describes the results for the other patients. This lar filtration rate. suggests that the manifestations of the disease in Dissociation of the amino acid transport defects this patient represent an extreme degree of the in cystinuria. There are striking, differences in same genetic abnormality as exists in the other the ratios Ccystine/GFR, Clysine/GFR, Cornithine/ patients rather than a separate genetic variant. GFR, and Carginine/GFR from patient to patient Extrapolation of the regression line for Carginine/ and in the relative magnitudes of these ratios GFR on Clysine/GFR cuts the latter axis at a finite within a given patient. These differences were value. These results indicate that at low rates of not related to the administration of D-penicillamine. basic amino acid clearance the defect in lysine Thus, it appears that within a group of cystinuric transport is more marked than the defect in argi- patients there exist variations in the degree of ex- nine transport, although at high rates of basic pressivity of the abnormal genetic mechanism, amino acid clearance the arginine clearance can which does not affect the transport of the four be greater than the lysine clearance (Figure 1). amino acids equally. Such variations of expres- This is compatible with Harris and Robson's (24) sivity with respect to a single structural gene di- generalization that when the reabsorptive capacity recting the synthesis of a carrier for cystine and for the four amino acids is partially limited, argi- the basic amino acids in the kidney would be ex- nine is reabsorbed preferentially to cystine and pected to give rise to variations in the degree of lysine. the amino acid transport defect in which all four A few preliminary studies suggest that the pa- amino acids would be affected to the same extent. tients with Ccystine/GFR greater than unity may 170 CRAWHALL, SCOWEN, THOMPSON, AND WATTS or may not have parents with detectable abnor- References malities of cystine and basic amino acid excretion. 1. Crawhall, J. C., E. F. Scowen, and R. W. E. Watts. Similarly, the occurrence of high basic amino acid Effect of penicillamine on cystinuria. Brit. med. clearances does not appear to be correlated with J. 1963, 1, 588. the types of genetic pattern previously described 2. Crawhall, J. C., E. F. Scowen, and R. W. E. Watts. by Harris and his colleagues (21, 23). Further observations on use of D-penicillamine in cystinuria. Brit. med. J. 1964, 1, 1411. 3. Dent, C. E., B. Senior, and J. M. Walshe. The Appendix pathogenesis of cystinuria. II. Polarographic Clinical data on the patients studied studies of the metabolism of sulphur-containing amino-acids. J. clin. Invest. 1954, 33, Patient 1 is a female with 1216. twenty-three-year-old bi- 4. V. and R. lateral renal calculi for 4 years. Her urinary tract was Arrow, K., G. Westall. Amino acid clearances in cystinuria. J. Physiol. (Lond.) radiologically normal. 1958, 141. Patient 2 is a twenty-five-year-old female who had 142, right nephrectomy for recurrent calculi 15 years ago. 5. Frimpter, G. W., M. Horwith, E. Furth, R. E. Fel- Her left kidney and lower renal tract were radiologically lows, and D. D. Thompson. Inulin and endoge- normal. nous amino acid renal clearances in cystinuria: Patient 3 is an eleven-year-old female who had right evidence for tubular secretion. J. clin. Invest. nephrolithotomy and left renal colic 6 and 3 years ago, 1962, 41, 281. respectively. Her right kidney contained a small calcu- 6. Dent, C. E., and B. Senior. Studies on the treat- lus and had just detectable function on excretion pyelog- ment of cystinuria. Brit. J. Urol. 1955, 27, 317. raphy. 