271
Clinica Chimica Acta, 79 (1977) 271-275 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 8704
AGE AND SEX RELATED RIBONUCLEASE
REFERENCE
VALUES FOR SERUM
E.J. COOMBES a*, P.G. SHAKESPEARE b and G.F. BATSTONE B 0 Department of Chemical Pathology, Salisbury General Infirmary, Salisbury, Wiltshire SP2 7SX (U.K.) and b Burns Research Unit, Odstock Hospital, Salisbwy, Wiltshire (U.K.) (Received January 6th, 1977)
Introduction Ribonuclease, an enzyme which catalyses the hydrolysis of polyribonucleic acids to their component nucleotides, may be involved in cell death and tissue catabolism. Sigulem et al. [l] have shown that serum alkaline ribonuclease is a sensitive marker in the assessment of the nutritional status of children with varying degrees of malnutrition, in that all malnourished children had elevated enzyme activities. In human nutritional experiments Albanese et al. [2] have established an inverse relationship between nitrogen balance and serum ribonuclease activities. Elevated ribonuclease activities correlated with a negative nitrogen balance. As a result of this research, Albanese et al. [Z] suggest that ribonuclease may serve as an index of the degree of tissue catabolism. In a continuing laboratory investigation into parameters which may indicate the extent of tissue catabolism, some properties of serum ribonuclease have been investigated. In this paper we present the results of ribonuclease assays on normal subjects. These results show that there are significant sex differences at different ages. In an initial investigation into one catabolic condition, thyrotoxicosis, serum alkaline ribonuclease activity has been shown to be significantly elevated when compared with the values for normal females aged 30-70 years. Materials and methods Controls Serum for ribonuclease assay was taken from 50 male and 63 female subjects comprising laboratory staff and volunteers between the ages of 10 and 85. All sub-
a.
* To whom correspondence should be addressed.
212
jects were in good health and eating a normal diet. All subjects were screened for liver and renal disorders and found to be within the acceptable normal reference limits of this laboratory. b. Patients Serum from 35 women with clinical symptoms of th~otoxicosis was assayed for alkaline ribonuclease and thyroxine (T4). Thyroxine was measured by radioimmunoassay (Radiochemical Centre, Amersham RIA T4 method) and the mean + 1 standard deviation in thyrotoxic women was 225 + 40 nmol/l. All thyroxine levels exceeded the laboratory upper limit of normal (160 nmol/l). c. Method Acid and alkaline ribonuclease were measured using the method of Biswas and Hindocha [ 31 with several minorSvariations. Tubes cont~ning highly polyme~s~ ~bonucleic acid (B.D.H. Ltd., Poole, 1 mg/ml prepared freshly before use) in 0.066 M phosphate buffer (pII 6.5 or 7.6) were incubated at 37°C for 5 min before addition of the serum. Blanks were treated similarly but cold precipitating reagent added immediately before addition of serum. Chromic acid-washed glassware was found to be essential for reproducible results as unwashed glassware resulted in high blanks. Fig. 1 shows the pH curve for serum ribonuclease from a normal subject. Serum alkaline ribonuclease was found to have a pH optimum of 7.6 (Biswas and Hindocha [3] 7.5) and the acid ribonuclease pH 6.5 (Biswas and Hindocha [ 31 did not measure acid ribonuclease) using phosphate buffered ribonucleic acid.
O-8
0.8 A o-5' 0.4 o-3 ' o-2 ' o-1
’
I
.
.’ 4-o
s-o
I
8.0
I
FO
I
8.0
I
9.0
I 30.0
jE$ Fig. 1. Variation of serum ribonuelease activity with PH in normal subject.
