•
Nuclear Medicine
, Noninvasive Determination of Glomerular Filtration Rate Using X-Ray Fluorescence 1 Naomi Alazraki, M.D.,2 John W. Verba, Ph.D., James E. Henry, B.S., Richard Becker, B.S., Andrew Taylor Jr., M.D., and Samuel E. Halpern, M.D. Accurate glomerular filtration rates (GFR)can be calculated based on the infusion of small amounts of nonradioactive iothalamate and collection of plasma samples for assay by x-ray fluorescence. This innovation permits frequent clearance determinations in patients without the hazard of repeated radiation exposure and without the necessity of catheterization of the bladder for urine samples. Thus. the technique becomes feasible in children, transplant patients, and others needing accurate and frequent GFR determinations. INDEX TERM:
Kidneys, physiology
Radiology 122:183-186. January 1977
• HE COMMONLY
USED
indicators of renal function
C = UV /P
T in clinical medical practice yield only rough estimates.
AT EQUILIBRIUM:
Blood urea nitrogen (BUN) and plasma creatinine levels may not be indicative of renal dysfunction until the glomerular filtration rate (GFR) is reduced to less than 40 ml/min. Individuals on a low-protein diet may achieve normal BUN values when the GFR is as low as 5 ml/min. (1). Various investigators have also documented a wide range of plasma creatinine values for any given GFR
RATE OF INFUSION = RATE OF EXCRETION IV
=
UV
•• C=IV/P
Fig. 1.
GFR calculations.
inulin (1,400-1,800 mg inulin) were administered over a period of 2-3 hours either intravenously by infusion pump, or subcutaneously (iothalamate only following local infiltration with xylocaine 1 %) to simulate infusion. Six-milliliter samples of blood were drawn at 20-minute intervals after the injection of iothalamate for 1-2 hours, or until an equilibrium level was reached in the blood. For the next hour, blood samples were drawn every 10 minutes. In those experiments which required urine measurements, samples were collected every 20 minutes and the volume recorded. Calculation of quantity of iothalamate used: The loading dose was determined by estimating the extracellular fluid (ECF)volume of the dog to be 16 % of the body weight (3); 0.2 mg/ml was considered to be the minimum level of iodine which could accurately be measured by the x-ray fluorescence technique. Thus, for a 25-kg dog with an estimated ECF volume of 4,000 ml, 800 mg of iodine (2 ml Conray 400) was administered as the loading dose. The amount of iothalamate given after the loading dose, either by intravenous infusion over 2-3 hours, or in the form of at least two simultaneous subcutaneous injections, was calculated by first estimating the GFR for the dog. For dogs who were approximately normal or near normal, 80 ml/min. was used as an estimated clearance. This figure
(2).
Radionuclide methods have been used to determine the GFR, but they have not achieved popularity in clinical use. The best known of these methods has utilized 1251-labeled iothalamate as the radiopharmaceutical. The purpose of this report is to present a method for determining GFR utilizing x-ray fluorescence of nonradioactive iothalamate. MATERIALS AND METHODS
Animal studies: Two groups of dogs (20-25 kg) were studied. The first group had normal kidneys, while the second group had markedly diminished renal function due to a bilateral nephrectomy and a preserved renal allograft. Both groups were anesthetized with 2 ml Inovar (intramuscular), 0.4 mg atropine sulfate (intramuscular) and 12 mg/kg Nembutal (pentobarbital sodium) (intravenous). After anesthesia, the animals were hydrated with approximately 500 ml dextrose (5 %) and water. 1.0-2.0 ml iothalamate (Conray 400, containing 400 mg iodine per ml) and 2.6-3.2 j.LCi of 14C inulin 3 (300-360 mg inulin) were administered intravenously as a loading dose. An additional 2-6 ml of iothalamate (800-2,400 mg iodine) and 12-16 /-LCi 14C
1 From the Nuclear Medicine Service, VA Hospital, and the Department of Radiology, University of California, Medical School, San Diego, Calif. Accepted for publication in May 1976. Supported by VA Research funds MRIS 0306-01. 2 James Picker Foundation Scholar. 3 Carboxyl(14C}-inulin, New England Nuclear Co. elk
183
184
NAOMI ALAZRAKI AND OTHERS
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was multiplied by the desired level of 0.2 mg/ml of iodine in the extracellular fluids to determine the rate of infusion. Thus, 80 ml/min. X 0.2 mg/ml iodine or 16 mg/min. iodine (0.04 ml Conray 400) were infused during an average 21/2 hour experiment. This rate of infusion approximated the rate of excretion, a condition necessary for rapid equilibration (Fig 1).
