Clinica Chimica Acta. 210 (1992) 41-54 0 1992 Elsevier Science Publishers B.V. All rights reserved.
47 0009-8981/92/$05.00
CCA 05359
Comparison of urinary excretion of four lysosomal hydrolases in healthy elderly and young adults* Dorothy J. VanderJagt, Brenda R. Steinberg and Robert H. Glew Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, NM 87131 (USA) (Received
26 December
Key words; Lysosomal
hydrolases;
1991; revision
Renal
received
1 June 1992; accepted
function; Fractional Aging
enzyme
10 June
excretion;
1992)
fi-Hexosaminidase;
Summary The activities of four lysosomal enzymes and creatinine levels were measured in the plasma and urine of 17 healthy elderly and 7 young adults. Fractional enzyme excretion (FE ENZ) values for fl-hexosaminidase (IV-acetylglucosaminidase), Qgalactosidase, &galactosidase and &glucuronidase were calculated and compared between the two groups of subjects. FE ENZ was calculated as the ratio of enzyme clearance to creatinine clearance. The FE ENZ values for a-galactosidase, flgalactosidase and /3-glucuronidase between the elderly and young populations were not statistically different; however, relative to the young control group, the FE ENZ value for fl-hexosaminidase was elevated approximately 2-fold in the elderly population (P = 0.06). The mean urinary a-galactosidase activity for the elderly population, when expressed on the basis of creatinine, was 50% lower than that of the control group (P = 0.03), whereas the mean urinary &hexosaminidase activity for the elderly was significantly higher compared to the control group (P = 0.008). When data for all subjects was analyzed, no correlation was observed between the urinary excretion of /3-hexosaminidase or a-galactosidase and glomerular filtration rate. These data indicate that with advancing age there are changes in the tubular secretion or reabsorption of selective lysosomal enzymes, particularly fl-hexosaminidase and cr-galactosidase. These biochemical changes may provide a means of assessing subtle progressive deterioration of renal function.
Correspondence to: Robert H. Glew, Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. *This work was supported by a grant from the National Institutes of Health (AM 31357) to RHG.
48
Introduction The anatomic changes in the kidney which occur with aging have been thoroughly documented; between the fourth and ninth decades the kidneys lose as much as 30% of their mass and volume, the renal tubules decrease in number, and there is a decrease in the number of functional glomeruli [1,2]. In addition, age-dependent vascular changes occur in the glomerulus; the cortical afferent and efferent arterioles atrophy and the glomerular become sclerotic, resulting in reduced cortical blood flow and a decrease in the glomerular filtration rate [3]. Furthermore, the permeability of the glomerular basement membrane increases with aging resulting in increased urinary protein excretion. Although it is well known that creatinine and urea clearance decrease with age [ 11, little is known about the effect of aging on the excretion of lysosomal enzymes by the kidney. The concentration of these and other enzymes in urine is determined by several factors, such as their rate of escape from the blood into the glomerular filtrate and the rates at which the enzymes are secreted or degraded by renal tubular cells. Urinary excretion of P-hexosaminidase and several other lysosomal enzymes have been shown to be increased in humans with well-characterized renal disease, especially when activities are expressed on the basis of the urinary creatinine concentration [4,5]. In light of the age-dependent deteriorative changes in the glomerulus and reduction in the tubule cell mass, the question arises as to what effects these changes might have on the urinary excretion of lysosomal enzymes. We previously reported on the urinary excretion of three lysosomal hydrolases, P-hexosaminidase, a-galactosidase