entirely by glomerular filtration and was neither reabsorbed nor secreted

6I2.46I.63 INORGANIC SULPHATE EXCRETION BY THE HUMAN KIDNEY. BY CUTHBERT L. COPE. (From the Biochemistry Department, Oxford, and the Radcliffe Infirmary, Oxford.) IN putting forward his modern theory of renal function, Cushny [1917] pointed out that if any substance could be found which was eliminated entirely by glomerular filtration and was neither reabsorbed nor secreted by the cells of the renal tubules, then that substance could be employed to determine the actual rate of glomerular filtration. The truth of this can scarcely be questioned unless we suppose that the process occurring in the glomeruli is not one of simple filtration. For thus estimating glomerular filtration rate, Cushny originally suggested that urea fulfilled the requirements, but later, in view of the accumulating evidence against an uncomplicated filtration of urea, abandoned that substance in favour of inorganic sulphate [1926]. Mayrs [1922] and White [1923] had already employed sulphate excretion for the same purpose in animals. In 1926 Rehberg pointed out the apparent relative advantages of creatinine excretion as an index of filtration rate, and in more recent papers has published observations which he interprets as supporting the view that creatinine fulfils the necessary requirements, i.e. is filtered off in the glomerular filtrate in the same concentration as in the plasma, and is neither reabsorbed nor secreted by the tubule cells. For any substance fulfilling these conditions the mode of calculation of the filtration rate is the same. Thus if Xp and X. be the concentrations of the substance in plasma and in urine respectively, expressed as mg. per 100 c.c., and V be the volume of urine excreted in c.c. per unit of time, then the glomerular filtration rate will be 100 X c.c. in the same unit of time. This ratio, determined for creatinine, Rehberg calls the "glomerular filtration rate," but in the present paper it is considered preferable to employ the ratio XV. and to call this the "clearance for substance X in keeping with the nomenclature now in frequent use in discussing urea excretion.

330 C. L. COPE. The terms "clearance for substance X" and "glomerular filtration rate as estimated by substance X" thus refer to essentially the same ratio, but differ in that the former attempts no interpretation of the physiological significance of the ratio, whilst the latter ascribes to it a meaning which as yet is not strictly justified. For this reason the former term is considered preferable. Of the various substances occurring naturally in the urine, two, viz. creatinine and inorganic sulphate, have been held to afford reliable indices of the extent of glomerular filtration. If the claims for both substances are to be justified it must be shown that the results obtained by the use of creatinine are the same as those obtained with sulphate, and that this agreement holds under all conditions both physiological and pathological. In other words, the clearance for sulphate must always be the same as that for creatinine. Accordingly Mayrs [1922] and White [1923] showed that when the blood sulphate concentration was raised to abnormally high levels by intravenous injection of sodium sulphate, the concentration ratios for creatinine and for sulphate were practically the same. Earlier technical difficulties in the accurate estimation of inorganic sulphate in the low concentrations, encountered in the plasma of normal animals and of men, prevented these observations from being extended to include the normal subject. Nevertheless Poulsson [1930] sought to show that the two clearances were the same in man. He assumed the concentration of plasma inorganic sulphate in his experiments to lie within the limits of normal given by Wakefield [1929]. These limits were considerably below those obtained by other workers using different methods, and more recently Wakefield himself, in collaboration with Power and Keith [1931], and using an improved method, has obtained higher normal values in good agreement with those of other investigators. The deductions of Poulsson are thus based on figures which Wakefield himself has discarded as too low. It is clear therefore, that Po ulss on's suggestion that the creatinine and sulphate clearances are of the same magnitude in man cannot be accepted on the evidence he provides. In the present paper results of a number of direct determinations of the sulphate clearance in normal and in nephritic men are presented in which this claim of Poulsson could not be confirmed. The theoretical bearing of this fact is also considered.