January 1977
X-ray fluorescence measurements: Samples of plasma and urine were drawn into standard plastic tuberculin syringes. Each sample was counted for approximately 5 minutes, resulting in sufficient counts that the statistical error was less than 2 %. The x-ray fluorescence apparatus used consisted of two 1-curie sources of americium-24 1 (Fig. 2), and a semiconductor detector (silicon Iithiumdrifted crystal) so positioned that x-rays emitted from the sample being irradiated could be efficiently detected. Comparisons of methods of obtaining GFR: The following three types of comparative approaches to obtaining GFR were studied: (a) A loading dose of iothalamate and 14C inulin was injected intravenously, followed by a 2-3 hour infusion of both compounds. Plasma and urine were collected as described. The GFR was then determined by the x-ray fluorescence of plasma and urine for iodine levels, and by liquid scintillation counting of these fluids for 14C inulin. The clearances of both were then calculated in the classical manner of Smith (4-6) and the GFR obtained with 14C-inulin measurements were compared to those calculated from x-ray fluorescence of iothalamate. (b) A loading dose of iothalamate and 14C inulin was injected intravenously followed by their infusion over a 2-3 hour period. Plasma and urine were collected as described. 14C inulin was measured in the urine and plasma for calculation of GFR. This result was compared to the GFR derived by x-ray fluorescence measurements of iodine levels In plasma alone. (c) A loading dose of iothalamate and 14C inulin was injected intravenously, followed by an infusion of 14C inulin. Urine and plasma levels of 14C were measured. lothalamate was not continuously infused, but rather injected subcutaneously. Iodine levels were again measured in the plasma and urine by x-ray fluorescence. The GFR calculated from plasma and urine iodine levels sustained by subcutaneous absorption of iothalamate (Fig. 1) were correlated with that calculated by the classical approach utilizing 14C-inulin infusion. RESULTS
The correlation of the clearance determinations by x-ray fluorescence technique vs. 14C-inulin clearances are shown in Figures 3-5. Comparison of clearances obtained with x-ray fluorescence of plasma and urine to those obtained with simultaneous 14C-inulin infusions (Fig. 3) resulted in a correlation coefficient of 0.99 in normal and low-GFR dogs (results based on 10 experiments). Clearances determined solely from plasma measurements of iothalamate with x-ray fluorescence are compared with those obtained using classical 14C-inulin technique in Figure 4. The correlation coefficient (R) of 0.98 is based on 10 dog experiments. Figure 5 shows the results of subcutaneous injections of iothalamate performed to verify the postulate that this route of administration can simulate the intravenous infusion of this agent. The subcutaneous iothalamate injected after xylocaine infiltration seemed to be well tolerated by the dogs with no marked inflammatory reaction noted. The clearance determinations, based on subcuta-
Nuclear Vol. 122
DETERMINATION OF GLOMERULAR FILTRATION RATE
neous absorption of iothalamate, correlated well with the infusion method (R = 0.96, 18 experiments).
