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Serum ribonuclease levels in patients with cystic fibrosis
BIOCHEMICAL
MEDICINE
Serum
19, 294-297
(1978)
SHORT
COMMUNICATION
Ribonuclease with Cystic
Levels in Patients Fibrosis
INTRODUCTION
Ribonucleases (RNases) are enzymes which cleave the 3’-5’ phosphodiester bond of RNA to yield either 5’ or 3’ nucleoside phosphates. Each of the many eukaryotic and prokaryotic RNases displays a characteristic site of attack (exonucleolytic or endonucleolytic) of the nucleic acid chain, a direction of cleavage (3’ to 5’ or 5’ to 3’), pH optimum, stability, and specificity for substrates and inhibitors (1). Human serum and urine RNase activity has been shown to be of two distinct types: one resembling the RNase found in the pancreas and duodenal contents, and the other resembling the RNase of the liver and spleen (2). It has been suggested that the source of the pancreatic-type RNase (P-type RNase) is ;he pancreas itself (3), through secretion either into the blood directly or into the duodenum with subsequent reabsorption through the gastrointestinal mucosa (4). The functioning of this latter pathway would imply that a reduced level of P-type RNase should occur in the serum of humans with pancreatic insufficiency. Cystic fibrosis (CF) is an autosomal recessive disease, usually fatal before the third decade, characterized by chronic obstructive lung disease, elevated sweat electrolyte concentrations, and pancreatic insufficiency. The present investigation was carried out to determine if P-type serum RNase activity was reduced in CF patients 3 to 24 years of age. MATERIALS
AND METHODS
Crystalline bovine serum albumin (BSA) was obtained from Calbiochem, Tris buffer from GIBCO, and polycytidylic acid [poly (C)l from Miles Laboratories, Inc. Poly (C) was chosen because of its specificity for P-type RNase (3). Serum was obtained by venipuncture from patients with CF who were receiving pancreatic enzyme supplements for pancreatic insufficiency and their heterozygous parents. Normal children were out-patients or admitted for elective surgery. Serum was frozen at -20°F until use. 294 0006-2944178/0192-0294$02.00/O Copyright All rights
@ 1978 by Academic Press. Inc. of reproduction in any form resewed.
SHORT COMMUNICATION
295
RNase was assayed by modification of the procedure described by Schmukler et al. (5). The standard assay system contained 0.5 hmol of poly (C), 0.5 mg of BSA, 100 kmol of Tris-HCl buffer, pH 7.5, and enzyme in 1 ml on ice. The substrate. poly (C), was added last, followed by vortexing. The mixture was incubated at 25°C for 15 min and the reaction was stopped with 1 ml of 12% perchloric acid. After vortexing, the cloudy mixture was chilled in an ice bath for 10 min and clarified by centrifugation at 50,OOOg for 15 min. The concentration of acid-soluble nucleotides was measured at 260 nm. An enzyme unit is defined as that amount of enzyme causing an increase in absorbance of 1.O OD under the conditions of the assay. Enzyme samples were diluted when necessary such that the assay gave absorbances of less than 1.5 OD in order to assure that the optical density changes remained a linear function of enzyme concentration. Under these conditions the usual assay contained 10 or 20 ~1 of a I : 10 dilution in H,O of a serum sample. In comparing RNase determinations for both CF and normal individuals, an extra serum sample provided an internal standard to correct for unrecognized variations in the conditions of each assay. RESULTS
Table I shows RNase activity, using poly (C) as substrate, in the sera of individuals homozygous for CF and normal individuals of different age groups. RNase activity in normal children 3- 12 years of age does not TABLE 1 RIBONUCLEASEACTIVITYINTHESERUMOFPATIENTSHOMOZYGOUSANDHETEROZYGOUS FORTHE CF GENE AND IN NORMAL PATIENTS" Homozygote Age (years) 3-12 13-24 31-3.5 All ages
Number of cases 16 5 21
RNase activity (units/ml) 443 k 79 509 2 132 459 2 95
Heterozygote Number of cases 5 5
RNase activity (units/ml) 490 + 52 490 r 52
Normal Number of cases 6 II 17
RNase activity (units/ml) 428 + 66 488 ” 118 467 k 10s
u Results shown are means & SD. The mean RNase activity of each serum sample was determined by averaging the activity per milliliter derived from the assay of two different serum volumes (10 and 20 *I of a I: 10 dilution of serum). Blank tubes contained no added serum. Serum in the absence of substrate had an activity of less than 3.0 units/ml. Results were corrected for variations in activity through an internal standard. See Materials and Methods section.