273
Enzyme units were expressed as absorbance (A) units per ml of serum. One unit of enzyme is equivalent to the amount of enzyme that produces an increase of 1.0 absorbance unit per hour at 260 nm,. The “Student” t-test was used to evaluate the probability (P) of significant differences between groups. Results and discussion Table I shows the overall mean L- 1 standard deviation (S.D.) for males and females for both acid and alkaline ribonuclease. The means for alkaline ribonuclease in this study bear favourable comparison with those obtained by Biswas and Hindocha [ 31. Table II presents results obtained by me~~ement of alkaline ribonuclease in the serum of normal males and females. Patients are grouped in lO-yearage blocks. Results (expressed as mean + 1 SD.) demonstrate that alkaline ribonuclease in males appea& to ‘rise gradually throughout the life span. Alkaline ribonuclease in females increases initially, reaches a plateau in middle age”an’d increases rapidly in old age. Significant sex differences were found in the 5160 year age group (P < 0.05) and the 70+ group (P < 0.05). Table III shows the variation between male and female acid ribonuclease in IO-year age blocks. A si~ific~t sex difference was found in the 70+ age group (P < 0.05). Fig. 2 presents results obtained by measurement of alkaline ribonuclease in 35 thyrotoxic women and the reference range (mean + 1 S.D.) for normal women. Table IV shows the variation in alkaline ribonuclease between thyrotoxic and normal females in lo-year age blocks. Significant differences were found in the 31-40 year age group (P < O.Ol), 41-50 {P < O.Ol), 51-60 (P< 0.001) and 61-70 year age group (P < 0.05). These results support the previous work of Albanese et al. [2] and Sved et al. [4] who demonstrated that serum alkaline ribonuclease is age related. This study has demonstrated that serum acid ribonuclease is age related also. It has also been demonstrated that there are significant male/female differences at various age groups in both acid and alkaline ribonuclease. These variations are important when investigating enzyme activity in disease states.
TABLE I ALKALINE
AND ACID RIBONUCLEASE
IN NORMAL SUBJECTS
Enzyme activity expressed as absorbance units/ml of serum. n, sample number. Activity Alkaline ribonuelease
Overal) m,ean 1 S.D. n
Acid ribonuclease
Male
FemaIe
Male
FemaIe
311.5 44 50
317 62 63
240.5 39 44
251.5 71 36
274 TABLE II VARIATION
IN MALE AND FEMALE
ALKALINE
RIBONUCLEASE
IN lo-YEAR
AGE BLOCKS
Enzyme activity expressed as A units/ml of serum. P, probability; N.S., not significant. P
Activity (mean + 1 S.D.)
Age
Female
Male 11-20 21-30 31-40 41-50 51-60 61-70 70+
227 277 293 323 339 337 351
+ f * f + * *
28 24 19 28 25 36 30
245 294 283 304 306 355 423
+ 25 + 20 f 28 + 25 ?- 25 f 28 f 74
N.S. N.S. N.S. N.S.
Clinica Chimica Acta, 79 (1977) 271-275 @ Elsevier/North-Holland Biomedical Press
SHORT COMMUNICATION CCA 8704
AGE AND SEX RELATED RIBONUCLEASE
REFERENCE
VALUES FOR SERUM
E.J. COOMBES a*, P.G. SHAKESPEARE b and G.F. BATSTONE B 0 Department of Chemical Pathology, Salisbury General Infirmary, Salisbury, Wiltshire SP2 7SX (U.K.) and b Burns Research Unit, Odstock Hospital, Salisbwy, Wiltshire (U.K.) (Received January 6th, 1977)
Introduction Ribonuclease, an enzyme which catalyses the hydrolysis of polyribonucleic acids to their component nucleotides, may be involved in cell death and tissue catabolism. Sigulem et al. [l] have shown that serum alkaline ribonuclease is a sensitive marker in the assessment of the nutritional status of children with varying degrees of malnutrition, in that all malnourished children had elevated enzyme activities. In human nutritional experiments Albanese et al. [2] have established an inverse relationship between nitrogen balance and serum ribonuclease activities. Elevated ribonuclease activities correlated with a negative nitrogen balance. As a result of this research, Albanese et al. [Z] suggest that ribonuclease may serve as an index of the degree of tissue catabolism. In a continuing laboratory investigation into parameters which may indicate the extent of tissue catabolism, some properties of serum ribonuclease have been investigated. In this paper we present the results of ribonuclease assays on normal subjects. These results show that there are significant sex differences at different ages. In an initial investigation into one catabolic condition, thyrotoxicosis, serum alkaline ribonuclease activity has been shown to be significantly elevated when compared with the values for normal females aged 30-70 years. Materials and methods Controls Serum for ribonuclease assay was taken from 50 male and 63 female subjects comprising laboratory staff and volunteers between the ages of 10 and 85. All sub-
a.
* To whom correspondence should be addressed.