185
.s 0
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Inulin is considered an ideal substance for the measurement of GFR (7); however, inulin GFR determinations have not become a routine clinical tool because of the tedious chemical analyses required. In addition, variations in inulin clearances secondary to high or varying levels of blood glucose have been reported (8). Thus, the search to find a reliable substitute for inulin which would permit easy, accurate determinations of GFR has continued. Agents studied include radiolabeled inulins, labeled vltamtn 8 12 , labeled radio-opaque contrast media, and various labeled chelatinq agents (6, 11, 12). lodine-125-iothalamate has been selected by several investigators as the best available agent for the practical measurement of GFR (9). Griep and Nelp (10) showed that radioiodinated sodium iothalamate was not secreted by renal tubules of aglomerular fishes. Thus, the labeled substance offers the advantage of easier measurements of GFR, but it exposes the patient to radiation. The rad dose is minimal for a single study, however it can become significant if many studies are performed, as might be expected in a renal transplant patient. In addition, there is always the possibility of elution of the radioiodine atom from the parent compound which will alter the calculated GFR (11, 12). Our proposed technique would eliminate the disadvantages of using radioactive compounds for GFR determinations, while preserving the advantages of clinical suitability and accuracy. The fluorescent technique described (Fig. 3) is based on the use of an americium source. 241 Americium produces a 60-keV photon which, upon interaction with an electron from the K-shell of an iodine atom within the sample, results in emission of characteristic x rays (28.5 and 32.4 keV) which are detected and measured by the semiconductor detector. The semiconductor detector is required to give the necessary energy resolution to separate iodine from other elements which may be present in the sample. The detector is interfaced to an 8K dedicated computer which quantitates the characteristic x rays produced and converts them (based on previous measurement with known standards) into milligram per cent of iodine, enabling calculation of the GFR. Various investigators have tried different techniques to simplify the determination of GFR and render it more useful clinically (13-18). Our approach to determining GFR using relatively small amounts of iothalamate and measuring iodine levels via x-ray fluorescence technique can be applied to almost any of these simplified methods. Furthermore, the samples can be counted by x-ray fluorescence immediately or they can be placed in storage for counting at a more convenient time without influencing the result. The high sensitivity of x-ray fluorescence for measuring iodine, approaching one part per million (0.1 mg %), permits the injected iothalamate doses to be less than 10 cm 3 for an average adult. Conray 400 was selected
Medicine
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Fig. 4. lothalamate clearances based on the plasma equilibrium method compared with clearances utilizing classical 14 C-inulin procedure. Correlation coefficient 0.986.
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Fig. 5. lothalamate clearances based on a subcutaneous !njection of iothalamate and measurement of plasma arid urine fluorescence compared with 14C-inulin clearances determined by the lnfuslon method. Correlation coefficient 0.965.
because it contains more milligrams of Iodine per ml of iothalamate (400 mg/ml) than most other available preparations. With further improvements in the x-ray fluorescent system, the sensitivity could be reduced to less than one part per million and the injected dose lowered. The data from a single dog study (Fig. 6, A and B) show that an equulbrlum state occurs about 2-3 hours after the start of an infusion. In this experiment the iothalamate and 14C inulin were administered in the same infusion, and blood samples were drawn at regular time intervals. The time-concentration curves are the same for both substances. Our rationale for calculating clearances utiliZing only measurements of plasma samples is based on the fact that at eqUilibrium (that is when the rate of infusion is equal to the rate of excretion of the test compound) the clearance
186
NAOMI ALAZRAKI AND OTHERS
