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Comparison of glomerular filtration rate measurements using inulin, 51CrEDTA, and a phosphate infusion technique
351
CLINICA CHIMICA ACTA
COMPARISON
OF GLOMERULAR
USING INULIN,
T. C. B. STAMP,
FILTRATION
51CrEDTA, AND A PHOSPHATE
T. E. STACEY
AND
RATE
MEASUREMENTS
INFUSION
TECHNIQUE
G. A. ROSE
The Medical Unit, University College Hosflital Medical School, University Street, London, and The Institute of Urology, Henrietta Street, London, W.C.r. (U.K.)
W.C.1.
SUMMARY I. Measurements of glomerular filtration rate (GFR) using inulin, %rEDTA, and a phosphate infusion technique were compared in order to assess their relative accuracy. 2. GFR was determined from phosphate data in a series of 66 infusions in 50 subjects. Comparison with simultaneous inulin clearance (180 measurements during 38 infusions) gave a ratio of GFR derived from phosphate data (GFR,)/mean inulin clearance of 1.00 & 0.03 (S.E.). Comparison with simultaneous 51CrEDTA clearance (189 measurements in 33 infusions) gave a ratio of %rEDTA clearance/GFR, of 0.94 & 0.03 (S.E.). 3. Separate direct comparison of renal inulin clearance with 51CrEDTA clearance in another series of 65 measurements from 15 infusions gave a ratio of %rEDTA clearance/inulin clearance of 0.96 Jr 0.02 (S.E.). 4. The method of determining GFR without urine collection was also used to compare inulin with WrEDTA. In 13 infusions this urineless GFR ratio of 51CrEDTA/ inulin was 0.92 & 0.04 (S.E.). The urineless GFR gave significantly higher absolute values than mean standard clearances, in the ratio of 1.07 for both inulin and 51CrEDTA; the discrepancy was probably due to incomplete equilibration resulting from purposely shortened infusion times. 5. It was concluded that an infusion technique based on the measurement of phosphate alone gives an accurate measure of GFR, that GFR is consistently underestimated by the use of 51CrEDTA, and that the method without urine collection overestimates renal clearance when infusion times are shorter than 4 h.
INTRODUCTION
Determination of the renal clearance of accurate measurement of glomerular filtration chemical determination of inulin has, however, precluded its adoption for routine purposes and
inulin has long been accepted as an rate (GFR), (see refs. I and 2). The always posed difficulties which have encouraged the search for more easily C&z. Chim. Acta, 30 (1970)
351-358
352
STAMP
et 641.
measured compounds. One of these compounds recently introduced is the Q&romiumlabelled chelate of ethylenediamine tetraacetic acid (SlCrEDTA). While original work suggested that 51CrEDTA clearance was identical with that of in&n+5, further studies have indicated that it may consistently underestimate GFR as judged by inulin measurement@. Anderson7 introduced a technique of phosphate infusion, for the determination of tubular maximal reabsorptive capacity (TmP), from which GFR could be directly determined. By achieving a linear rise in plasma phosphorus levels with time, once Tvn has been reached, the rate of rise in urinary phosphorus excretion relative to the rise in plasma phosphorus should be determined only by GFR. Thus GFR should be measurable as the slope of the straight line given by plotting urinary phosphorus excretion against plasma phosphorus levels, above the plasma level required to achieve Tm. While based on sound theoretical principles, the validity of this method relies in part on the accuracy of GFR determinations made from phosphate data alone. A further method of GFR determination which obviates the necessity for urine collections was first introduced by Earle and Berliner8 and Berger et aln, and has been recently revived by Rose 10.It is based on the principle that if a substance is eliminated from the extracellular fluid only by renal excretion, then after equilibration the achievement of constant plasma levels during a continuous infusion signifies that renal excretion is exactly equal to the rate of infusion. Thus the standard expression UVjP can then be rewritten as intravenous infusion rate (~‘I~“~)~constant plasma level. The present study was undertaken in order to evaluate the use of the phosphate infusion method as a sole measure of GFR by comparison both with inulin and 5lCrEDTA clearances, and in addition to establish more firmly any discrepancy between %rEDTA and inulin clearances by direct comparison of clearances obtained by both standard and “urineless” methods. Certain problems inherent in the urineless method were also clarified. MATERIALS
