Clin. Biochem. 11 (4) 133-134 (1978)
Serum Ribonuclease of Normal Persons I and Patients with Renal Impairment K. K. REDDI Department of Neoplastic Diseases Mount Sinai School of Medicine, New York 10029 (Accepted November 29, 1977) MATERIALS AND METHODS CLBIA, 11 (4) 133-134 (1978)
Clin. Biochem. Reddi, K. K.
Department of Neoplastic Diseases, Mount Sinai School of Medicine, New York, New York, 10029 S E R U M R I B O N U C L E A S E OF NORMAL P E R SONS AND P A T I E N T S W I T H R E N A L IMPAIRMENT Serum ribonuclease of normal persons and of patients with renal impairment was determined with polycytidylic acid as substrate. There was a pronounced rise in the serum ribonuclease of patients with renal impairment. Average serum ribonuclease values of 25 normal persons and 25 patients with renal impairment, respectively were 110 and 2329 units per ml of serum. Serum ribonuclease, because of its unique specificity, stability and abnormal elevation in the sera of patients with renal failure, might serve as an additional indicator in the assessment of renal function.
RIBONUCLEASE IS ONE OF SEVERAL ENZYMES e l a b o r a t e d by the pancreas. Recent studies revealed t h a t h u m a n p a n c r e a t i c ribonuclease, s e r u m ribonuclease and u r i n e ribonuclease are s i m i l a r in t h e i r chemical nature, elect r o p h o r e t i c mobility, isoelectric point, molecular w e i g h t , t h e r m o s t a b i l i t y , pH o p t i m u m , r e q u i r e m e n t of phosphate or c i t r a t e for a c t i v i t y , i n h i b i t i o n by polyadenylic acid and polyguanylic acid, specificity t o w a r d secondary phosphate esters of c y t i d i n e 3'-phosphates, mode of action and i n a b i l i t y to hydrolyze c y t i d i n e 2':3'-cyclic and u r i d i n e 2 ' : 3 ' - c y c l i c p h o s p h a t e s ''~'. F u r t h e r m o r e , h u m a n s e r u m and urine ribonuclease activity, which hydrolyzes secondary phosphate e s t e r s of c y t i d i n e 3'-phosphates, can be e n t i r e l y p r e c i p i t a t e d w i t h antibodies r a i s e d a g a i n s t h u m a n p a n c r e a t i c ribonuclease (unpublished data, Reddi). These s i m i l a r i t i e s s u g g e s t t h a t the ribonuclease o r i g i n a t e s in the pancreas, e n t e r s the blood and is e x c r e t e d in the urine. D u r i n g the course of our studies c o r r e l a t i n g s e r u m ribonuclease levels w i t h the p a n c r e a t i c ailments, we observed an a b n o r m a l i n c r e a s e in the s e r u m ribonuclease in p a t i e n t s w i t h renal i n s u f f i c i e n c y cs'. The p r e s e n t study h e r e i n described c o n f i r m s and e x t e n d s this observation.
Mailing address: Dr. K. K. Reddi, Department of NeoPlastic Diseases, Mount Sinai School of Medicine, New York, N.Y. 10029 1This investigattion was aided in p a r t by the United
States Public Health Service Grant CA 1593-04.