SULPHATE EXCRETION BY KIDNEY. 331 METHODS. Creatinine in urine was determined by the usual method of Folin [1914]. Creatinine in plasma was determined colorimetrically in a F o lin-w ui filtrate. Details of the precautions employed have been given elsewhere; [Cope, 1931 a]. Sulphate in urine. The gravimetric method of Folin [1905] was found entirely satisfactory and was used throughout. Sulphate in plasma and serum. This has been estimated by two entirely independent methods: (i) Nephelometrically, by a modified Denis technique giving an accuracy of about 10 p.c. [Cope, 1931 b]. (ii) By benzidine precipitation and microtitration [Cope, 1931 b], a, method in which the error does not exceed 5 p.c. in the concentrations estimated. In the tables of results these methods are designated by the letters N and B respectively. Urea in blood and in urine was determined by the urease and aeration methods of Van Slyke and Cullen [1914]. RESULTS. Table I shows the results of a series of inorganic sulphate clearance determinations made on healthy young adults, in which the nephelometric method of serum sulphate estimation was used. Observations were made during the morning hours on subjects who had had no breakfast and who were during the observational period moving about the laboratories. The condition was, therefore, not one of complete rest, nor was a diuresis provoked. Urine was collected over a period of one hour, in the middle of which a sample of venous blood was taken for sulphate analysis in the serum. The results are divided into two groups. In the first, plasma sulphate concentration was normal; in the second it had been raised somewhat by the oral ingestion of 10 g. of sodium sulphate the previous evening. It will be seen that, with one exception, the sulphate clearances lie between 26*0 and 51-3, with a mean value for each group of about 35-5. Although, unfortunately, simultaneous creatinine clearance determinations were not made in this series, there can be little doubt that these would be considerably higher than 35-5. Holten and Rehberg [1931] give a minimum normal value of about 60 with a mean of about 90. PH. LXXVI. 22

332 C. L. COPE. TABLE I. Sulphate clearances in normal human subjects. (Nephelometric method.) A. Normal plasma sulphate concentration. Urine Inorganic inorganic sulphate Volume of sulphate excretion Serum Sulphate urine per mg. S per mg. S per inorganic clearance Subject hour 100 c.c. hour sulphate (hourly) Hunt 39-5 99 00 39 00 1 11 35-4 Osborn 51-5 50-86 26-20 0-72 36-1 Osborn 860 46*50 40 00 0-78 51-3 Cope 39-0 61-60 24-00 0-80 30 0 Palin 47.5 107-00 50-88 1.15 44-2 Cope 41*2 55-25 22*76 1-17 19-5 Starling 18*5 284*80 52-68 1F60 32-9 Mean 1*05 35-6 B. 15-18 hours after oral ingestion of sodium sulphate (10 g.). Osborn 57 0 118-60 67-64 1-76 38-2 Talbot 61-0 115-30 70 30 1-76 40 0 Cope 54 0 160-60 86-90 1-87 46-6 Watson 27-0 249*20 67-05 1-92 34.9 Palin 38-0 156-70 59-57 2-24 26-6 Bosworth 189*0 39-48 74-63 2-26 33 0 Cope 46-0 133-60 61-45 2-27 27-0 Swan 66-1 134-10 88-52 2-42 36-3 Mean 2-06 35.3 Poulsson [1930] obtained creatinine clearance values from 80 to 108 per hour, and the present writer has also obtained similar figures. A series in which simultaneous determinations of urea, creatinine and sulphate clearances were all made on the same subject is shown in Table II. With the exception of (10 a) and (11 a), these determinations were all made with the subject at rest, and in the presence of a large diuresis provoked by combined water and urea ingestion, i.e. the so-called Addis conditions. That such conditions do not materially influence the sulphate clearance is indicated by experiments (10 a) and (11 a) which were performed on the same mornings as (10) and (11) respectively, but during moderate exercise and in the absence of diuresis. Here a very considerable change in conditions has had no significant effect on the sulphate clearance. In this series the sulphate clearance, far from equalling the creatinine clearance, seldom exceeds one-third of the value of the latter, and tends, indeed, to be slightly lower than the maximum urea clearance. That the same essential ratio between the three clearances tends to persist when the functional activity of the kidney is reduced by disease, is shown in the observations on nephritic human subjects given in Table III.