AXD
XETHODS
56 subjects were studied of whom 15 were volunteers with no evidence of abnormal renal function, and the remainder were largely patients with disorders of calcium and phosphorus metabolism who have been described in more detail elsewherell. 87 infusions were performed of which 66 consisted of phosphate combined either with inulin (38 infusions), 5lCrEDTA (33 infusions), or both (5 infusions). The remaining 21 tests were combined with glucose infusions for the determination of maximal tubular reabsorptive capacity (TmG) ; in IO of these latter both 51CrEDTA and inulin were infused in addition to glucose, while in the remainder only one substance was administered for the detestation of mean clearance and comparison with “urineless” GFR. All subjects were studied after an overnight fast and tests were begun at approximately 9 a.m. Subjects were given 2-3 1 of water to drink during the course of the infusions. A solution of 0.1 M sodium phosphate buffered at pH 7.4 was used (Na,HPO,.zH,O 13.8 g; NaH,PO,.aH,O 3.6 g; water to I 1). Inulin was obtained from Thomas Kerfoot and Co. Ltd., Vale of Bardsley Lancashire, -and 51CrEDTA was obtained from the Radiochemical Centre, Amersham (Code Ch.I3P), In all tests C&Z.Chi~.Acfa,~O(1970)
351-358
GFR BY INULIN, %rEDTA,
AND
PHOSPHATE
353
after a loading dose of inulin and/or WrEDTA, a sustaining infusion in 0.9% NaCl was given using a constant infusion pump (Buchler Polystaltic pump, Buchler Instrument Co. Ltd., Fort Lee, N.J., U.S.A.), and 45-60 min were allowed for equilibration. Phosphate (or glucose) infusions were delivered from a second pump through a single indwelling venous cannula via an open z-way tap. In the phosphate infusions, the flow rate was raised by a constant increment every 15 min by altering a simple dial setting, thus producing a linear rise in plasma phosphorus with time. In some of the infusions equilibration of inulin or WrEDTA was awaited before beginning the phosphate infusion, and 6 or 7 full clearance periods were measured; in others phosphate, inulin and/or WrEDTA were begun simultaneously and only the last 4 collection periods were analysed for %rEDTA and inulin (out of 7 phosphate clearance periods). Bladder catheterisation was unjustifiable, and nearly all patients could void on request. Blood samples could thus be collected from an indwelling catheter in an opposite forearm vein at the planned midpoint of each urine collection. Collection periods were usually of 24 min duration (range 22-32 min). GFR was determined from phosphate data by calculating the slope of the regression of urine phosphorus excretion (mg/min) on plasma phosphorus level (mg/ml) . Only the last 5 collection periods (out of 6 obtained during the infusion) were used to calculate this regression; it was assumed in this way that phosphate reabsorption was constant throughout (i.e. TmP had been reached). Inulin was determined by Dawborn’s12 Auto Analyzer modification of Heyrovsky’s13 3-indolylacetic acid methodl”. 51CrEDTA was counted in a Packard Auto-Gamma spectrometer, and at least 3000 counts were made on each sample. Phosphate was estimated in duplicate or triplicate by a standard Auto Analyzer adaptation of Gomorri’sls method. RESULTS
il. Phos$hate infusions combined with &din and/or WrEDTA The mean of from 4-8 measured inulin clearances was obtained for each of 38 combined phosphate infusions (180 inulin clearance periodsj. The ratio of GFR derived from phosphate data (GFR,)/ mean inulin clearance was 1.00 & 0.03 (SE.). Similarly the mean of from 4-8 measured WrEDTA clearances was obtained in each of 33 combined phosphate infusions. The ratio of %rEDTA clearance/GFR, was 0.94 & 0.03 (SE.). The correlation coefficient between GFRp and mean inulin clearance r = 0.92, and regression analysis gave the equation GFR, = 0.99 GIN + 1.9 The correlation coefficient between GFR,, and mean WrEDTA clearance Y = 0.91, and regression analysis gave the. equation GFR, = 0.99 CEDTA+ 6.9 Combination of the above equations produces the equation Cm’CEDTA= 5.05 The plot of GFR, against mean inulin ond WrEDTA clearances, together with the regression lines of GFR, on inulin and WrEDTA clearance; are shown in Fig. I. B. Simultaneous inuli~..and WrEDTA infusions 65 simultaneous inulin and %rEDTA clearance periods from 15 infusions
Cl&. Chim. Acta, 30 (1970) 351-358
STAMP et al.