and /3-glucuronidase by healthy adults, aged 40-52 and a 46-year-old patient with Fabry’s disease who had received a heterologous renal graft [6]. We obtained evidence that the renal graft of the CYgalactosidase deficient patient with Fabry’s disease was capable of secreting prodigious quantities of ol-galactosidase to the extent that the fractional enzyme excretion (FE ENZ) value for this particular enzyme exceeded 2,000. FE ENZ reflects the urinary clearance of a particular enzyme relative to creatinine clearance. In the present study we determined FE ENZ values for four different lysosomal hydrolases - namely fl-hexosaminidase (EC 3.2.1.30), &galactosidase (EC 3.2.1.23) cY-galactosidase (EC 3.2.1.22) and &glucuronidase (EC 3.2.1.3 1) in 17 healthy elderly men and women and seven young men and women in an effort to inquire if urinary excretion of these enzymes provides a useful, non-invasive method of evaluating changes in renal function with age. Materials and Methods Subjects
Plasma and urine samples for enzyme analyses were obtained from subjects who were participating in a vitamin clearance study. The elderly subjects were recruited from a population of healthy individuals enrolled in the ongoing New Mexico Aging Process Study at the University of New Mexico (Albuquerque, NM). The young healthy controls were recruited from the staff and students at the University of New Mexico School of Medicine. These subjects were free of kidney disease and were not
49
taking any medications which might affect their renal function. Prior to the study, each of the subjects received a physical examination at which time a medical history was obtained and a blood sample was drawn for the determination of plasma creatinine to rule out renal insufficiency. This study was approved by the Human Research Review Committee of the University of New Mexico School of Medicine. As summarized in Table I, the elderly population was comprised of 8 males and 9 females whose average age was 77.6 years (range 71-84 years). The control subjects comprised 3 women and 4 men, ages 27-40 years. Determination
of glomerular
filtration
rate (GFR)
The GFRs were dete~ined using inufin clearance. All subjects, in~fuding the young controls, spent the night preceding the clearance test at the General Clinical Research Center of the University School of Medicine. On the day the clearance test was performed, subjects were awakened at 06:OOh at which time a urine sample was collected, its volume determined and then discarded. They were then given a volume of water to drink which was equal to the quantity of urine voided..At 07:OOh blood and urine specimens were obtained from each subject for the dete~ination of creatinine and lysosomaf enzyme activities. Blood was collected into heparinized tubes and centrifuged immediately to obtain the plasma fraction. Plasma and urine samples were stored at -30°C until analyzed. At this time, each subject ingested 10 ml water/kg body weight. After 1 h, an intravenous infusion of 5% (w/v) dextrose was begun with a sustaining infusion of inufin. A primary dose of inufin designed to achieve a plasma inufin concentration of approximately 300 mg/f was given by slow infusion over a 5-lomin period. The purpose of the sustaining infusion of inufin was to maintain the plasma inufin concentration at 300 mg/f. The infusion was continued for 225 min at 2 mf/min (approx. 450 ml solution). Urine was collected at 30-min intervals after the 45-min period which allowed for equilibration of the priming dose of inufin. Blood
TABLE I Summary of the characteristics of the young and elderly subjects
Age (years) (range) Male/Female GFR (ml/s) (range) Serum creatinine (mmolil) Urine creatinine (mmoW
Elderly (n = 17)
Young controk (n = 7)
77.6 f 3.3 71-84 8/9 I.12 f 0.298 0.740-1.88 0.082 zt 0.025
34.4 f 4.9 27-40 413 1.56 L+:0.224* 1.24-1.83 0.067 f 0.008
4.97 f 4.31
6.52 2t: 5.41
Results are given as the mean f 1 S.D. *P = 0.003.