Reagents - -
polycytidylic acid was purchased from Schwartz/Mann, 0rangehurg, New York. All other reagents used in this investigation were of reagent grade. Normal persons -- These were volunteer laboratory workers with apparently normal renal function. Patients -- Those included in this study were on maintenance haemodialysis at Mount Sinai Hospital due to renal failure. Blood samples were taken before dialysis. In a few cases pre- and postdialysis blood samples were also obtained from the same patients. Serum -- Venous blood drawn from volunteer laboratory workers or patients, with their consent, was allowed to clot at room temperature for 1 hr and centrifuged at 750 x g for 15 min at room temperature. The serum was removed with a capillary pipette and assayed immediately or stored at-20 °. Ribonuclease Assay -- Because h u m a n serum ribonuclease is highly specific to secondary phosphate esters of cytidine 3'-phosphate, its assay with polycytidylic acid as a substrate is more sensitive than with ribonucleic acid, which is hydrolized only partially. Cleavage of polycytidylic acid by serum ribonuclease was followed by the formation of mono- and oligonucleotides, which were separated from partially digested polycytidylic acid fragments by acid precipitation. Reaction mixtures consisting of 0.05 ml of polycytidylic acid (100 ~g), 0.15 ml of 0.08 M sodium phosphate-borate buffer, pH 6.5 and 0.05 ml of serum that had been diluted to 200-fold in the case of normal serum and 2000-fold in the case of patients' sera, were incubated at 37 ° for 15 rain and then transferred to an ice bath. To each was added with mixing, 0.25 ml of cold 1.2 M HCI04 containing 0.02 M lanthanum nitrate. After 20 rain at 0 °, the precipitates were removed by centrifugation in the cold at 12,000 g for 30 rain. The supernatants were diluted with H20 and their absorptions were measured at 278 nm. Enzyme and substrate blanks were always run side by side. One ribonuclease unit is defined as that amount which renders 1 ~mole of polycytidylic acid acid-soluble in 1 rain at 37 ° and at pH 6.5. Creatinine - - This was determined in the Clinical Chemistry Department of Mount Sinai Hospital with Autoanalyser. RESULTS A N D DISCUSSION A S shown in the accompanying Table, serum ribonuclease values of 25 normal persons and 25 patients with renal impairment respectively ranged from 62 to 146 and 751 to 3756 units per ml of serum with an average of 110 and 2329 units per ml of serum. Thus, the serum ribonuclease of patients increased by about 21-fold, while their serum creatinine values increased by about 15-fold. A s reported earlier, the increased serum ribonuclease was also observed in patients with hypernephroma ''~' and hence, the high serum ribonuclease is not specifically related to haemodialysis, but to the renal impairment. The serum ribonuclease has a molecular weight of about 21,000 daltons '~''' and is nondialysable. This is further confirmed by
K. K. REDDI
184
TABLE 1 SERUM RIBONUCLEASE AND CREATININE OF NORMAL PERSONS AND PATIENTS WITH RENAL IMPAIRMENT
Source of Serum
Number of Subjects
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
Patients on maintenance haemodialysis . . . . . . . .
25
e x a m i n i n g t h e pre- and p o s t d i a l y s i s s e r u m samples, w h i c h r e v e a l e d no d i f f e r e n c e s in t h e i r r i b o n u c l e a s e levels. In persons with normal renal function, ribonuclease o r i g i n a t i n g f r o m t h e p a n c r e a s e n t e r s t h e blood a n d is f i n a l l y e x c r e t e d in t h e u r i n e p r e s u m a b l y b y g l o m e r u l a r f i l t r a t i o n . I n cases of r e n a l i m p a i r m e n t , t h i s enzyme a c c u m u l a t e s in t h e blood. T h e p h y s i o l o g i c a l e f f e c t s o f such h i g h levels of r i b o n u c l e a s e r e s u l t i n g f r o m r e n a l f a i l u r e , a r e not, a t p r e s e n t , known. B e c a u s e of i t s unique s p e c i f i c i t y a n d o f its a b n o r m a l i n c r e a s e in the s e r a o f p a t i e n t s w i t h r e n a l a i l m e n t s , t h i s enzyme m i g h t s e r v e as an a d j u n c t t e s t in a s s e s s i n g t h e r e n a l f u n c t i o n . F u r t h e r m o r e , only 0.25 /zl of t h e s e r u m of n o r m a l p e r s o n s a n d 0.025/zl of t h e s e r u m o f p a t i e n t s w i t h r e n a l i n s u f f i c i e n c y is n e e d e d to p e r f o r m a ribonuclease assay.
Ribonuclease unit.s/ml serum Range 62 - -
146
751 - - 3 7 5 6
Creatinine mg/100 ml serum
Mean ± SD 110 ±
20.9
2329 ± 862.4
Range 0.6 - 7.5-
Mean ± SD
1.4
1.0 ± 0.21
27.3
15 ± 4.78
ACKNOWLEDGMENT
I g r a t e f u l l y a c k n o w l e d g e t h e help of Dr. Sheldon G l a b m a n in o b t a i n i n g t h e p a t i e n t s ' s e r a used in t h i s investigation. REFERENCES
1. Reddi, K. K. (1975). Biochem. Biophys. Res. Commun., 67, 110-118. 2. Reddi, K. K. and Holland, J. F., In Third Int. Symp. for Detection and Prevention of Cancer (edited by H. Nieburgs) ; in press, New York, 1977. 3. Reddi, K. K., and Holland, J. F. (1976) Proc. Natl. Acad. Sci. U.S.A., 73, 2308-2310. 4. Reddi, K. K. (1977). Preparative Biochem., 7, 283-299. 5. Reddi, K. K. (1976). Biochem. Biophys. Res. Commun., 68, 1119-1125.