354 180
EDTA 16C
14c
1x
100 Phosphate DerivedGFR (ml/min )
‘t 80I-
. , 0
20
I
I
I
1
1
40
60
80
100
120
I
140
,
160
I
180
Fig. I. Correlation between phosphate-derived GFR (GFR,) and mean inulin (0) or WrEDTA (0) clearances with fitted regression lines of GFR, on mean clearances: GFR, = 6.9 + o.gg x CEDTA; GFR, = I.9 + 0.99 X Cm.
were compared (Fig. 2) and the ratio of mean WrEDTA clearance/mean inulin clearance was 0.96 + 0.02 (SE.). The correlation coefficient between Cm and CEDMA, Y = 0.91, and regression analysis gave the equation CIN
=
0.98 CEDTA
+
6.5
Thus at theoretical zero WrEDTA clearance, GFRr, and inulin clearance measure 6.9 and 6.5 ml/min, respectively, further indicating the persistent similar discrepancy of WrEDTA clearance. C. GFR without urine collectiom Plasma levels of inulin and WrEDTA were measured at the end of simultaneous infusions, and from knowledge of infusion rate and concentration, paired values for GFR in 13 tests were derived from the equation GFR = infusion rate of inulin and/or WrEDTA/constant plasma level The average length of these infusions was 172 f 25 (S.D.) min. The ratio of WrEDTA/inulin GFR values obtained without urine collection was 0.92 f 0.04 (S.E.), and the correlation coefficient, Y = 0.92. The relationships were studied between urineless GFR and respective mean clearances during 4g inulin infusions and 32 %rEDTA infusions. The final inulin level (range o.zg-0.42 mg/ml with isolated outside values) was judged constant provided it was identical with one or more immediately preceding levels or if it did not differ by more than 0.01 mg/ml from the mean of the 3 preceding levels. Constancy Clin. Chim. Acta, 30 (1970) 351-358
GFR BY INULIN, 51CrEDTA, AND PHOSPHATE
355
c Inulin
(ml/min
I
0
20
I
I
I
/
I
I
/
40
60
80
100
120
140
160
’
5’Cr.EDTA
(ml_ ,m,n
)
Correlation between simultaneous renal clearance regression of GIN on CEDTA: CIE = 6.5 + 0.98 x CEDTA.
Fig.
2.
I 180
of inulin and WrEDTA,
with fitted
of 51CrEDTA levels was judged similarly. 33 of the 49 inulin infusions and 22 of the 32 SICrEDTA infusions fulfilled these criteria, and only these tests were analysed further. The mean length of the infusions was 170 + 20 (SD.) min for inulin and 194 & 36 (S.D.) min for 51CrEDTA. With inulin, urineless GFR was higher than mean clearance in the ratio 1.07 & 0.03 (SE.) and with 51CrEDTA this ratio was identical, 1.07 & 0.04 (S.E.). Individual values are shown in Fig. 3 and the regressions of urineless GFR on mean clearance were given by the equationsy = 8.5 + 0.96 x, and y = 10.0 + 0.94 x, for XrEDTA and inulin, respectively. With inulin, the mean difference between urineless GFR and clearance was 4..4 + 2.5 (S.E.) m I/ min, and with XrEDTA the mean difference was 4.9 + 2.6 (SE.). These differences were also significant ($ < 0.05). There was no correlation between length of infusion and size of discrepancy with either of the two methods. Understandably, those tests in which plasma levels were rising at the end of the infusion tended to give still greater positive differences between the urineless method and mean clearances, and conversely when plasma levels were falling the positive differences were less. DISCUSSION
The present study has shown the virtual identity of inulin clearance with GFR measured by a simplified phosphate infusion technique11 and thus confirms the original Clin.
Chim.
Acta,
30 (1970)
351-358
STAMP
356
et cd.
Urineless GFR ('IV/p')
Fig. 3. Correlation between GFR obtained without urine collection and respective mean clearances of inulin (a) and WrEDTA (0). Theoretical line given for IV/P = UV/P.