296
SHORT
COMMUNICATION
differ from RNase activity in children the same age with CF (N = 20, P > 0.30) nor from normal patients 13-24 years old (N = 15, P > 0.05). Considering all ages, the mean RNase activity of patients heterozygous for the CF gene is the same as that of CF patients (N = 24, P > 0.15). which does not differ from RNase activities in normal sera (A’ = 36, P > 0.40). DISCUSSION
The experiments reported in this paper provide information on the levels of P-type RNase activity present in the serum of normal and CF patients 3-24 years of age. Individuals heterozygous for the CF gene were also studied. No difference in serum RNase activity was found among the groups of 17 normals, 21 CF patients, and 5 CF heterozygotes. Bardon et al. (2) did report a difference in serum P-type RNase activity between CF and normal patients. However, her group studied a population of patients age 2 to 7 years from whom pancreatic supplements were withheld for 1 week. Our subjects were considerably older, and we were not able to withhold the pancreatic enzyme preparations. In addition, Bardon used uridine 3’ (a-naphthylphosphate) as substrate for the enzyme, while we employed poiycytidiiic acid. Both are actively cleaved by pancreatic-type RNase (3, 6-8). It should be mentioned that no decrease in serum RNase activity was seen by Bardon’s group in CF patients with purely pulmonary manifestations (9). Only patients with symptoms of pancreatic insufficiency from whom pancreatic extract was withheld exhibited the serum P-type RNase deficiency. It appears that observations of decreased RNase activity in the serum of young children with pancreatic insufficiency cannot be extended to older children, at least without withholding pancreatic supplements. SUMMARY
The levels of serum pancreatic-type ribonuclease (RNase) activity in normal individuals and in individuals homozygous and heterozygous for the cystic fibrosis (CF) gene have been studied, In 21 CF patients, ages 3-24 years, RNase levels of 459 -+ 95 units/ml did not differ significantly from the RNase levels of 467 -+ 105 units/ml in 17 normal individuals of the same age group nor from those of 490 -+ 52 units/ml in 5 individuals heterozygous for the CF gene. ACKNOWLEDGMENTS The authors thank Dr. E. McCabe and the staff of the Cystic Fibrosis Clinic of the University of Wisconsin Medical School as well as the staff of the clinical laboratory at Burnett General Hospital (Grantsburg. Wisconsin) for their assistance in collection of serum. We are also grateful to Ann Vale for her excellent technical assistance. This work was supported in part by a grant from the National Institute of Arthritis and Metabolism (AM-13380).
297
SHORT COMMUNICATION
REFERENCES I. 2. 3. 4. 5. 6.
Levy. C. C., Life Sci. 17, 311 (1975). Bardon, A., Sierakowska, H., and Shugar, D., C/in. Chim. Acta 67, 231 (1976). Reddi, K. K., Biochem. Biophys. Res. Commun. 67, 110 (1975). Alpers. D. H., and Isselbacher, K. J., J. Biol. Chem. 242, 5617 (1967). Schmukler, J., Jewett, P. B., and Levy, C. C., J. Biol. Chem. 250, 2206 (1974). Zan-Kowalczewska, J., Sierakowska, H., Bardon. A., and Shugar. D., Biochim. Biophys. Acta 341, 138 (1974). 7. Bardon, A., Sierakowska, H., and Shugar, D., Biochim. Biophys. Acta 438,461 (1976). 8. Zimmerman, S. B., and Sandeen, G., Anal. Biochem. 10, 444 (1965). 9. Bardon, A., Institute of Mother and Child, Warszawa, Poland. Personal communication, March 9, 1976. WILLIAM A. GAHL JOAN CHESNEY HENRY C. PITOT’
Departments of Oncology, Pathology, Canter for Health Sciences I/%iversity of Wisconsin Madison, Wisconsin 53706 Received October 7. 1977
and Pediatrics
’ Address reprint requests to Henry C. Pitot, McArdle Laboratories, Health Sciences, University of Wisconsin, Madison, Wisconsin 53706.