Serum Ribonuclease of Normal Persons I and Patients with Renal Impairment K. K. REDDI Department of Neoplastic Diseases Mount Sinai School of Medicine, New York 10029 (Accepted November 29, 1977) MATERIALS AND METHODS CLBIA, 11 (4) 133-134 (1978)
Clin. Biochem. Reddi, K. K.
Department of Neoplastic Diseases, Mount Sinai School of Medicine, New York, New York, 10029 S E R U M R I B O N U C L E A S E OF NORMAL P E R SONS AND P A T I E N T S W I T H R E N A L IMPAIRMENT Serum ribonuclease of normal persons and of patients with renal impairment was determined with polycytidylic acid as substrate. There was a pronounced rise in the serum ribonuclease of patients with renal impairment. Average serum ribonuclease values of 25 normal persons and 25 patients with renal impairment, respectively were 110 and 2329 units per ml of serum. Serum ribonuclease, because of its unique specificity, stability and abnormal elevation in the sera of patients with renal failure, might serve as an additional indicator in the assessment of renal function.
RIBONUCLEASE IS ONE OF SEVERAL ENZYMES e l a b o r a t e d by the pancreas. Recent studies revealed t h a t h u m a n p a n c r e a t i c ribonuclease, s e r u m ribonuclease and u r i n e ribonuclease are s i m i l a r in t h e i r chemical nature, elect r o p h o r e t i c mobility, isoelectric point, molecular w e i g h t , t h e r m o s t a b i l i t y , pH o p t i m u m , r e q u i r e m e n t of phosphate or c i t r a t e for a c t i v i t y , i n h i b i t i o n by polyadenylic acid and polyguanylic acid, specificity t o w a r d secondary phosphate esters of c y t i d i n e 3'-phosphates, mode of action and i n a b i l i t y to hydrolyze c y t i d i n e 2':3'-cyclic and u r i d i n e 2 ' : 3 ' - c y c l i c p h o s p h a t e s ''~'. F u r t h e r m o r e , h u m a n s e r u m and urine ribonuclease activity, which hydrolyzes secondary phosphate e s t e r s of c y t i d i n e 3'-phosphates, can be e n t i r e l y p r e c i p i t a t e d w i t h antibodies r a i s e d a g a i n s t h u m a n p a n c r e a t i c ribonuclease (unpublished data, Reddi). These s i m i l a r i t i e s s u g g e s t t h a t the ribonuclease o r i g i n a t e s in the pancreas, e n t e r s the blood and is e x c r e t e d in the urine. D u r i n g the course of our studies c o r r e l a t i n g s e r u m ribonuclease levels w i t h the p a n c r e a t i c ailments, we observed an a b n o r m a l i n c r e a s e in the s e r u m ribonuclease in p a t i e n t s w i t h renal i n s u f f i c i e n c y cs'. The p r e s e n t study h e r e i n described c o n f i r m s and e x t e n d s this observation.
Mailing address: Dr. K. K. Reddi, Department of NeoPlastic Diseases, Mount Sinai School of Medicine, New York, N.Y. 10029 1This investigattion was aided in p a r t by the United
States Public Health Service Grant CA 1593-04.