observations of Anderson7 in a smaller series using a more cumbersome technique. Similar results have also been reported by Bijvoet et al., le. It is of theoretical interest that a measurement of GFR can be derived from studying the renal excretion of a substance which is handled by the kidney in a different way from inulin. In comparison with inulin and phosphate WrEDTA clearance underestimates GFR by approximately 4% and 6%, respectively, and the similarity of these differences supports the identity of results obtained with phosphate and inulin. Comparison between WrEDTA and inulin alone has produced rather widely differing data in different centres. Thus results have ranged from an average WrEDTA/inulin ratio of 1.02 (ref. 4) to one of unity5, and one of 0.956 (ref. 17). This last figure is virtua.lly identical with the findings of the present study. Finally Heath et al.6 have indicated that the clearance of WrEDTA may be some 14-167~ lower than that of inulin. As Chantler et al. l7 have pointed out it is difficult to visualize how variations could occur in radioactivity measurements of WrEDTA. Thus these differences more probably reflect problems in the difficult chemical determination of inulin arising from the requirement for stron, n acid hydrolysis, high temperatures, the need for constancy of both timin, Q and temperature, and the interference from substances such as glucose13 or Auto Analyzer acid-flex tubinglo, before estimation of a proportional fructose product. Somel have resisted the use of inulin because of the use of a nonhomogeneous polysaccharide, but these dispelled by the recent demonstration that inulin in possesses identical Sephadex gel filtration patterns lg. It Glin. Chim. Acta,
30 (1970) 351-358
the theoretical objections to objections have been largely plasma and resulting urine is thus of particular interest
GFR
BY INULIN,
51CrEDTA,
AND
357
PHOSPHATE
that the inulin and phosphate results agree so closely and that the XrEDTA results are lower. It would therefore seemreasonable to assume that the phosphate method and inulin clearances correspond with GFR and that 51CrEDTA underestimates GFR by about 5 ml/min. GFR determination which obviates urine collections-l0 has much to recommend it since it requires only a measurement of concentrations in plasma and the sustaining infusion, and of infusion rate. A disadvantage is the uncertainty as to whether constant plasma levels have been reached until the test is finished. In this study the urineless method gave slightly higher results for GFR than did the classical method both with inulin and XrEDTA, and the possible cause for this must be considered. Firstly, Robson et CZZ.~Oraised the objection that even the achievement of constant plasma levels does not necessarily signify that the equilibrium between plasma and ECF has been reached, and Rose lo stressed that at least 4 h should be allowed for this even in nonoedematous subjects. The infusions in this study were all less than 4 h so that it is quite possible that this would explain the discrepancy. Secondly, bladder emptying may give rise to discrepancies which would tend to alter the apparent “classical GFR”. Thus, although it is unlikely that residual urine would increase during the course of a study a constant large residual urine combined with a steadily changing rate of urinary excretion would give erroneous values for UVjP. Finally, for the urineless method to be effective, plasma levels of the substance under study must become constant. This was not so in 32% of the infusions despite preliminary calculations, using an assumed GFR, of the infusion rate required to maintain constant ECF dilutions of the priming dose: plasma levels were rising in g tests, falling in 8. Thus it can be reaffirmed, that an infusion time of 4 h is the minimal length of time on which the measurement of GFR should be based. ACKNOWLEDGEMENTS
We are indebted to Mrs. H. Durbin for having performed all the inulin measurements, and to Sister Butterworth and the nursing staff of the Metabolic Ward, U.C.H., for their continuous help. The patients studied were under the care of Professor C. E. Dent and Dr. Lyal Watson for whose interest we are grateful. T.B.C.S. and G.A.R. have received grants from the Medical Research Council. REFERENCES I 2 3 4 5 6
7 8 g 10 II IZ 13 14 15
J. H. B. H. E. D. J. D. E. G. T. J. A. G. G.
A. SHANNON, Ann. Rev. Physiol., 4 (1942) 297. W. SMITH, Pvinci$les qf Renal Physiology, Oxford Univ. Press, New York, 1956. D. STACY AND G. D. THORBURN, Science, 152 (1966) 1076. R. FAVRE AND A. J. WING, Brit. Med. J., i (1968) 84. S. GARNETT. V. PARSONS AND N. VEALL, Lancet, i (1967) 818. A. HEATH, M. S. KNAPP AND W. H. C. WALKER, Lancet, ii (1968) IIIO. ANDERSON, J. Physiol., 130 (1955) 268. P. EARLE AND R. W. BERLINER. Pvoc. Sot. Exbtl. Biol. Med., 62 (Ia. 262. Y. BERGER, S. J. FARBER AND h. P. EARLE, J: Clin. Inuest.,~27 (;gi8)‘71o. A. ROSE, Sit. Med. I., ii (1969) 91. C. B. STAMP AND T. I?. STACEY, Clin. Sci., 39 (1970) in press. K. DAWBORN, C&n. Chim. A&, 12 (1965) 63. HEYROVSKY, C&n. Chim. Acta, I (1956) 470. A. ROSE AWD H. DURBIN, Lance& ii (1967) 159. GOMORRI, J. Lab. Clin. Med., 27 (1942) 955. Clin. Chim.