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can be calculated from the rate of infusion. By substituting in the equation in Figure 1, accurate clearances were obtained for the above dog. Two normal dogs were studied on several different days to establish reproducibility. Based on four experiments, the first dog had a measured mean iothalamate-fluorescent GFR of 77.0 ± 5.2 and a mean inulin measured GFR of 83.4 ± 8.8. The second dog, based on three experiments, has a measured mean iothalamate-fluorescent GFR of 69.6 ± 7.8, and mean inulin measured GFR of 76.4 ± 12.6. These results indicate close reproducibility of measured GFR by both iothalamate-fluorescence and inulin methods. It is noteworthy that the iothalamate-fluorescent technique resulted in a smaller standard deviation for both normal dogs than the inulin method, perhaps indicating slightly closer reproducibility. As emphasized by Cole et st. (15), the infusion or subcutaneous injected approach is only reliable during equilibrium, where the rate of infusion, or blood absorption, equals the rate of excretion. If the loading dose used is too small because of underestimation of the extracellular fluids, or the GFR is overestimated and the infusion rate is too rapid, plasma levels of iothalamate may rise with delay in achievement of equilibrium. In our experience this has not been a problem, but we recognize that none of our dog subjects had any additional extracellular fluid compartment such as ascites or edema which would prolong the achievement of equilibrium. In the absence of those conditions, this approach to GFR determination could be of true clinical importance since it appears to be both accurate and reproducible. ACKNOWLEDGMENT: We thank Dr. Nicholas Halasz for having provided the animal subjects with impaired renal function and for his review of the manuscript. Nuclear Medicine Service (115) VA Hospital 3350 La Jolla Village Drive San Diego, Calif. 92161
REFERENCES 1. Monasterio G, Giovanetti S. Maggiore Q: Le Traitement dietetique de I'uremie chronique. Actualites Nephrologiques de I'Hopital Necker, Paris Editions Medicales Flammarion, 1965, pp 31-42 2. Bianchi C, Coli A, Palla R, et al: Serum creatinine in the evaluation of renal function. Abstr Proc 4th Int Cong Nephrol, Stockholm, 1969, p 48 3. Documenta Geigy, Scientific Tables, Ardsley, New York, 6th ed., Geigy Pharmaceuticals, p 538 4. Smith HW: The Kidney, Structure and Function in Health and Disease. New York, Oxford University Press, 1951, p 39-40 5. Smith HW: Principles of Renal Physiology, New York, Oxford University Press, 1956, p 27 6. Smith HW, Goldring W, Chasis H: The measurement of the tubular excretory mass, effective blood flow and filtration rate in the normal human kidney. J Clin Invest 17:263-278, May 1938 7. Bianchi C: Measurement of the Glomerular Filtration Rate, [In] Evaluation of Renal Function and Disease with Radionuclides, eel by MD Blaufox, Baltimore, University Park Press, 1972, p 21-48 8. Liewendahl K, Tallgren LG, Rusk J: Separation of inulin from chemically interfering dextrans by gel filtration. Scan J Clin Lab Invest 18:553-556, 1966 9. Sigman EM, Elwood CM, and Knox F: The measurement of glomerular filtration rate in man with sodium iothalamate 131 1(Conray). J Nucl Med 7:60-68, Jan 1966 10. Griep RJ, Nelp WB: Mechanism of excretion of radioiodinateel sodium iothalamate. Radiology 93:807-811, Oct 1969 11. Schmid HE. Muehlbaecher CA, Hutchins PM: Continuous determination of the clearance of 131-I-diatrizoate, an inulin substitute. J Appl Physiol 25:294-300, Sep 1968 12. Cohen M: Radionuclide clearance techniques. Sem Nucl Med 4:23-28, Jan 1974 13. Blaufox MD: Nuclear medicine and renal disease. Hospital Practice 7:137-144, 1972 14. Rose GA: Measurement of glomerular filtration rate by inulin clearance without urine collection. Br Med J 2:91-93, 12 Apr 1969 15. Cole BR, Giangiacomo J, Inglefinger JR, et al: Measurement of renal function without urine collection. N Engl J Mad 287:1109-1114, 30 Nov 1972 16. Blaufox MD, Cohen A: Single-injection clearances of iothalamate- 1311 in the rat. Am J Physiol 218:542-544, Feb 1970 17. Findley T, White HL: Measurement of diodrast and inulin clearances after subcutaneous administration. Proc Soc Exp Bioi 45:623, 1940 18. Israelit AH, Long DL, White MG, et al: Measurement of glomerular filtration rate utilizing a single subcutaneous injection of 1251-iothalamate. Kidney Int 4:346-349, Nov 1973
Nuclear Medicine
, Noninvasive Determination of Glomerular Filtration Rate Using X-Ray Fluorescence 1 Naomi Alazraki, M.D.,2 John W. Verba, Ph.D., James E. Henry, B.S., Richard Becker, B.S., Andrew Taylor Jr., M.D., and Samuel E. Halpern, M.D. Accurate glomerular filtration rates (GFR)can be calculated based on the infusion of small amounts of nonradioactive iothalamate and collection of plasma samples for assay by x-ray fluorescence. This innovation permits frequent clearance determinations in patients without the hazard of repeated radiation exposure and without the necessity of catheterization of the bladder for urine samples. Thus. the technique becomes feasible in children, transplant patients, and others needing accurate and frequent GFR determinations. INDEX TERM:
Kidneys, physiology
Radiology 122:183-186. January 1977
• HE COMMONLY
USED
indicators of renal function
C = UV /P
T in clinical medical practice yield only rough estimates.