SULPHATE EXCRETION BY KIDNEY. 333 TABLE II. Comparison of urea, sulphate and creatinine clearances in human subject C. L. C. Creatinine Urine Excretion clearance Volume inorganic inorganic Plasma Inorganic A Urea urine sulphate sulphate sulphate sulphate Un- clearance per mg. S per mg. S per mg. S per clearance No. corrected Corrected (hourly) hour 100 c.c. hour 100 c.c. (hourly) Method 1 59-3 90-8 - 662 6-21 41-1 1-34 30-9 N 2 66-9 113-3 656 5-17 33-9 0-97 34-8 N 3 101-0 121-0 42-2 478 7-31 34-9 0-90 38-8 N 4 120-2 135-1 45-0 564 6-48 36-6 1-14 32-1 N 5 119-4 137-7 42-1 442 7-87 34-8 1-34 25-9 N 6 100-5 120-8 36-0 438 7-48 32-8 1-78 18-4 N 7 100-0 115-0 35-0 734 3-98 29-3 1-11 26-4 B 8 109-2 125-0 - 490 8-28 40-56 1-18 34-4 B 9 1046 117-5 820 4-35 35-64 1-00 35-6 B 10 110-0 125-0 667 4-96 33-08 0-905 36-5 B 10 a 115-4 126-0 83 40-1 33-23 0-88 37-8 B 11 107-0 113-0 748 4-24 31-7 1-01 31-4 B 11 a 97-8 109-5 40-6 94-4 38-3 1-11 34-6 B Mean - 119-2 40-0 1-13 32-1 Note. The letters N and B in the last column indicate respectively the nephelometric and benzidine methods of estimating plasma sulphate. Corrected creatinine clearances are obtained by subtracting 0-5 mg. from the estimated plasma creatinine value and recalculating the ratio [see Cope, 1931 a]. TABLE III. Comparison of sulphate clearances with urea and creatinine clearances in nephritis. Plasma Excretion Un- Volume inorganic Urinary inorganic Maximum corrected Corrected of urine sulphate sulphate sulphate Sulphate urea creatinine creatinine per mg. S per mg. S per mg. S per clearance No. clearance clearance clearance hour 100 c.c. 100 c.c. hour (hourly) Method 1 21-9 59-5 67-3 105 0-525 13-3 14-0 26-6 B 2 35-6 95-0 110-0 670 1-87 8-99 41-5 22-2 N 3 69-9 78-2 471 3-14 13-96 65-8 20-9 4 27-1 29-0 52-7 424 2-90 14-15 60-0 20-7 N 5 20-0 59-9 65-4 124 2-44 40-3 50-0 20-5 6 56-8 61-7 296 1-67 11-12 32-6 19-6 B 7 55-2 60-1 114 2-47 40-3 45-9 18-6 8-74-2 81-3 166 0-99 10-0 16-7 16-8 B 9 22-0 39-6 52-3 184 1-90 15-4 28-3 14-9 N 10 17-2 41-2 45-4 190 2-44 19-06 36-2 14-8 N 11 78-5 88-8 148 0-82 8-22 12-2 14-8 12 21-3 54-9 62-4 128 1-04 10-22 13-1 12-6 B 13 4-47 11-9 12-85 50 5-80 93-1 46-5 8-0 N 14 6-53 14-3 17-7 61 5-00 53-7 32-6 6-5 N 15 4-07 7-6 8-14 61 8-00 52-2 32-0 4-0 B 16 25 16-00 151-8 37-9 2-27 N In the final column the letters N and B indicate respectively the nephelometric and benzidine methods of estimating plasma inorganic sulphate. 22-2

334 C. L. COPE. DIsCUSSION. The important point which it is desired to stress is that the value of the sulphate clearance in the human subject, healthy or nephritic, is consistently below that for the creatinine clearance, and is usually only about 30 p.c. of the latter. In no case has the sulphate clearance even approached the value for the creatinine clearance, and this remains true even if uncorrected creatinine clearances are considered. It is evident therefore, that both substances cannot be regarded as indices of glomerular filtration rate. Several alternative explanations of the difference are possible. If it be supposed that the creatinine clearance does indeed represent the glomerular filtration rate, then we must conclude either that about two-thirds of the plasma inorganic sulphate exists in the circulating blood in a non-diffusible form, or else that a similar fraction is reabsorbed in the tubules. The former possibility is unlikely since blood may be completely freed of its inorganic sulphate by dialysis through collodion. It must be conceded, however, that as this process takes some considerable time, the possibility that an indiffusible compound is gradually converted into a diffusible form during dialysis cannot be definitely excluded. The assumption that two-thirds of the sulphate filtered in the glomeruli is reabsorbed during its passage down the tubules, also presents difficulties, for if it were so, it would be reasonable to suppose that the induction of a heavy diuresis, by affording less opportunity for reabsorption, would lead to an increase in the sulphate excretion rate and so in the sulphate clearance. Such an increase apparently does not occur. Moreover, the fact reported by Mayrs [1922] and by others, that ureteric obstruction reduces urea excretion much more than it does that of sulphate, has been interpreted by holders of the filtration-reabsorption view as evidence that urea is more readily reabsorbed than is sulphate. If this conclusion be true, then the sulphate clearance should be higher than that for urea. For although maximal urea clearances are being considered, the reabsorption theory must suppose that even from such dilute urines 50 p.c. or more of the filtered urea is reabsorbed, in order to account for the fact that the creatinine clearance is so much higher than the urea clearance. Yet in the instances here reported, the sulphate clearance tends rather to be lower than the urea clearance, and the experience of Sager [1930] in dogs would appear to be similar, for his results show sulphate clearances sometimes slightly below, and sometimes rather above the urea