Acta,
30 (1970)
351-358
STAMP et al.
358 16 0.
L.M. BIJVOET,A.P. JANSEN, H. PRENEN AND C. L. H.MAJOOR,~ J.DE GRAEFF AND B. LEYNSE (Eds),Water and Electrolyte Metabolism, Vol.II,Elsevier, Amsterdam, pp. I~I168.
17 18 19 20
6. &ANTLER, E. S. GARNETT,~. PARSONS AND N. VEALL,C&. Sci., 37 (1969)169. C. F. PHELPS,Biochem. J., g5 (1965) 41. C. E. MOGENSEN, Stand.J. Clin. Lab. Invest., 22 (1968) 203. J. S. ROBSON, M. H. FERGUSON, 0. OLBRICH AND C. P. STEWART, Quart. J. Exptl. Phyzysiol., 35 (1950) 111.
Clin. Chinz. Acta,
30 (1970) 351-358
CLINICA CHIMICA ACTA
COMPARISON
OF GLOMERULAR
USING INULIN,
T. C. B. STAMP,
FILTRATION
51CrEDTA, AND A PHOSPHATE
T. E. STACEY
AND
RATE
MEASUREMENTS
INFUSION
TECHNIQUE
G. A. ROSE
The Medical Unit, University College Hosflital Medical School, University Street, London, and The Institute of Urology, Henrietta Street, London, W.C.r. (U.K.)
W.C.1.
SUMMARY I. Measurements of glomerular filtration rate (GFR) using inulin, %rEDTA, and a phosphate infusion technique were compared in order to assess their relative accuracy. 2. GFR was determined from phosphate data in a series of 66 infusions in 50 subjects. Comparison with simultaneous inulin clearance (180 measurements during 38 infusions) gave a ratio of GFR derived from phosphate data (GFR,)/mean inulin clearance of 1.00 & 0.03 (S.E.). Comparison with simultaneous 51CrEDTA clearance (189 measurements in 33 infusions) gave a ratio of %rEDTA clearance/GFR, of 0.94 & 0.03 (S.E.). 3. Separate direct comparison of renal inulin clearance with 51CrEDTA clearance in another series of 65 measurements from 15 infusions gave a ratio of %rEDTA clearance/inulin clearance of 0.96 Jr 0.02 (S.E.). 4. The method of determining GFR without urine collection was also used to compare inulin with WrEDTA. In 13 infusions this urineless GFR ratio of 51CrEDTA/ inulin was 0.92 & 0.04 (S.E.). The urineless GFR gave significantly higher absolute values than mean standard clearances, in the ratio of 1.07 for both inulin and 51CrEDTA; the discrepancy was probably due to incomplete equilibration resulting from purposely shortened infusion times. 5. It was concluded that an infusion technique based on the measurement of phosphate alone gives an accurate measure of GFR, that GFR is consistently underestimated by the use of 51CrEDTA, and that the method without urine collection overestimates renal clearance when infusion times are shorter than 4 h.
INTRODUCTION
Determination of the renal clearance of accurate measurement of glomerular filtration chemical determination of inulin has, however, precluded its adoption for routine purposes and
inulin has long been accepted as an rate (GFR), (see refs. I and 2). The always posed difficulties which have encouraged the search for more easily C&z. Chim. Acta, 30 (1970)
351-358
352
STAMP
et 641.