Reagents - -
polycytidylic acid was purchased from Schwartz/Mann, 0rangehurg, New York. All other reagents used in this investigation were of reagent grade. Normal persons -- These were volunteer laboratory workers with apparently normal renal function. Patients -- Those included in this study were on maintenance haemodialysis at Mount Sinai Hospital due to renal failure. Blood samples were taken before dialysis. In a few cases pre- and postdialysis blood samples were also obtained from the same patients. Serum -- Venous blood drawn from volunteer laboratory workers or patients, with their consent, was allowed to clot at room temperature for 1 hr and centrifuged at 750 x g for 15 min at room temperature. The serum was removed with a capillary pipette and assayed immediately or stored at-20 °. Ribonuclease Assay -- Because h u m a n serum ribonuclease is highly specific to secondary phosphate esters of cytidine 3'-phosphate, its assay with polycytidylic acid as a substrate is more sensitive than with ribonucleic acid, which is hydrolized only partially. Cleavage of polycytidylic acid by serum ribonuclease was followed by the formation of mono- and oligonucleotides, which were separated from partially digested polycytidylic acid fragments by acid precipitation. Reaction mixtures consisting of 0.05 ml of polycytidylic acid (100 ~g), 0.15 ml of 0.08 M sodium phosphate-borate buffer, pH 6.5 and 0.05 ml of serum that had been diluted to 200-fold in the case of normal serum and 2000-fold in the case of patients' sera, were incubated at 37 ° for 15 rain and then transferred to an ice bath. To each was added with mixing, 0.25 ml of cold 1.2 M HCI04 containing 0.02 M lanthanum nitrate. After 20 rain at 0 °, the precipitates were removed by centrifugation in the cold at 12,000 g for 30 rain. The supernatants were diluted with H20 and their absorptions were measured at 278 nm. Enzyme and substrate blanks were always run side by side. One ribonuclease unit is defined as that amount which renders 1 ~mole of polycytidylic acid acid-soluble in 1 rain at 37 ° and at pH 6.5. Creatinine - - This was determined in the Clinical Chemistry Department of Mount Sinai Hospital with Autoanalyser. RESULTS A N D DISCUSSION A S shown in the accompanying Table, serum ribonuclease values of 25 normal persons and 25 patients with renal impairment respectively ranged from 62 to 146 and 751 to 3756 units per ml of serum with an average of 110 and 2329 units per ml of serum. Thus, the serum ribonuclease of patients increased by about 21-fold, while their serum creatinine values increased by about 15-fold. A s reported earlier, the increased serum ribonuclease was also observed in patients with hypernephroma ''~' and hence, the high serum ribonuclease is not specifically related to haemodialysis, but to the renal impairment. The serum ribonuclease has a molecular weight of about 21,000 daltons '~''' and is nondialysable. This is further confirmed by
K. K. REDDI
184
TABLE 1 SERUM RIBONUCLEASE AND CREATININE OF NORMAL PERSONS AND PATIENTS WITH RENAL IMPAIRMENT
Source of Serum
Number of Subjects
Normal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
Patients on maintenance haemodialysis . . . . . . . .
25
e x a m i n i n g t h e pre- and p o s t d i a l y s i s s e r u m samples, w h i c h r e v e a l e d no d i f f e r e n c e s in t h e i r r i b o n u c l e a s e levels. In persons with normal renal function, ribonuclease o r i g i n a t i n g f r o m t h e p a n c r e a s e n t e r s t h e blood a n d is f i n a l l y e x c r e t e d in t h e u r i n e p r e s u m a b l y b y g l o m e r u l a r f i l t r a t i o n . I n cases of r e n a l i m p a i r m e n t , t h i s enzyme a c c u m u l a t e s in t h e blood. T h e p h y s i o l o g i c a l e f f e c t s o f such h i g h levels of r i b o n u c l e a s e r e s u l t i n g f r o m r e n a l f a i l u r e , a r e not, a t p r e s e n t , known. B e c a u s e of i t s unique s p e c i f i c i t y a n d o f its a b n o r m a l i n c r e a s e in the s e r a o f p a t i e n t s w i t h r e n a l a i l m e n t s , t h i s enzyme m i g h t s e r v e as an a d j u n c t t e s t in a s s e s s i n g t h e r e n a l f u n c t i o n . F u r t h e r m o r e , only 0.25 /zl of t h e s e r u m of n o r m a l p e r s o n s a n d 0.025/zl of t h e s e r u m o f p a t i e n t s w i t h r e n a l i n s u f f i c i e n c y is n e e d e d to p e r f o r m a ribonuclease assay.
Ribonuclease unit.s/ml serum Range 62 - -
146
751 - - 3 7 5 6
Creatinine mg/100 ml serum
Mean ± SD 110 ±
20.9
2329 ± 862.4
Range 0.6 - 7.5-
Mean ± SD
1.4
1.0 ± 0.21
27.3
15 ± 4.78
ACKNOWLEDGMENT
I g r a t e f u l l y a c k n o w l e d g e t h e help of Dr. Sheldon G l a b m a n in o b t a i n i n g t h e p a t i e n t s ' s e r a used in t h i s investigation. REFERENCES
1. Reddi, K. K. (1975). Biochem. Biophys. Res. Commun., 67, 110-118. 2. Reddi, K. K. and Holland, J. F., In Third Int. Symp. for Detection and Prevention of Cancer (edited by H. Nieburgs) ; in press, New York, 1977. 3. Reddi, K. K., and Holland, J. F. (1976) Proc. Natl. Acad. Sci. U.S.A., 73, 2308-2310. 4. Reddi, K. K. (1977). Preparative Biochem., 7, 283-299. 5. Reddi, K. K. (1976). Biochem. Biophys. Res. Commun., 68, 1119-1125.
