THE RENAL THRESHOLD OF BILIRUBIN. II*

Transcription

1 THE RENAL THRESHOLD OF BILIRUBIN. II* BY E. H. BENSLEY (From the Department of Metabolism, the Montreal General Hospital, Montreal, Canada) (Received for publication, August 9, 1933) Shortly after the introduction of the van den Bergh method for the quantitative estimation of bilirubin in blood, data accumulated which tended to support the view that there is a renal threshold for this pigment. Solution of the problem was made difficult by the fact that the available tests for bilirubin in urine were relatively unsatisfactory. This accounted largely for the multiplicity of procedures (Gmelin, Rosenbach, Huppert-Cole, etc.), the underlying principle of which is oxidation of bilirubin, with the formation of a series of colored derivatives. It is now fairly well established that there are at least two types of bilirubin in human blood; one gives both the direct and indirect reactions with the van den Bergh reagent and is usually referred to as direct bilirubin; the other gives the indirect reaction only, and is known as indirect bilirubin. The exact chemical compositions of these pigments and their physiological significances are, as yet, imperfectly understood. As will presently be shown, however, their identification is important in renal threshold studies. Thus, correlation of blood and urinary data led to the formulation of the following rules. In conditions in which the serum bilirubin is elevated but the serum gives the indirect reaction only, bilirubinuria never occurs; whereas, when the serum gives the direct reaction, bilirubinuria occurs as soon as the bilirubin in the blood reaches about 4 units; below this level bilirubinuria is absent. In other words, the kidneys are impermeable to indirect bilirubin at all times; whereas, they are permeable to direct bilirubin when its concentration in the blood reaches the threshold value of 4 units. * This work was done with the aid of a grant from Mr. W. S. Fallis of Montreal, a Governor of this hospital. 71

2 72 Renal Threshold of Bilirubin. II These rules, i%st formulated by van den Bergh (1) and con6rmed by McNee (2) and Lepehne (3), have been generally accepted and are quoted widely in the literature. That the renal threshold may vary is, however, suggested by the observations of Andrewes (4) and Retzlaff (5). Though the findings of these authors agree in general with van den Bergh s rules, threshold values are recorded both above and below 4 units. Opposed to the above observations are the tidings of Sch8 and El&berg (6). These workers reported a number of cases of catarrhal jaundice and one of complete congenital atresia of the bile ducts in which there was marked bilirubinuria, though the sera gave the indirect reaction only. They, t,herefore, concluded that bilirubmuria is not dependent upon the type of bilirubin in the blood. In 1930, Hunter (7) reported a very satisfactory method for the detection of bilirubin in urine. It is a diaza procedure and is very sensitive. Rabinowitch (8) has shown that bilirubin, when added to urine, can be detected in aa high a dilution as 1 in 666,000. The test is also highly specific. This test, combined with the procedure to be described presently, has therefore made possible a more satisfactory method of determining whether bilirubin is or is not a threshold substance. In a previous study Rabinowitch (8), using Hunter s test, obtained data which were not in accord with those previously reported; bilirubin was found in urine when the concentration of bilirubin in the blood was much below the threshold level; in some instances, bilirubinuria was found when the bilirubin content of the blood was normal. Because of these findings, Rabinowitch concluded that, Other than the fact that bilirubin could not be detected in normal urine by the method employed, the combined data of this preliminary study fail to support the generally accepted view that there is a renal threshold for bilirubin. It w&s also suggested that failure to detect bilirubin in normal urine may be due not to its absence, but to the limit of sensitivity of the test. Because of the chemical and physical differences between the two types of pigment in blood, Rabinowitch suggested that excretion might depend upon the type of pigment. If so, measurement of total bilirubin in blood would afford no indication of the amount available for excretion. In this study, no observations were made