measured compounds. One of these compounds recently introduced is the Q&romiumlabelled chelate of ethylenediamine tetraacetic acid (SlCrEDTA). While original work suggested that 51CrEDTA clearance was identical with that of in&n+5, further studies have indicated that it may consistently underestimate GFR as judged by inulin measurement@. Anderson7 introduced a technique of phosphate infusion, for the determination of tubular maximal reabsorptive capacity (TmP), from which GFR could be directly determined. By achieving a linear rise in plasma phosphorus levels with time, once Tvn has been reached, the rate of rise in urinary phosphorus excretion relative to the rise in plasma phosphorus should be determined only by GFR. Thus GFR should be measurable as the slope of the straight line given by plotting urinary phosphorus excretion against plasma phosphorus levels, above the plasma level required to achieve Tm. While based on sound theoretical principles, the validity of this method relies in part on the accuracy of GFR determinations made from phosphate data alone. A further method of GFR determination which obviates the necessity for urine collections was first introduced by Earle and Berliner8 and Berger et aln, and has been recently revived by Rose 10.It is based on the principle that if a substance is eliminated from the extracellular fluid only by renal excretion, then after equilibration the achievement of constant plasma levels during a continuous infusion signifies that renal excretion is exactly equal to the rate of infusion. Thus the standard expression UVjP can then be rewritten as intravenous infusion rate (~‘I~“~)~constant plasma level. The present study was undertaken in order to evaluate the use of the phosphate infusion method as a sole measure of GFR by comparison both with inulin and 5lCrEDTA clearances, and in addition to establish more firmly any discrepancy between %rEDTA and inulin clearances by direct comparison of clearances obtained by both standard and “urineless” methods. Certain problems inherent in the urineless method were also clarified. MATERIALS
AXD
XETHODS
56 subjects were studied of whom 15 were volunteers with no evidence of abnormal renal function, and the remainder were largely patients with disorders of calcium and phosphorus metabolism who have been described in more detail elsewherell. 87 infusions were performed of which 66 consisted of phosphate combined either with inulin (38 infusions), 5lCrEDTA (33 infusions), or both (5 infusions). The remaining 21 tests were combined with glucose infusions for the determination of maximal tubular reabsorptive capacity (TmG) ; in IO of these latter both 51CrEDTA and inulin were infused in addition to glucose, while in the remainder only one substance was administered for the detestation of mean clearance and comparison with “urineless” GFR. All subjects were studied after an overnight fast and tests were begun at approximately 9 a.m. Subjects were given 2-3 1 of water to drink during the course of the infusions. A solution of 0.1 M sodium phosphate buffered at pH 7.4 was used (Na,HPO,.zH,O 13.8 g; NaH,PO,.aH,O 3.6 g; water to I 1). Inulin was obtained from Thomas Kerfoot and Co. Ltd., Vale of Bardsley Lancashire, -and 51CrEDTA was obtained from the Radiochemical Centre, Amersham (Code Ch.I3P), In all tests C&Z.Chi~.Acfa,~O(1970)
351-358
GFR BY INULIN, %rEDTA,
AND
PHOSPHATE
353
after a loading dose of inulin and/or WrEDTA, a sustaining infusion in 0.9% NaCl was given using a constant infusion pump (Buchler Polystaltic pump, Buchler Instrument Co. Ltd., Fort Lee, N.J., U.S.A.), and 45-60 min were allowed for equilibration. Phosphate (or glucose) infusions were delivered from a second pump through a single indwelling venous cannula via an open z-way tap. In the phosphate infusions, the flow rate was raised by a constant increment every 15 min by altering a simple dial setting, thus producing a linear rise in plasma phosphorus with time. In some of the infusions equilibration of inulin or WrEDTA was awaited before beginning the phosphate infusion, and 6 or 7 full clearance periods were measured; in others phosphate, inulin and/or WrEDTA were begun simultaneously and only the last 4 collection periods were analysed for %rEDTA and inulin (out of 7 phosphate clearance periods). Bladder catheterisation was unjustifiable, and nearly all patients could void on request. Blood samples could thus be collected from an indwelling catheter in an opposite forearm vein at the planned midpoint of each urine collection. Collection periods were usually of 24 min duration (range 22-32 min). GFR was determined from phosphate data by calculating the slope of the regression of urine phosphorus excretion (mg/min) on plasma phosphorus level (mg/ml) . Only the last 5 collection periods (out of 6 obtained during the infusion) were used to calculate this regression; it was assumed in this way that phosphate reabsorption was constant throughout (i.e. TmP had been reached). Inulin was determined by Dawborn’s12 Auto Analyzer modification of Heyrovsky’s13 3-indolylacetic acid methodl”. 51CrEDTA was counted in a Packard Auto-Gamma spectrometer, and at least 3000 counts were made on each sample. Phosphate was estimated in duplicate or triplicate by a standard Auto Analyzer adaptation of Gomorri’sls method. RESULTS
il. Phos$hate infusions combined with &din and/or WrEDTA The mean of from 4-8 measured inulin clearances was obtained for each of 38 combined phosphate infusions (180 inulin clearance periodsj. The ratio of GFR derived from phosphate data (GFR,)/ mean inulin clearance was 1.00 & 0.03 (SE.). Similarly the mean of from 4-8 measured WrEDTA clearances was obtained in each of 33 combined phosphate infusions. The ratio of %rEDTA clearance/GFR, was 0.94 & 0.03 (SE.). The correlation coefficient between GFRp and mean inulin clearance r = 0.92, and regression analysis gave the equation GFR, = 0.99 GIN + 1.9 The correlation coefficient between GFR,, and mean WrEDTA clearance Y = 0.91, and regression analysis gave the. equation GFR, = 0.99 CEDTA+ 6.9 Combination of the above equations produces the equation Cm’CEDTA= 5.05 The plot of GFR, against mean inulin ond WrEDTA clearances, together with the regression lines of GFR, on inulin and WrEDTA clearance; are shown in Fig. I. B. Simultaneous inuli~..and WrEDTA infusions 65 simultaneous inulin and %rEDTA clearance periods from 15 infusions
Cl&. Chim. Acta, 30 (1970) 351-358
STAMP et al.