212
jects were in good health and eating a normal diet. All subjects were screened for liver and renal disorders and found to be within the acceptable normal reference limits of this laboratory. b. Patients Serum from 35 women with clinical symptoms of th~otoxicosis was assayed for alkaline ribonuclease and thyroxine (T4). Thyroxine was measured by radioimmunoassay (Radiochemical Centre, Amersham RIA T4 method) and the mean + 1 standard deviation in thyrotoxic women was 225 + 40 nmol/l. All thyroxine levels exceeded the laboratory upper limit of normal (160 nmol/l). c. Method Acid and alkaline ribonuclease were measured using the method of Biswas and Hindocha [ 31 with several minorSvariations. Tubes cont~ning highly polyme~s~ ~bonucleic acid (B.D.H. Ltd., Poole, 1 mg/ml prepared freshly before use) in 0.066 M phosphate buffer (pII 6.5 or 7.6) were incubated at 37°C for 5 min before addition of the serum. Blanks were treated similarly but cold precipitating reagent added immediately before addition of serum. Chromic acid-washed glassware was found to be essential for reproducible results as unwashed glassware resulted in high blanks. Fig. 1 shows the pH curve for serum ribonuclease from a normal subject. Serum alkaline ribonuclease was found to have a pH optimum of 7.6 (Biswas and Hindocha [3] 7.5) and the acid ribonuclease pH 6.5 (Biswas and Hindocha [ 31 did not measure acid ribonuclease) using phosphate buffered ribonucleic acid.
O-8
0.8 A o-5' 0.4 o-3 ' o-2 ' o-1
’
I
.
.’ 4-o
s-o
I
8.0
I
FO
I
8.0
I
9.0
I 30.0
jE$ Fig. 1. Variation of serum ribonuelease activity with PH in normal subject.
273
Enzyme units were expressed as absorbance (A) units per ml of serum. One unit of enzyme is equivalent to the amount of enzyme that produces an increase of 1.0 absorbance unit per hour at 260 nm,. The “Student” t-test was used to evaluate the probability (P) of significant differences between groups. Results and discussion Table I shows the overall mean L- 1 standard deviation (S.D.) for males and females for both acid and alkaline ribonuclease. The means for alkaline ribonuclease in this study bear favourable comparison with those obtained by Biswas and Hindocha [ 31. Table II presents results obtained by me~~ement of alkaline ribonuclease in the serum of normal males and females. Patients are grouped in lO-yearage blocks. Results (expressed as mean + 1 SD.) demonstrate that alkaline ribonuclease in males appea& to ‘rise gradually throughout the life span. Alkaline ribonuclease in females increases initially, reaches a plateau in middle age”an’d increases rapidly in old age. Significant sex differences were found in the 5160 year age group (P < 0.05) and the 70+ group (P < 0.05). Table III shows the variation between male and female acid ribonuclease in IO-year age blocks. A si~ific~t sex difference was found in the 70+ age group (P < 0.05). Fig. 2 presents results obtained by measurement of alkaline ribonuclease in 35 thyrotoxic women and the reference range (mean + 1 S.D.) for normal women. Table IV shows the variation in alkaline ribonuclease between thyrotoxic and normal females in lo-year age blocks. Significant differences were found in the 31-40 year age group (P < O.Ol), 41-50 {P < O.Ol), 51-60 (P< 0.001) and 61-70 year age group (P < 0.05). These results support the previous work of Albanese et al. [2] and Sved et al. [4] who demonstrated that serum alkaline ribonuclease is age related. This study has demonstrated that serum acid ribonuclease is age related also. It has also been demonstrated that there are significant male/female differences at various age groups in both acid and alkaline ribonuclease. These variations are important when investigating enzyme activity in disease states.
TABLE I ALKALINE
AND ACID RIBONUCLEASE
IN NORMAL SUBJECTS
Enzyme activity expressed as absorbance units/ml of serum. n, sample number. Activity Alkaline ribonuelease
Overal) m,ean 1 S.D. n
Acid ribonuclease
Male
FemaIe
Male
FemaIe
311.5 44 50
317 62 63
240.5 39 44
251.5 71 36
274 TABLE II VARIATION
IN MALE AND FEMALE
ALKALINE
RIBONUCLEASE
IN lo-YEAR
AGE BLOCKS
Enzyme activity expressed as A units/ml of serum. P, probability; N.S., not significant. P
Activity (mean + 1 S.D.)
Age
Female
Male 11-20 21-30 31-40 41-50 51-60 61-70 70+
227 277 293 323 339 337 351
+ f * f + * *
28 24 19 28 25 36 30
245 294 283 304 306 355 423
+ 25 + 20 f 28 + 25 ?- 25 f 28 f 74
N.S. N.S. N.S. N.S.