The Center for
MEDICINE
Serum
19, 294-297
(1978)
SHORT
COMMUNICATION
Ribonuclease with Cystic
Levels in Patients Fibrosis
INTRODUCTION
Ribonucleases (RNases) are enzymes which cleave the 3’-5’ phosphodiester bond of RNA to yield either 5’ or 3’ nucleoside phosphates. Each of the many eukaryotic and prokaryotic RNases displays a characteristic site of attack (exonucleolytic or endonucleolytic) of the nucleic acid chain, a direction of cleavage (3’ to 5’ or 5’ to 3’), pH optimum, stability, and specificity for substrates and inhibitors (1). Human serum and urine RNase activity has been shown to be of two distinct types: one resembling the RNase found in the pancreas and duodenal contents, and the other resembling the RNase of the liver and spleen (2). It has been suggested that the source of the pancreatic-type RNase (P-type RNase) is ;he pancreas itself (3), through secretion either into the blood directly or into the duodenum with subsequent reabsorption through the gastrointestinal mucosa (4). The functioning of this latter pathway would imply that a reduced level of P-type RNase should occur in the serum of humans with pancreatic insufficiency. Cystic fibrosis (CF) is an autosomal recessive disease, usually fatal before the third decade, characterized by chronic obstructive lung disease, elevated sweat electrolyte concentrations, and pancreatic insufficiency. The present investigation was carried out to determine if P-type serum RNase activity was reduced in CF patients 3 to 24 years of age. MATERIALS
AND METHODS
Crystalline bovine serum albumin (BSA) was obtained from Calbiochem, Tris buffer from GIBCO, and polycytidylic acid [poly (C)l from Miles Laboratories, Inc. Poly (C) was chosen because of its specificity for P-type RNase (3). Serum was obtained by venipuncture from patients with CF who were receiving pancreatic enzyme supplements for pancreatic insufficiency and their heterozygous parents. Normal children were out-patients or admitted for elective surgery. Serum was frozen at -20°F until use. 294 0006-2944178/0192-0294$02.00/O Copyright All rights
@ 1978 by Academic Press. Inc. of reproduction in any form resewed.
SHORT COMMUNICATION
295
RNase was assayed by modification of the procedure described by Schmukler et al. (5). The standard assay system contained 0.5 hmol of poly (C), 0.5 mg of BSA, 100 kmol of Tris-HCl buffer, pH 7.5, and enzyme in 1 ml on ice. The substrate. poly (C), was added last, followed by vortexing. The mixture was incubated at 25°C for 15 min and the reaction was stopped with 1 ml of 12% perchloric acid. After vortexing, the cloudy mixture was chilled in an ice bath for 10 min and clarified by centrifugation at 50,OOOg for 15 min. The concentration of acid-soluble nucleotides was measured at 260 nm. An enzyme unit is defined as that amount of enzyme causing an increase in absorbance of 1.O OD under the conditions of the assay. Enzyme samples were diluted when necessary such that the assay gave absorbances of less than 1.5 OD in order to assure that the optical density changes remained a linear function of enzyme concentration. Under these conditions the usual assay contained 10 or 20 ~1 of a I : 10 dilution in H,O of a serum sample. In comparing RNase determinations for both CF and normal individuals, an extra serum sample provided an internal standard to correct for unrecognized variations in the conditions of each assay. RESULTS
Table I shows RNase activity, using poly (C) as substrate, in the sera of individuals homozygous for CF and normal individuals of different age groups. RNase activity in normal children 3- 12 years of age does not TABLE 1 RIBONUCLEASEACTIVITYINTHESERUMOFPATIENTSHOMOZYGOUSANDHETEROZYGOUS FORTHE CF GENE AND IN NORMAL PATIENTS" Homozygote Age (years) 3-12 13-24 31-3.5 All ages
Number of cases 16 5 21
RNase activity (units/ml) 443 k 79 509 2 132 459 2 95
Heterozygote Number of cases 5 5
RNase activity (units/ml) 490 + 52 490 r 52
Normal Number of cases 6 II 17
RNase activity (units/ml) 428 + 66 488 ” 118 467 k 10s
u Results shown are means & SD. The mean RNase activity of each serum sample was determined by averaging the activity per milliliter derived from the assay of two different serum volumes (10 and 20 *I of a I: 10 dilution of serum). Blank tubes contained no added serum. Serum in the absence of substrate had an activity of less than 3.0 units/ml. Results were corrected for variations in activity through an internal standard. See Materials and Methods section.