3 E. H. Bensley 73 with respect to the different types of bilirubin. The writer was, therefore, prompted to extend the investigation by correlating bilirubinuria with the type of bilirubin in the blood. Method of Investigation Selection of Cases-Two types of cases were chosen; namely, (a) those in which bloods gave positive direct van den Bergh reactions with bilirubin values less than 4 units, and (b) those in which bloods gave negative direct reactions with bilirubin values of 1 unit or more. Bloods which gave a positive direct reaction with bilirubin values of more than 4 units were excluded, since bilirubinuria has been demonstrated repeatedly under these conditions. Bloods which gave a negative direct reaction and contained less than 1 unit of bilirubin were also excluded, since such cases are normal, or nearly so, with respect to bile pigment metabolism. Absence of bilirubinuria has been demonstrated repeatedly in such cases. Rabinowitch (8) has shown that, with very few exceptions, the concentration of bilirubin in the blood neither rises nor falls very rapidly; it tends to remain constant for hours at least. This, the the writer found, applies also to the types of bilirubin. Renal threshold studies are thus simplified, since one problem in this type of work is the uncertainty that any given sample of urine corresponds to a given sample of blood. The few exceptions to this rule, such as a rapid hemolytic process, were excluded from this investigation. Cases with polyuria were also excluded for, in spite of the sensitivity of Hunter s test, it appears reasonable that, with highly dilute urines, small quantities of pigment might be overlooked. Apart from this precaution, no attempt was made to regulate urine volume by food or fluid intake. The writer has observed repeatedly that the bilirubin content of the urine was remarkably constant in any one case in spite of variations of urine volume, provided the latter was below the polyuric level. A variety of clinical material was used. The cases included cirrhosis of the liver, chronic passive congestion of the liver due to heart failure, obstructive jaundice due to stone, catarrhal jaundice, pernicious anemia, jaundice due to Llibothriocephalus latus, congenital hyperbilirubinemia, and hepatitis associated with cholecystitis, severe infections, and vomiting of pregnancy.

4 74 Renal Threshold of Bilirubin. II Colleclion of Blood and Urine--In each case, the subject voided 1 to 2 hours prior to the collection of blood. This specimen was discarded. Blood was then withdrawn from a vein and transferred to a tube containing a few crystals of sodium oxalate, care being taken to avoid hemolysis. A sample of urine was then obtained as soon as possible; the time varied between 15 minutes and 2 hours. Since bilirubin in blood and urine tends to deteriorate rapidly, all analyses were made not later than 45 minutes after the specimens were obtained. For.purposes of uniformity, all analyses were made by the writer; no technical assistance was employed. As the procedures are calorimetric, test-tubes were carefully selected with respect to uniformity of thickness of wall, composition of glass, and internal diameter. Examinatkrn of Urine-In Hunter s test, special precautions are described for highly pigmented urines (7). For purposes of uniformity, however, these precautions were taken in all cases, regardless of the amount of pigment. Examination of Blood. Direct van den Bergh Reaction-1 cc. of plasma was placed in each of two tubes. To one tube was added 0.5 cc. of distilled water, and plasma and water were well mixed. This tube served as a control. To the other was added 0.5 cc. of freshly prepared diazo reagent, and plasma and reagent were well mixed. The appearance, in this tube, of the slightest trace of pink within 30 seconds was regarded as evidence of a Kphaaic direct reaction. If the pink colorzdeveloped later, the reaction was regarded as delayed bloods in this series gave either a biphasic or delayed direct reaction; the true negative direct and the so called immediate direct reactions were not observed. In accord with the generally acceptedjinterpretation, a biphasic direct reaction was taken to indicate the presence of the direct type of bilirubin and, when a delayeddirect reaction only was observed, it was assumed that this type of bilirubin was absent. Assuming the intensity of color at the end of 30 seconds to be roughly proportional to concentration ofidirect bilirubin, the amounts of this type of bilirubin were recorded as (a) very faint trace, (b) trace, or (c) plus.1 1 No attempt WM made to use color standards for more exact measurement. Thevalue of such an estimationwould be doubtful since it hasnever been proved that the intensity of color is directly proportional to concen-