50
samples were drawn before the inulin prime and at the midpoints of the 30-min urine collection periods. Inulin concentrations in plasma and urine were determined using the method of Heyrovsky [7]. Determination of creatinine
The plasma and urine samples were analyzed for creatinine using a manual colorimetric determination (Kit no. 555: Sigma Diagnostics, St. Louis, MO). Measurement of lysosomal enzyme activities
The four lysosomal enzymes were assayed by the fluorometric measurement of 4-methyl-umbelliferone liberated from the appropriate glycoside. The /3-hexosaminidase incubation medium contained 2.0 mM 4-methyl-umbelliferyl-~-D-glucosaminide in 0.1 M sodium citrate buffer (pH 4.6) with bovine serum albumin (1 mg/ml). The a-galactosidase activity was determined using an incubation medium which contained 5.0 mM 4-methyl-umbelliferyl-o-galactopyranoside in 0.1 M sodium acetate buffer (pH 4.5). The fl-galactosidase activity was determined using an incubation medium which contained 3.0 mM 4-methyl-umbelliferyl-/3-D-galactopyranoside in 0.06 M sodium citrate-phosphate buffer with 0.06 M sodium chloride (pH 4.8). The B-glucuronidase activity was determined in a reaction medium that contained 4.0 mM 4-methyl-umbelliferyl-O-D-glucuronide in 0.02 M sodium acetate buffer (pH 4.6). Plasma or urine (0.01 ml) was added to 0.09 ml of incubation mixture for each enzyme assay. All assays were performed at 37°C for 30 min and the reactions were terminated by adding 0.3 M glycine-ammonium hydroxide buffer (pH 10.5) to a final volume of 3.0 ml. The 4-methyl-umbelliferone liberated was measured fluorometrically (Turner Model III fluorometer; Sequoia-Turner Corp., Mountain View, CA) as described elsewhere [6] with excitation at 360 nm and emission being measured at 520-580 nm. The four hydrolase assays are highly reproducible and have a standard error of less than 4%. Plasma and urine activities are expressed as units/mm01 creatinine (U/mmol Cr). One unit of enzyme activity equals 1 pmol of substrate hydrolyzed per min at 37°C. The FE ENZ is calculated as the ratio of enzyme clearance to creatinine clearance: (U/P) enzyme activity
x loo
(U/P) creatinine where U = urine and P = plasma. Results for both the elderly population and the control population are expressed as the mean f 1 standard deviation (SD.). Statistical analyses
Statistical analyses were performed with the aid of the Number Cruncher Statistical System software program (version 5; Kaysville, UT). Group comparisons were made using the Mann-Whitney two sample test. A significance level of P C 0.05 was used for all analyses.
51 TABLE II Comparison of the serum lysosomal hydrolase levels in the young and elderly populations with activities expressed on the basis of serum volume. Enzyme
Activity/l serum Elderly
NAG c&al &Gal &Glu
17.9 0.139 0.229 1.15
Young controls f f f f
4.32 0.043 0.180 0.528
13.0 f 1.74* 0.133 f 0.020 0.207 l 0.106 1.18 f 1.04
Enzyme activities are expressed as pmollmin per I serum. NAG = fl-hexosaminidase; o-Gal = ogalactosidase; @-Gal = fl-galactosidase; @-Glu = /%glucuronidase. Results are given as the mean f 1 SD. *I’ = 0.006.
Results As shown in Table I, the average glomerular filtration rate for the elderly group was 1.12 ml/s, whereas that of the control population was 1.56 ml/s. The difference between the GFR values of the two groups was significant (P = 0.003). The mean plasma creatinine level was 22% higher in the elderly population (0.082 f 0.025 vs. 0.067 f 0.008 mmol/l), but this difference was not significant. When the levels of the four lysosomal enzymes in the plasma of the two groups were compared on a volume basis, the differences were significant only for NAG (P = 0.006) (Table II). Comparison of FE ENZ values for four enzymes in elderly and young populations By measuring the plasma and urine activities of each of the four hydrolases and the corresponding creatinine concentrations in the two fluids, we were able to calculate FE ENZ values for these enzymes. As shown in Table III, if one focuses
TABLE III Comparison of the FE ENZ values for four lysosomal hydrolases in young and elderly subjects Enzyme
NAG o-Gal P-Gal P-Glu
Fractional enzyme’ excretion (FE ENZ) Elderly
Young controls
0.518 0.836 7.97 1.26
0.277 1.50 5.42 1.55
f 0.311 f 0.738 f 12.70 f 0.973
f f f f
0.152* 1.07 7.88 1.75
Results are given as the mean k 1 S.D. Enzyme abbreviations are given in Table II. *P = 0.06.