SULPHATE EXCRETION BY KIDNEY. 335 clearances. Furthermore, if we postulate such a large reabsorption of sulphate then it is necessary to suppose that through the tubule cells sulphate escapes with ease but creatinine with extreme difficulty, although into other tissues of the body creatinine is readily diffusible whilst sulphate only passes with great difficulty [Denis and Leche, 1925]. If, however, the possibility of secretion into the lumen through the tubule cells be conceded, then the explanation becomes relatively simpler. We may now suppose that the actual glomerular filtration rate is equal to, or somewhat less than, the value indicated by the average sulphate clearance, i.e. about 3-5 litres per hour in the normal human adult. Let us assume that the actual glomerular filtration be only 3 litres per hour. This will correspond to an hourly glomerular clearance activity of 30 for both creatinine and sulphate. If, further, we assume that the sulphate from 500 c.c. of plasma and the creatinine from 9 litres of plasma are eliminated through the tubules, then the observed values can be accounted for, for these figures correspond to tubular clearing activities of 5 and 90 respectively, bringing the total clearing activity of glomeruli and tubules combined, i.e. of the kidneys as a whole, up to 35 and 120 respectively. Such a view requires a behaviour of sulphate and of creatinine towards the tubule cells which is similar to that shown towards other tissues, and consequently would appear preferable to the filtrationreabsorption view which necessitates relative rates of passage of these substances through the tubule cells which are the reverse of those through other tissues of the body. Rehberg bases his hypothesis that creatinine is filtered off, but neither reabsorbed nor secreted in the tubules, upon the following points [Holten and Rehberg, 1931]. (i) Its concentration ratio is higher than that of any other substance. (ii) The excretion rate is independent of the volume of diuresis. (iii) Change in osmotic pressure of the plasma proteins is associated with a change in rate of creatinine excretion in the same direction as would be anticipated if the excretory process were one of ultrafiltration in the glomeruli. Of these, the high concentration ratio and the constancy of excretion with changing urine volume are equally compatible with a secretory mechanism. The relation to plasma protein osmotic pressure, whilst affording evidence suggestive that some creatinine is filtered off in the glomeruli, cannot be taken as evidence that all the creatinine is excreted in this manner.

336 C. L. COPE. In a recent study of the action of cyanide on the isolated kidney, Bayliss and Lundsgaard [1932] observed a very marked drop in the creatinine clearance. Being unwilling to believe that the glomerular filtration rate really fell to such an extent under the influence of cyanide alone, these writers postulated a progressively increasing leakage of fluid and of creatinine outwards from the tubule lumen. Their results, however, are equally compatible with the view that cyanide inhibits a creatinine secretory mechanism in the tubules, so that at the end of the experiment only that fraction of the creatinine which is filtered off in the glomeruli appears in the urine. Whether simultaneously any reduction in the true filtration rate did occur in their experiments can scarcely be decided, but it would seem probable. More direct evidence pointing to a secretory elimination of creatinine is contained in the experiments of Edwards and Condorelli [1928] on the aglomerular kidneys of certain fishes. Nor can the similarity of behaviour of phenol red and creatinine during excretion which was shown by Marshall and Kolls [1919] be brought forward as suggesting a filtration, for although Oliver and Shevky [1929] present evidence that phenol red is eliminated solely by glomerular filtration, yet H6ber [1930] maintains that it is secreted by the tubules, and the more recent work of Chambers on the behaviour of tissue cultures of embryonic tubules suggests strongly a secretory mechanism for the dye. As regards the mechanism of excretion of inorganic sulphate, the situation is scarcely more clear. White [1923], from his experiments on the concentration ratios, concluded that sulphate comes in part through the glomeruli and in part through the tubules by secretion. Starling and Verney [1924] interpreted the fall in rate of excretion and in concentration of sulphate in the urine from a kidney poisoned by cyanide, as evidence of an active secretory process. Such an interpretation was, however, based on the belief that cyanide had no effect on the volume of glomerular filtrate, an assumption which must be considered doubtful in view of the work of H6ber and Mackuth [1927] and of Bayliss and Lundsgaard [1932]. Of the attempts to dissociate glomerular from tubular elimination of sulphate in the frog, the earlier ones of Cullis [1906] and of Atkinson, Clark and Menzies [1921] must be interpreted with caution in view of the possibility of access of the tubular perfusion fluid to the vessels of the glomerular system. In the experiments of Yoshida [1924], this danger would appear to have been lessened. Yoshida found that when sulphate was supplied to the glomeruli of the frog's kidney it was well excreted and