5 E. H. Bensley 75 Quantitative Estimation of Bilirmbin-In the majority of cases, simultaneous determinations were made by two methods; namely, the original van den Bergh procedure (9) and the Thannhauser and Andersen modification (10). The van den Bergh technique alone was used in a few cases only. All values are expressed in terms of the van den Bergh unit (1 unit = 0.4 mg. of bilirubin in 100 cc. of plasma (11)). The writer made a large series of simultaneous determinations by these two methods (unpublished data). No significant differences were found when the bihrubin content of the plasma was low with one exception; namely, individuals who were previously deeply jaundiced and in whom the jaundice was subsiding. In such cases the original van den Bergh procedure was not quantitative. There were losses of from 40 to 80 per cent of bilirubin, due to occlusion of pigment during protein precipitation. As cases of subsiding jaundice have often been used in renal threshold studies, the importance of the more quantitative Thannhauser and Andersen procedure is obvious. When both methods were used and the values differed, the higher of the two was accepted as correct. DISCUSSION In this investigation 80 tests were made among forty-six individuals. The cases were grouped according to the presence or absence of direct bilirubin in the blood: Group 1, direct bilirubin; Group 2, no direct bilirubin. Of 52 tests in which the plasma contained direct bilirubin (Group l), bilirubin was found in the urine of forty-seven-an incidence of approximately 90 per cent. Bilirubinuria was apparently not dependent upon total bilirubin in blood (Table I). t&ion of direct bilirubin. Furthermore, accurate calorimetric comparison would have been diflicult, since it would have been necessary to make the comparisonezactlv 30 seconds after addition of the diaz oreagent. If plasma and reagent are allowed to react for more than 30 seconds the color may be intensified owing to coupling of the diazo compoundwith indirect bilirubin. The terms very faint trace, trace, and plus, although approximations, are not entirely without value, in view of the fact that all readings were made by the one observer. * The highest plasma bilirubin value in this series was 4.9 units.

6 76 Renal Threshold of Bilirubin. II The latter ranged between 0.3 and 3.8 units. In fourteen cases with bihrubinuria it was less than 1 unit, and in one case the blood contained 0.3 unit only. The findings, therefore, do not support the view that the direct type of bilirubin behaves as a threshold substance; the kidneys appear to be permeable to it regardless of its concentration in the blood. TABLE Showing Absence of Relationship between Total Concentration of Bilirubin in Blood and Bilirubinuria in Cases in Which Plasma Contained Direct Bilirubin Plasma bilimbin units No. of CasCa Absent TABLE I II Bilirubin in urine PreaCd Showing Relationship between Concentration of Direct Bilirubin in Blood and Bilirubinuria Direct biliiubin No. of basks Absent Very faint trace Trace Plus It will be observed (Table I) that there is no relationship between total concentration of bilirubin in the blood and bilirubinuria. The data in Table II, however, clearly indicate that there is a relationship between concentration of direct bilirubin and bilirubinuria. Thus, of seven tests in which there were very faint traces only of direct bilirubin in the plasma, three only

7 E. H. Bensley 77 showed bilirubinuria; of thirty-seven tests with traces, thirty-six showed bilirubinuria; and of the eight tests with large quantities (plus) of direct bilirubin, all showed bilirubin in the urine. That the kidneys may, at times, offer a barrier to the excretion of the direct type of bilirubm is suggested from failure to 6nd bilirubin in the urine in five of the 52 tests mentioned above. The direct color reactions noted in these five cases were faint. In view, therefore, of the relationship between concentration of direct bilirubin and bilirubin in urine, it is highly probable that these urines did contain bilirubin, but failure to detect it was due to the limit of sensitivity of the test. Of twenty-eight tests in which no direct bilirubin was found in the blood (Group 2), bilirubinuria was detected in four instances TABLE Cases in Which Plasma Contained Plasma birubin No. of cama III No Direct Bilirubin Biliiubin in urine Absent very faint trace unit only, in spite of the fact that the bilirubin content of the plasma ranged between 1.0 and 4.9 units (Table III). This group of tests suggests that, in general, the kidneys are impermeable to the indirect type of bilirubin. This finding agrees with the prevalent view, although there are exceptions. The latter will be dealt with presently. In view of these data, failure to find bilirubin in the urine of normal individuals appears to be due, not to lackof sensitivity of the test as suggested by Rabinowitch (8), but to true absence of bilirubin in urine, because of absence of direct bilirubin in the blood. Very faint traces of bilirubin were found in four urines (Table III) in spite of no direct bilirubin in the blood. This may have been due to a number of conditions. First, it may be possible that the kidneys are occasionally permeable to indirect bilirubin. It is