354 180
EDTA 16C
14c
1x
100 Phosphate DerivedGFR (ml/min )
‘t 80I-
. , 0
20
I
I
I
1
1
40
60
80
100
120
I
140
,
160
I
180
Fig. I. Correlation between phosphate-derived GFR (GFR,) and mean inulin (0) or WrEDTA (0) clearances with fitted regression lines of GFR, on mean clearances: GFR, = 6.9 + o.gg x CEDTA; GFR, = I.9 + 0.99 X Cm.
were compared (Fig. 2) and the ratio of mean WrEDTA clearance/mean inulin clearance was 0.96 + 0.02 (SE.). The correlation coefficient between Cm and CEDMA, Y = 0.91, and regression analysis gave the equation CIN
=
0.98 CEDTA
+
6.5
Thus at theoretical zero WrEDTA clearance, GFRr, and inulin clearance measure 6.9 and 6.5 ml/min, respectively, further indicating the persistent similar discrepancy of WrEDTA clearance. C. GFR without urine collectiom Plasma levels of inulin and WrEDTA were measured at the end of simultaneous infusions, and from knowledge of infusion rate and concentration, paired values for GFR in 13 tests were derived from the equation GFR = infusion rate of inulin and/or WrEDTA/constant plasma level The average length of these infusions was 172 f 25 (S.D.) min. The ratio of WrEDTA/inulin GFR values obtained without urine collection was 0.92 f 0.04 (S.E.), and the correlation coefficient, Y = 0.92. The relationships were studied between urineless GFR and respective mean clearances during 4g inulin infusions and 32 %rEDTA infusions. The final inulin level (range o.zg-0.42 mg/ml with isolated outside values) was judged constant provided it was identical with one or more immediately preceding levels or if it did not differ by more than 0.01 mg/ml from the mean of the 3 preceding levels. Constancy Clin. Chim. Acta, 30 (1970) 351-358
GFR BY INULIN, 51CrEDTA, AND PHOSPHATE
355
c Inulin
(ml/min
I
0
20
I
I
I
/
I
I
/
40
60
80
100
120
140
160
’
5’Cr.EDTA
(ml_ ,m,n
)
Correlation between simultaneous renal clearance regression of GIN on CEDTA: CIE = 6.5 + 0.98 x CEDTA.
Fig.
2.
I 180
of inulin and WrEDTA,
with fitted
of 51CrEDTA levels was judged similarly. 33 of the 49 inulin infusions and 22 of the 32 SICrEDTA infusions fulfilled these criteria, and only these tests were analysed further. The mean length of the infusions was 170 + 20 (SD.) min for inulin and 194 & 36 (S.D.) min for 51CrEDTA. With inulin, urineless GFR was higher than mean clearance in the ratio 1.07 & 0.03 (SE.) and with 51CrEDTA this ratio was identical, 1.07 & 0.04 (S.E.). Individual values are shown in Fig. 3 and the regressions of urineless GFR on mean clearance were given by the equationsy = 8.5 + 0.96 x, and y = 10.0 + 0.94 x, for XrEDTA and inulin, respectively. With inulin, the mean difference between urineless GFR and clearance was 4..4 + 2.5 (S.E.) m I/ min, and with XrEDTA the mean difference was 4.9 + 2.6 (SE.). These differences were also significant ($ < 0.05). There was no correlation between length of infusion and size of discrepancy with either of the two methods. Understandably, those tests in which plasma levels were rising at the end of the infusion tended to give still greater positive differences between the urineless method and mean clearances, and conversely when plasma levels were falling the positive differences were less. DISCUSSION
The present study has shown the virtual identity of inulin clearance with GFR measured by a simplified phosphate infusion technique11 and thus confirms the original Clin.
Chim.
Acta,
30 (1970)
351-358
STAMP
356
et cd.