AT EQUILIBRIUM:
Blood urea nitrogen (BUN) and plasma creatinine levels may not be indicative of renal dysfunction until the glomerular filtration rate (GFR) is reduced to less than 40 ml/min. Individuals on a low-protein diet may achieve normal BUN values when the GFR is as low as 5 ml/min. (1). Various investigators have also documented a wide range of plasma creatinine values for any given GFR
RATE OF INFUSION = RATE OF EXCRETION IV
=
UV
•• C=IV/P
Fig. 1.
GFR calculations.
inulin (1,400-1,800 mg inulin) were administered over a period of 2-3 hours either intravenously by infusion pump, or subcutaneously (iothalamate only following local infiltration with xylocaine 1 %) to simulate infusion. Six-milliliter samples of blood were drawn at 20-minute intervals after the injection of iothalamate for 1-2 hours, or until an equilibrium level was reached in the blood. For the next hour, blood samples were drawn every 10 minutes. In those experiments which required urine measurements, samples were collected every 20 minutes and the volume recorded. Calculation of quantity of iothalamate used: The loading dose was determined by estimating the extracellular fluid (ECF)volume of the dog to be 16 % of the body weight (3); 0.2 mg/ml was considered to be the minimum level of iodine which could accurately be measured by the x-ray fluorescence technique. Thus, for a 25-kg dog with an estimated ECF volume of 4,000 ml, 800 mg of iodine (2 ml Conray 400) was administered as the loading dose. The amount of iothalamate given after the loading dose, either by intravenous infusion over 2-3 hours, or in the form of at least two simultaneous subcutaneous injections, was calculated by first estimating the GFR for the dog. For dogs who were approximately normal or near normal, 80 ml/min. was used as an estimated clearance. This figure
(2).
Radionuclide methods have been used to determine the GFR, but they have not achieved popularity in clinical use. The best known of these methods has utilized 1251-labeled iothalamate as the radiopharmaceutical. The purpose of this report is to present a method for determining GFR utilizing x-ray fluorescence of nonradioactive iothalamate. MATERIALS AND METHODS
Animal studies: Two groups of dogs (20-25 kg) were studied. The first group had normal kidneys, while the second group had markedly diminished renal function due to a bilateral nephrectomy and a preserved renal allograft. Both groups were anesthetized with 2 ml Inovar (intramuscular), 0.4 mg atropine sulfate (intramuscular) and 12 mg/kg Nembutal (pentobarbital sodium) (intravenous). After anesthesia, the animals were hydrated with approximately 500 ml dextrose (5 %) and water. 1.0-2.0 ml iothalamate (Conray 400, containing 400 mg iodine per ml) and 2.6-3.2 j.LCi of 14C inulin 3 (300-360 mg inulin) were administered intravenously as a loading dose. An additional 2-6 ml of iothalamate (800-2,400 mg iodine) and 12-16 /-LCi 14C
1 From the Nuclear Medicine Service, VA Hospital, and the Department of Radiology, University of California, Medical School, San Diego, Calif. Accepted for publication in May 1976. Supported by VA Research funds MRIS 0306-01. 2 James Picker Foundation Scholar. 3 Carboxyl(14C}-inulin, New England Nuclear Co. elk
183
184
NAOMI ALAZRAKI AND OTHERS
_ _ RING SOURCE (241Am) .-:.:.:..... -COLLIMATOR
SIGNALS TO PULSE-HEIGHT ANALYZER
B
IODINE ATOMIC SHELLS
CHARACTERISTIC X -RA YS
EXCITING RADIA TlON
IODINE NUCLEUS VACANCY FILLED
Fig. 2. A. The syringe which holds plasma or urine sample is being bombarded by exciting radiation originating from 241 americium ring-shaped sources placed above and below the syringe. The characteristic x rays emitted from the sample are detected by the semiconductor detector and the amount of iodine quantitated from the signals to the pulse height analyzer. B. The atomic processes are depicted. showing the iodine nucleus and atomic electron shells which are bombarded by the exciting radiation. A K-electron is knocked out of orbit creating a vacancy which is filled by an electron from an outer shell with resulting emission of characteristic x rays from the iodine atom.