7. Crawhall, J. C., and C. J. Thompson. Cystinuria: Patient 4 is a twenty-one-year-old male with an 18- effect of D-penicillamine on plasma and urinary year history of bilateral renal colic who needed surgical cystine concentrations. Science 1965, 147, 1459. treatment on several occasions. Calculi were present in 8. Crawhall, J. C., C. J. Thompson, and K. H. Bradley. his left kidney. Separation of cystine, penicillamine disulfide and Patient 5 is a thirty-year-old male with a 16-year his- cysteine-penicillamine mixed disulfide by auto- tory of recurrent bilateral renal calculi who required matic amino acid analysis. Analyt. Biochem. 1966, surgical treatment on several occasions. Bilateral cal- 14, 405. culi were present. Pretreatment renal clearance studies 9. Edwards, K. D. G., and H. M. Whyte. The meas- were performed while he had thyrotoxicosis, which was urement of creatinine in plasma and urine. Aust. treated by subtotal thyroidectomy before D-penicillamine J. exp. Biol. med. Sci. 1958, 36, 383. was given. 10. Schreiner, G. E. Determination of inulin by means Patient 6 is a nineteen-year-old male who had been of resorcinol. Proc. Soc. exp. Biol. (N. Y.) 1950, passing stones and "gravel" for 5 years. His renal tract 74, 117. was radiologically normal. 11. Brigham, M. P., W. H. Stein, and S. Moore. The Patient 7 is a twenty-nine-year-old male. He had renal concentrations of cysteine and cystine in human colic and an impacted urethral calculus 5 years ago; his blood plasma. J. clin. Invest. 1960, 39, 1633. left kidney was not functioning then. He had repeated 12. Frimpter, G. W. Cystinuria: metabolism of the di- right renal colic with an impacted calculus and uremia sulfide of cysteine and . J. clin. In- on one occasion. No calculi were present at the time of vest 1963, 42, 1956. the present studies. Patient 13. Dickinson, J. C., H. Rosenblum, and P. B. Hamilton. 8 is a twenty-two-year-old female (sister of Ion Patient 6). She had symptoms of renal calculi for 4 exchange chromatography of the free amino acids in the years and bilateral nephrolithotomies. No calculi are now plasma of the newborn infant. Pedi- present. atrics 1965, 36, 2. Patient 9 is a ten-year-old female who had bilateral re- 14. Spackman, D. H., W. H. Stein, and S. Moore. Au- nal calculi and an impacted left ureteric calculus. The tomatic recording apparatus for use in the chro- latter was removed surgically 5 months before the present matography of amino acids. Analyt. Chem. 1958, studies. 30, 1190. 15. Gerritsen, T., M. L. Rehberg, and H. A. Waisman. Acknowledgments On the determination of free amino acids in se- We acknowledge our indebtedness to the Board of rum. Analyt. Biochem. 1965, 11, 460. Governors of St. Bartholomew's Hospital for their gen- 16. Dent, C. E., M. Friedman, H. Green, and L. A. C. erous research grants. We also appreciate the help of P. Watson. Treatment of cystinuria. Brit. med. J. Woodward, J. Gluck, D. I. Chapman, and P. Purkiss, 1965, 1, 403. as well as Sister J. Parke and the dieticians on our meta- 17. Fox, M., S. Thier, L. E. Rosenberg, W. Kiser, and S. bolic ward. Segal. Evidence against a single renal transport RENAL CLEARANCE OF AMINO ACIDS IN CYSTINURIA 1171

defect in cystinuria. New Engl. J. Med. 1964, 270, glycine in health and in familial hyperprolinemia. 556. J. dlin. Invest. 1964, 43, 374. 18. Crawhall, J. C., and S. Segal. The intracellular cys- 21. Crawhall, J. C., P. Purkiss, E. P. Saunders, and teine/cystine ratio in kidney cortex. Biochem. J. R. W. E. Watts. Unpublished data. 1966, 99, 19C. 22. Crawhall, J. C., E. P. Saunders, and C. J. Thompson. 19. Rosenberg, L. E., J. L. Durant, and J. M. Holland. Heterozygotes for cystinuria. Ann. hum. Genet. 1966, 29, 257. Intestinal absorption and renal excretion of cys- 23. Harris, H., U. Mittwoch, E. B. Robson, and F. L. tine and cysteine in cystinuria. New Engl. J. Warren. Phenotypes and genotypes in cystinuria. Med. 1965, 273, 1239. Ann hum. Genet. 1955, 20, 57. 20. Scriver, C. R., M. L. Efron, and I. A. Schafer. Renal 24. Harris, H., and E. B. Robson. Cystinuria. Amer. tubular transport of proline, , and J. Med. 1957, 22, 774.