Urineless GFR ('IV/p')
Fig. 3. Correlation between GFR obtained without urine collection and respective mean clearances of inulin (a) and WrEDTA (0). Theoretical line given for IV/P = UV/P.
observations of Anderson7 in a smaller series using a more cumbersome technique. Similar results have also been reported by Bijvoet et al., le. It is of theoretical interest that a measurement of GFR can be derived from studying the renal excretion of a substance which is handled by the kidney in a different way from inulin. In comparison with inulin and phosphate WrEDTA clearance underestimates GFR by approximately 4% and 6%, respectively, and the similarity of these differences supports the identity of results obtained with phosphate and inulin. Comparison between WrEDTA and inulin alone has produced rather widely differing data in different centres. Thus results have ranged from an average WrEDTA/inulin ratio of 1.02 (ref. 4) to one of unity5, and one of 0.956 (ref. 17). This last figure is virtua.lly identical with the findings of the present study. Finally Heath et al.6 have indicated that the clearance of WrEDTA may be some 14-167~ lower than that of inulin. As Chantler et al. l7 have pointed out it is difficult to visualize how variations could occur in radioactivity measurements of WrEDTA. Thus these differences more probably reflect problems in the difficult chemical determination of inulin arising from the requirement for stron, n acid hydrolysis, high temperatures, the need for constancy of both timin, Q and temperature, and the interference from substances such as glucose13 or Auto Analyzer acid-flex tubinglo, before estimation of a proportional fructose product. Somel have resisted the use of inulin because of the use of a nonhomogeneous polysaccharide, but these dispelled by the recent demonstration that inulin in possesses identical Sephadex gel filtration patterns lg. It Glin. Chim. Acta,
30 (1970) 351-358
the theoretical objections to objections have been largely plasma and resulting urine is thus of particular interest
GFR
BY INULIN,
51CrEDTA,
AND
357
PHOSPHATE
that the inulin and phosphate results agree so closely and that the XrEDTA results are lower. It would therefore seemreasonable to assume that the phosphate method and inulin clearances correspond with GFR and that 51CrEDTA underestimates GFR by about 5 ml/min. GFR determination which obviates urine collections-l0 has much to recommend it since it requires only a measurement of concentrations in plasma and the sustaining infusion, and of infusion rate. A disadvantage is the uncertainty as to whether constant plasma levels have been reached until the test is finished. In this study the urineless method gave slightly higher results for GFR than did the classical method both with inulin and XrEDTA, and the possible cause for this must be considered. Firstly, Robson et CZZ.~Oraised the objection that even the achievement of constant plasma levels does not necessarily signify that the equilibrium between plasma and ECF has been reached, and Rose lo stressed that at least 4 h should be allowed for this even in nonoedematous subjects. The infusions in this study were all less than 4 h so that it is quite possible that this would explain the discrepancy. Secondly, bladder emptying may give rise to discrepancies which would tend to alter the apparent “classical GFR”. Thus, although it is unlikely that residual urine would increase during the course of a study a constant large residual urine combined with a steadily changing rate of urinary excretion would give erroneous values for UVjP. Finally, for the urineless method to be effective, plasma levels of the substance under study must become constant. This was not so in 32% of the infusions despite preliminary calculations, using an assumed GFR, of the infusion rate required to maintain constant ECF dilutions of the priming dose: plasma levels were rising in g tests, falling in 8. Thus it can be reaffirmed, that an infusion time of 4 h is the minimal length of time on which the measurement of GFR should be based. ACKNOWLEDGEMENTS
We are indebted to Mrs. H. Durbin for having performed all the inulin measurements, and to Sister Butterworth and the nursing staff of the Metabolic Ward, U.C.H., for their continuous help. The patients studied were under the care of Professor C. E. Dent and Dr. Lyal Watson for whose interest we are grateful. T.B.C.S. and G.A.R. have received grants from the Medical Research Council. REFERENCES I 2 3 4 5 6
7 8 g 10 II IZ 13 14 15
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6. &ANTLER, E. S. GARNETT,~. PARSONS AND N. VEALL,C&. Sci., 37 (1969)169. C. F. PHELPS,Biochem. J., g5 (1965) 41. C. E. MOGENSEN, Stand.J. Clin. Lab. Invest., 22 (1968) 203. J. S. ROBSON, M. H. FERGUSON, 0. OLBRICH AND C. P. STEWART, Quart. J. Exptl. Phyzysiol., 35 (1950) 111.
Clin. Chinz. Acta,
30 (1970) 351-358