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50
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14C-INULIN CLEARANCE (ml/minl, "CLASSICAL" METHOD
Fig. 3. lothalamate clearances measured by the classical procedure. Plasma and urine samples are collected and determinations performed by both x-ray fluorescence and 14C-inulin methods. Correlation coefficient 0.997.
was multiplied by the desired level of 0.2 mg/ml of iodine in the extracellular fluids to determine the rate of infusion. Thus, 80 ml/min. X 0.2 mg/ml iodine or 16 mg/min. iodine (0.04 ml Conray 400) were infused during an average 21/2 hour experiment. This rate of infusion approximated the rate of excretion, a condition necessary for rapid equilibration (Fig 1).
January 1977
X-ray fluorescence measurements: Samples of plasma and urine were drawn into standard plastic tuberculin syringes. Each sample was counted for approximately 5 minutes, resulting in sufficient counts that the statistical error was less than 2 %. The x-ray fluorescence apparatus used consisted of two 1-curie sources of americium-24 1 (Fig. 2), and a semiconductor detector (silicon Iithiumdrifted crystal) so positioned that x-rays emitted from the sample being irradiated could be efficiently detected. Comparisons of methods of obtaining GFR: The following three types of comparative approaches to obtaining GFR were studied: (a) A loading dose of iothalamate and 14C inulin was injected intravenously, followed by a 2-3 hour infusion of both compounds. Plasma and urine were collected as described. The GFR was then determined by the x-ray fluorescence of plasma and urine for iodine levels, and by liquid scintillation counting of these fluids for 14C inulin. The clearances of both were then calculated in the classical manner of Smith (4-6) and the GFR obtained with 14C-inulin measurements were compared to those calculated from x-ray fluorescence of iothalamate. (b) A loading dose of iothalamate and 14C inulin was injected intravenously followed by their infusion over a 2-3 hour period. Plasma and urine were collected as described. 14C inulin was measured in the urine and plasma for calculation of GFR. This result was compared to the GFR derived by x-ray fluorescence measurements of iodine levels In plasma alone. (c) A loading dose of iothalamate and 14C inulin was injected intravenously, followed by an infusion of 14C inulin. Urine and plasma levels of 14C were measured. lothalamate was not continuously infused, but rather injected subcutaneously. Iodine levels were again measured in the plasma and urine by x-ray fluorescence. The GFR calculated from plasma and urine iodine levels sustained by subcutaneous absorption of iothalamate (Fig. 1) were correlated with that calculated by the classical approach utilizing 14C-inulin infusion. RESULTS
The correlation of the clearance determinations by x-ray fluorescence technique vs. 14C-inulin clearances are shown in Figures 3-5. Comparison of clearances obtained with x-ray fluorescence of plasma and urine to those obtained with simultaneous 14C-inulin infusions (Fig. 3) resulted in a correlation coefficient of 0.99 in normal and low-GFR dogs (results based on 10 experiments). Clearances determined solely from plasma measurements of iothalamate with x-ray fluorescence are compared with those obtained using classical 14C-inulin technique in Figure 4. The correlation coefficient (R) of 0.98 is based on 10 dog experiments. Figure 5 shows the results of subcutaneous injections of iothalamate performed to verify the postulate that this route of administration can simulate the intravenous infusion of this agent. The subcutaneous iothalamate injected after xylocaine infiltration seemed to be well tolerated by the dogs with no marked inflammatory reaction noted. The clearance determinations, based on subcuta-
Nuclear Vol. 122
DETERMINATION OF GLOMERULAR FILTRATION RATE
neous absorption of iothalamate, correlated well with the infusion method (R = 0.96, 18 experiments).