52
lirst on the values for the young population and regards these data as the reference points, the four lysosomal hydrolases appear to fall into three categories. The first consists of enzymes whose FE EN2 values are close to unity and is exemplified by /3-glucuronidase. The other category is occupied by P-galactosidase for which the mean FE ENZ value is well above 1.00, in this case 5.42. The third category is typified by &hexosaminidase, an enzyme whose mean FE ENZ value is well below unity, for example 0.277 in the young population. When the two study groups were compared in terms of their FE EN2 values, a marginal difference between the groups was observed only for ~-hexosaminidase (P = 0.06), indicating that the elderly excreted 2.2-fold more ~-hexosaminidase in their urine than did the younger subjects. The mean FE ENZ values for /3-glucuronidase or &galactosidase for the two groups were nearly identical, 1.55 vs. 1.26 and 7.97 vs. 5.42, respectively (Table III). Noteworthy, though not attaining statistical significance, is the lower FE ENZ value for a-galactosidase in the elderly group (0.836 vs. 1.50). The increased urinary excretion of &hexosaminidase and the decreased excretion of cr-galactosidase with advancing age that was revealed through comparison of FE EN2 values (Table III) was also evident when urinary enzyme levels were expressed as activity per mmol creatinine (Table IV). Compared to the data for the young population, urinary ar-galactosidase activity was about 50% lower in the elderly but ~-hexo~minidase activity was 220% higher. Both differences were statistically significant (P = 0.03 and P = 0.008, respectively). We found that an even more effective way of distinguishing elderly from young subjects on the basis of the serum and urinary lysosomal hydrolase data was to divide the units/mm01 creatinine enzyme value for fl-hexosaminidase by the corresponding value for a-galactosidase. As shown in the Fig. 1, in which the units P-hexosaminidase per mmol creatinine/units cu-galactosidase per mmol creatinine ratios are plotted on a log scale, the ratios obtained for the elderly subjects are more variable than those obtained for the young controls. Furthermore, the difference between the means of these ratios in the elderly and young subjects was highly significant (P = 0.002).
TABLE IV Comparison of the urinary lysosomal hydrolase activities for young and elderly subjects based on the urine creatinine concentration. Enzyme
Activity, units/mmol creatinine Young controls
Elderly NAG a-Gal @-Gal @-Glu
1.11 0.0144 0.110 0.143
f f rt f
*P = 0 *008.I l*P = 0.03. Enzyme abbreviations are given in Table II.
0.579 0.013 0.123 0.069
0.510 0.0280 0.114 0.140
f f zt f
0.241r 0.016** 0.123 0.095
53 l
1529
.
5 0
400
-
-b ?
300
--
200
-
100
-
: 'C
47 0009-8981/92/$05.00
CCA 05359
Comparison of urinary excretion of four lysosomal hydrolases in healthy elderly and young adults* Dorothy J. VanderJagt, Brenda R. Steinberg and Robert H. Glew Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, NM 87131 (USA) (Received
26 December
Key words; Lysosomal
hydrolases;
1991; revision
Renal
received
1 June 1992; accepted
function; Fractional Aging
enzyme
10 June
excretion;
1992)
fi-Hexosaminidase;
Summary The activities of four lysosomal enzymes and creatinine levels were measured in the plasma and urine of 17 healthy elderly and 7 young adults. Fractional enzyme excretion (FE ENZ) values for fl-hexosaminidase (IV-acetylglucosaminidase), Qgalactosidase, &galactosidase and &glucuronidase were calculated and compared between the two groups of subjects. FE ENZ was calculated as the ratio of enzyme clearance to creatinine clearance. The FE ENZ values for a-galactosidase, flgalactosidase and /3-glucuronidase between the elderly and young populations were not statistically different; however, relative to the young control group, the FE ENZ value for fl-hexosaminidase was elevated approximately 2-fold in the elderly population (P = 0.06). The mean urinary a-galactosidase activity for the elderly population, when expressed on the basis of creatinine, was 50% lower than that of the control group (P = 0.03), whereas the mean urinary &hexosaminidase activity for the elderly was significantly higher compared to the control group (P = 0.008). When data for all subjects was analyzed, no correlation was observed between the urinary excretion of /3-hexosaminidase or a-galactosidase and glomerular filtration rate. These data indicate that with advancing age there are changes in the tubular secretion or reabsorption of selective lysosomal enzymes, particularly fl-hexosaminidase and cr-galactosidase. These biochemical changes may provide a means of assessing subtle progressive deterioration of renal function.