296
SHORT
COMMUNICATION
differ from RNase activity in children the same age with CF (N = 20, P > 0.30) nor from normal patients 13-24 years old (N = 15, P > 0.05). Considering all ages, the mean RNase activity of patients heterozygous for the CF gene is the same as that of CF patients (N = 24, P > 0.15). which does not differ from RNase activities in normal sera (A’ = 36, P > 0.40). DISCUSSION
The experiments reported in this paper provide information on the levels of P-type RNase activity present in the serum of normal and CF patients 3-24 years of age. Individuals heterozygous for the CF gene were also studied. No difference in serum RNase activity was found among the groups of 17 normals, 21 CF patients, and 5 CF heterozygotes. Bardon et al. (2) did report a difference in serum P-type RNase activity between CF and normal patients. However, her group studied a population of patients age 2 to 7 years from whom pancreatic supplements were withheld for 1 week. Our subjects were considerably older, and we were not able to withhold the pancreatic enzyme preparations. In addition, Bardon used uridine 3’ (a-naphthylphosphate) as substrate for the enzyme, while we employed poiycytidiiic acid. Both are actively cleaved by pancreatic-type RNase (3, 6-8). It should be mentioned that no decrease in serum RNase activity was seen by Bardon’s group in CF patients with purely pulmonary manifestations (9). Only patients with symptoms of pancreatic insufficiency from whom pancreatic extract was withheld exhibited the serum P-type RNase deficiency. It appears that observations of decreased RNase activity in the serum of young children with pancreatic insufficiency cannot be extended to older children, at least without withholding pancreatic supplements. SUMMARY
The levels of serum pancreatic-type ribonuclease (RNase) activity in normal individuals and in individuals homozygous and heterozygous for the cystic fibrosis (CF) gene have been studied, In 21 CF patients, ages 3-24 years, RNase levels of 459 -+ 95 units/ml did not differ significantly from the RNase levels of 467 -+ 105 units/ml in 17 normal individuals of the same age group nor from those of 490 -+ 52 units/ml in 5 individuals heterozygous for the CF gene. ACKNOWLEDGMENTS The authors thank Dr. E. McCabe and the staff of the Cystic Fibrosis Clinic of the University of Wisconsin Medical School as well as the staff of the clinical laboratory at Burnett General Hospital (Grantsburg. Wisconsin) for their assistance in collection of serum. We are also grateful to Ann Vale for her excellent technical assistance. This work was supported in part by a grant from the National Institute of Arthritis and Metabolism (AM-13380).
297
SHORT COMMUNICATION
REFERENCES I. 2. 3. 4. 5. 6.
Levy. C. C., Life Sci. 17, 311 (1975). Bardon, A., Sierakowska, H., and Shugar, D., C/in. Chim. Acta 67, 231 (1976). Reddi, K. K., Biochem. Biophys. Res. Commun. 67, 110 (1975). Alpers. D. H., and Isselbacher, K. J., J. Biol. Chem. 242, 5617 (1967). Schmukler, J., Jewett, P. B., and Levy, C. C., J. Biol. Chem. 250, 2206 (1974). Zan-Kowalczewska, J., Sierakowska, H., Bardon. A., and Shugar. D., Biochim. Biophys. Acta 341, 138 (1974). 7. Bardon, A., Sierakowska, H., and Shugar, D., Biochim. Biophys. Acta 438,461 (1976). 8. Zimmerman, S. B., and Sandeen, G., Anal. Biochem. 10, 444 (1965). 9. Bardon, A., Institute of Mother and Child, Warszawa, Poland. Personal communication, March 9, 1976. WILLIAM A. GAHL JOAN CHESNEY HENRY C. PITOT’
Departments of Oncology, Pathology, Canter for Health Sciences I/%iversity of Wisconsin Madison, Wisconsin 53706 Received October 7. 1977
and Pediatrics
’ Address reprint requests to Henry C. Pitot, McArdle Laboratories, Health Sciences, University of Wisconsin, Madison, Wisconsin 53706.
The Center for