185
.s 0
Eo E t-
100
~2
80
::::::1:
-w DISCUSSION
Inulin is considered an ideal substance for the measurement of GFR (7); however, inulin GFR determinations have not become a routine clinical tool because of the tedious chemical analyses required. In addition, variations in inulin clearances secondary to high or varying levels of blood glucose have been reported (8). Thus, the search to find a reliable substitute for inulin which would permit easy, accurate determinations of GFR has continued. Agents studied include radiolabeled inulins, labeled vltamtn 8 12 , labeled radio-opaque contrast media, and various labeled chelatinq agents (6, 11, 12). lodine-125-iothalamate has been selected by several investigators as the best available agent for the practical measurement of GFR (9). Griep and Nelp (10) showed that radioiodinated sodium iothalamate was not secreted by renal tubules of aglomerular fishes. Thus, the labeled substance offers the advantage of easier measurements of GFR, but it exposes the patient to radiation. The rad dose is minimal for a single study, however it can become significant if many studies are performed, as might be expected in a renal transplant patient. In addition, there is always the possibility of elution of the radioiodine atom from the parent compound which will alter the calculated GFR (11, 12). Our proposed technique would eliminate the disadvantages of using radioactive compounds for GFR determinations, while preserving the advantages of clinical suitability and accuracy. The fluorescent technique described (Fig. 3) is based on the use of an americium source. 241 Americium produces a 60-keV photon which, upon interaction with an electron from the K-shell of an iodine atom within the sample, results in emission of characteristic x rays (28.5 and 32.4 keV) which are detected and measured by the semiconductor detector. The semiconductor detector is required to give the necessary energy resolution to separate iodine from other elements which may be present in the sample. The detector is interfaced to an 8K dedicated computer which quantitates the characteristic x rays produced and converts them (based on previous measurement with known standards) into milligram per cent of iodine, enabling calculation of the GFR. Various investigators have tried different techniques to simplify the determination of GFR and render it more useful clinically (13-18). Our approach to determining GFR using relatively small amounts of iothalamate and measuring iodine levels via x-ray fluorescence technique can be applied to almost any of these simplified methods. Furthermore, the samples can be counted by x-ray fluorescence immediately or they can be placed in storage for counting at a more convenient time without influencing the result. The high sensitivity of x-ray fluorescence for measuring iodine, approaching one part per million (0.1 mg %), permits the injected iothalamate doses to be less than 10 cm 3 for an average adult. Conray 400 was selected
Medicine
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because it contains more milligrams of Iodine per ml of iothalamate (400 mg/ml) than most other available preparations. With further improvements in the x-ray fluorescent system, the sensitivity could be reduced to less than one part per million and the injected dose lowered. The data from a single dog study (Fig. 6, A and B) show that an equulbrlum state occurs about 2-3 hours after the start of an infusion. In this experiment the iothalamate and 14C inulin were administered in the same infusion, and blood samples were drawn at regular time intervals. The time-concentration curves are the same for both substances. Our rationale for calculating clearances utiliZing only measurements of plasma samples is based on the fact that at eqUilibrium (that is when the rate of infusion is equal to the rate of excretion of the test compound) the clearance
186
NAOMI ALAZRAKI AND OTHERS