8 Renal Threshold of Bilirubin. II difficult, a priori, to conceive of a living membrane being completely impermeable to any substance at all times. Secondly, the usual interpretation of the direct van den Bergh reaction is somewhat arbitrary. For example, it is assumed that the develop ment of a pink color within 30 seconds indicates the direct type of bilirubin. It may here be observed that if 60 seconds had been chosen arbitrarily as the proper time interval, instead of 30 seconds, at least two of the four cases of Group 2 showing bilirubiimria would have been included in Group 1. Lastly, one must consider the possibility of desquamation of epithelial cells containing bilirubin. Hyperbilirubinemia leads to deposition of bile pigment in the body tissues, including the renal tubular epithelium and epithelium lining the urinary tract in general. Desquamation of these cells is taking place continuously. The occurrence in the urine of such bilirubin-containing cells has been described in detail by Haessler, Rous, and Broun (12). Disintegration of such cells may account for the very faint traces of bilirubin found in the urine in five tests, in spite of the fact that the blood contained no direct bilirubm. That desquamation of bilirubin-containing cells does not explain the bilirubinuria in the 90 per cent of tests of Group 1 is indicated by failure to find bilirubin in the urine in twenty-four of the twenty-eight tests in which the plasma contained no direct bilirubin, and the iinding of no more than very faint traces in the remaining four. STJMMARY Simultaneous examinations of blood and urine for bilirubin were made in a variety of clinical conditions associated with disturbance of bile pigment metabolism. Hunter s diazo test was used to detect bilirubin in the urine. Urinary findings were correlated not only with total but also with the type of bilirubin in the blood. With few exceptions, bilirubin was found in the urine whenever the blood contained the direct type of bilirubin, regardless of concentration. With few exceptions, no bilirubin was found in the urine, when the blood contained indirect bilirubin only, regardless of concentration. Explanations are suggested to account for the exceptional findings.

9 E. H. Bensley 79 Assuming the explanations to be correct, there is no evidence that direct bilirubin behaves as a threshold substance; the kidneys offer no barrier to its excretion, regardless of its concentration in the blood. The data, in general, agree with the prevalent view that the kidneys are impermeable to the excretion of indirect bilirubin. BIBLIOGRAPHY 1. van den Bergh, A. A. H., Der Gallenfarbstoff im Blute, Leyden (1918), cited by Andrewes (4). 2. McNee, J. W., &n-t. J. Med., 16,390 ( ). 3. Lepehne, G., cited by McNee (2). 4. Andrewes, C. H., Quart. J. Med., 18,19 (192425). 5. Retslaff, K., 2. ges. exp. Med., 34,133 (1923). 6. Schiff, E., and Eliasberg, H., Klin. Woch., 1,189l (1922). 7. Hunter, G., Cunad. Med. ki8n. J., 38, 823 (1930). 8. Rabinowitch, I. M., J. Biol. Chem., 97, 163 (1932). 9. van den Bergh, A. A. H., cited by McNee, J. W., Brit. Med. J., 1, 716 (1922). 10. Thannhauser, J. S., and Andersen, E., Deutsch. Arch. Min. Med., 137, 179 (1921). 11. Hunter, G., Brit. J. Exp. Path., 11,407 (1930). 12. Haessler, H., ROUS, P., and Broun, G. O., J. Exp. Med., 36, 533 (1922).

10 THE RENAL THRESHOLD OF BILIRUBIN. II E. H. Bensley J. Biol. Chem. 1933, 103: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1