SULPHATE EXCRETION BY KIDNEY. concentrated, but that when applied to the tubules alone it appeared in the urine only in traces. More recently again, Kawasoe [1930], also working on frogs, has stated his conclusions that sulphate is excreted mainly by the glomeruli and possibly also slightly by the tubules. He also believed that no significant reabsorption of sulphate occurred. Such results as these would thus agree well with the view presented here that the glomerular filtration rate is equal to, or somewhat less than the figure indicated by the sulphate clearance. It seems to the writer highly desirable that this possibility should not be lost sight of. There is, strictly speaking, at present no real justification for supposing that the creatinine clearance is a more reliable index of glomerular filtration than is the sulphate clearance. There has of more recent years been a tendency to forget that Rehberg's suggestion was admittedly no more than a hypothesis based on an assumption concerning the behaviour of creatinine in the kidney. The rival claims of sulphate as a filtration index would seem to be at least equally strong. Possibly neither is a true index. Whilst this work was being prepared for the press, there appeared a report of similar work arising from the same line of thought by Haym an and Johnston [1932]. Using a different method of estimating plasma sulphate, they obtain essentially the same results. They find the concentration ratio for inorganic sulphate much below that for creatinine, and in most cases also below that for urea. With such results those presented here are in complete agreement. These workers interpret their findings as evidence of back diffusion in the tubules, a conclusion which, as pointed out above, the present writer considers the less likely possibility. SUMMARY. 337 The excretory activity for inorganic sulphate of the human kidney, either healthy or nephritic, is only about one-third of that for creatinine. Calculations of the glomerular filtration rate based on the excretion of inorganic sulphate thus give a value only about one-third of that indicated by the excretion of creatinine. The theoretical consequences of this fact are considered. This work was done under the tenure of a Beit Memorial Fellowship for Medical Research, and constitutes part of that submitted in a Thesis for the degree of Doctor of Medicine in the University of Oxford in 1931.

338 C. L. COPE. REFERENCES. Atkinson, M., Clark, G. A. and Menzies, J. A. (1921). J. Physiol. 55, 253. Bayliss, L. E. and Lundsgaard, E. (1932). Ibid. 74, 279. Cope, C. L. (1931 a). Quart. J. Med. 24, 567. Cope, C. L. (1931 b). Biochem. J. 25, 1183. Cullis, W. (1906). J. Physiol. 34, 250. Cushny, A. R. (1917). The Secretion of Urine. London. First Edition. Cushny, A. R. (1926). Ibid. Second Edition. Denis, W. and Leche, S. (1925). J. Biol. Chem. 65, 565. Edwards, J. G. and Condorelli, L. (1928). Amer. J. Physiol. 86,383. Folin, 0. (1905). J. Biol. Chem. 1, 131. Folin, 0. (1914). Ibid. 17,469. Hayman, J. M. and Johnston, S. M. (1932). J. Clin. Invest. ii, 607. Hober, R. (1930). Klin. Wochenschr. 9, 2065. Hober, R. and Mackuth, E. (1927). Pfluegers Arch. 216, 420. Holten, C. and Rehberg, P. B. (1931). Acta Med. Scand. 74, 479. Kawasoe, J. (1930). Jap. J. Med. Sci. 4 (Pharmacol.), 94*. Marshall, E. K. and Kolls, A. C. (1919). Amer. J. Physiol. 49, 302. Mayrs, E. B. (1922). J. Physiol. 56, 58. Oliver, J. and Shevky, E. (1929). J. Exp. Med. 50, 15, 601. Poulsson, L. T. (1930). Z. ges. exp. Med. 71, 577. Rehberg, P. B. (1926). Biochem. J. 20, 447. Sager, B. (1930). Arch. exp. Path. Pharmak. 153, 331. Starling, E. H. and Verney, E. B. (1924). Proc. Roy. Soc. B, 97, 321. Van Slyke, D. D. and Cullen, G. E. (1914). J. Biol. Chem. 19, 211. Wakefield, E. G. (1929). Ibid. 81, 713. Wakefield, E. G., Power, M. H. and Keith, N. M. (1931). J. Amer. Med. Assoc. 97,913. White, H. L. (1923). Amer. J. Physiol. 65, 537. Yoshida, H. (1924). Pfluegers Arch. 206, 274.