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can be calculated from the rate of infusion. By substituting in the equation in Figure 1, accurate clearances were obtained for the above dog. Two normal dogs were studied on several different days to establish reproducibility. Based on four experiments, the first dog had a measured mean iothalamate-fluorescent GFR of 77.0 ± 5.2 and a mean inulin measured GFR of 83.4 ± 8.8. The second dog, based on three experiments, has a measured mean iothalamate-fluorescent GFR of 69.6 ± 7.8, and mean inulin measured GFR of 76.4 ± 12.6. These results indicate close reproducibility of measured GFR by both iothalamate-fluorescence and inulin methods. It is noteworthy that the iothalamate-fluorescent technique resulted in a smaller standard deviation for both normal dogs than the inulin method, perhaps indicating slightly closer reproducibility. As emphasized by Cole et st. (15), the infusion or subcutaneous injected approach is only reliable during equilibrium, where the rate of infusion, or blood absorption, equals the rate of excretion. If the loading dose used is too small because of underestimation of the extracellular fluids, or the GFR is overestimated and the infusion rate is too rapid, plasma levels of iothalamate may rise with delay in achievement of equilibrium. In our experience this has not been a problem, but we recognize that none of our dog subjects had any additional extracellular fluid compartment such as ascites or edema which would prolong the achievement of equilibrium. In the absence of those conditions, this approach to GFR determination could be of true clinical importance since it appears to be both accurate and reproducible. ACKNOWLEDGMENT: We thank Dr. Nicholas Halasz for having provided the animal subjects with impaired renal function and for his review of the manuscript. Nuclear Medicine Service (115) VA Hospital 3350 La Jolla Village Drive San Diego, Calif. 92161
REFERENCES 1. Monasterio G, Giovanetti S. Maggiore Q: Le Traitement dietetique de I'uremie chronique. Actualites Nephrologiques de I'Hopital Necker, Paris Editions Medicales Flammarion, 1965, pp 31-42 2. Bianchi C, Coli A, Palla R, et al: Serum creatinine in the evaluation of renal function. Abstr Proc 4th Int Cong Nephrol, Stockholm, 1969, p 48 3. Documenta Geigy, Scientific Tables, Ardsley, New York, 6th ed., Geigy Pharmaceuticals, p 538 4. Smith HW: The Kidney, Structure and Function in Health and Disease. New York, Oxford University Press, 1951, p 39-40 5. Smith HW: Principles of Renal Physiology, New York, Oxford University Press, 1956, p 27 6. Smith HW, Goldring W, Chasis H: The measurement of the tubular excretory mass, effective blood flow and filtration rate in the normal human kidney. J Clin Invest 17:263-278, May 1938 7. Bianchi C: Measurement of the Glomerular Filtration Rate, [In] Evaluation of Renal Function and Disease with Radionuclides, eel by MD Blaufox, Baltimore, University Park Press, 1972, p 21-48 8. Liewendahl K, Tallgren LG, Rusk J: Separation of inulin from chemically interfering dextrans by gel filtration. Scan J Clin Lab Invest 18:553-556, 1966 9. Sigman EM, Elwood CM, and Knox F: The measurement of glomerular filtration rate in man with sodium iothalamate 131 1(Conray). J Nucl Med 7:60-68, Jan 1966 10. Griep RJ, Nelp WB: Mechanism of excretion of radioiodinateel sodium iothalamate. Radiology 93:807-811, Oct 1969 11. Schmid HE. Muehlbaecher CA, Hutchins PM: Continuous determination of the clearance of 131-I-diatrizoate, an inulin substitute. J Appl Physiol 25:294-300, Sep 1968 12. Cohen M: Radionuclide clearance techniques. Sem Nucl Med 4:23-28, Jan 1974 13. Blaufox MD: Nuclear medicine and renal disease. Hospital Practice 7:137-144, 1972 14. Rose GA: Measurement of glomerular filtration rate by inulin clearance without urine collection. Br Med J 2:91-93, 12 Apr 1969 15. Cole BR, Giangiacomo J, Inglefinger JR, et al: Measurement of renal function without urine collection. N Engl J Mad 287:1109-1114, 30 Nov 1972 16. Blaufox MD, Cohen A: Single-injection clearances of iothalamate- 1311 in the rat. Am J Physiol 218:542-544, Feb 1970 17. Findley T, White HL: Measurement of diodrast and inulin clearances after subcutaneous administration. Proc Soc Exp Bioi 45:623, 1940 18. Israelit AH, Long DL, White MG, et al: Measurement of glomerular filtration rate utilizing a single subcutaneous injection of 1251-iothalamate. Kidney Int 4:346-349, Nov 1973