Correspondence to: Robert H. Glew, Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque, NM 87131, USA. *This work was supported by a grant from the National Institutes of Health (AM 31357) to RHG.
48
Introduction The anatomic changes in the kidney which occur with aging have been thoroughly documented; between the fourth and ninth decades the kidneys lose as much as 30% of their mass and volume, the renal tubules decrease in number, and there is a decrease in the number of functional glomeruli [1,2]. In addition, age-dependent vascular changes occur in the glomerulus; the cortical afferent and efferent arterioles atrophy and the glomerular become sclerotic, resulting in reduced cortical blood flow and a decrease in the glomerular filtration rate [3]. Furthermore, the permeability of the glomerular basement membrane increases with aging resulting in increased urinary protein excretion. Although it is well known that creatinine and urea clearance decrease with age [ 11, little is known about the effect of aging on the excretion of lysosomal enzymes by the kidney. The concentration of these and other enzymes in urine is determined by several factors, such as their rate of escape from the blood into the glomerular filtrate and the rates at which the enzymes are secreted or degraded by renal tubular cells. Urinary excretion of P-hexosaminidase and several other lysosomal enzymes have been shown to be increased in humans with well-characterized renal disease, especially when activities are expressed on the basis of the urinary creatinine concentration [4,5]. In light of the age-dependent deteriorative changes in the glomerulus and reduction in the tubule cell mass, the question arises as to what effects these changes might have on the urinary excretion of lysosomal enzymes. We previously reported on the urinary excretion of three lysosomal hydrolases, P-hexosaminidase, a-galactosidase and /3-glucuronidase by healthy adults, aged 40-52 and a 46-year-old patient with Fabry’s disease who had received a heterologous renal graft [6]. We obtained evidence that the renal graft of the CYgalactosidase deficient patient with Fabry’s disease was capable of secreting prodigious quantities of ol-galactosidase to the extent that the fractional enzyme excretion (FE ENZ) value for this particular enzyme exceeded 2,000. FE ENZ reflects the urinary clearance of a particular enzyme relative to creatinine clearance. In the present study we determined FE ENZ values for four different lysosomal hydrolases - namely fl-hexosaminidase (EC 3.2.1.30), &galactosidase (EC 3.2.1.23) cY-galactosidase (EC 3.2.1.22) and &glucuronidase (EC 3.2.1.3 1) in 17 healthy elderly men and women and seven young men and women in an effort to inquire if urinary excretion of these enzymes provides a useful, non-invasive method of evaluating changes in renal function with age. Materials and Methods Subjects
Plasma and urine samples for enzyme analyses were obtained from subjects who were participating in a vitamin clearance study. The elderly subjects were recruited from a population of healthy individuals enrolled in the ongoing New Mexico Aging Process Study at the University of New Mexico (Albuquerque, NM). The young healthy controls were recruited from the staff and students at the University of New Mexico School of Medicine. These subjects were free of kidney disease and were not
49
taking any medications which might affect their renal function. Prior to the study, each of the subjects received a physical examination at which time a medical history was obtained and a blood sample was drawn for the determination of plasma creatinine to rule out renal insufficiency. This study was approved by the Human Research Review Committee of the University of New Mexico School of Medicine. As summarized in Table I, the elderly population was comprised of 8 males and 9 females whose average age was 77.6 years (range 71-84 years). The control subjects comprised 3 women and 4 men, ages 27-40 years. Determination
of glomerular
filtration
rate (GFR)
The GFRs were dete~ined using inufin clearance. All subjects, in~fuding the young controls, spent the night preceding the clearance test at the General Clinical Research Center of the University School of Medicine. On the day the clearance test was performed, subjects were awakened at 06:OOh at which time a urine sample was collected, its volume determined and then discarded. They were then given a volume of water to drink which was equal to the quantity of urine voided..At 07:OOh blood and urine specimens were obtained from each subject for the dete~ination of creatinine and lysosomaf enzyme activities. Blood was collected into heparinized tubes and centrifuged immediately to obtain the plasma fraction. Plasma and urine samples were stored at -30°C until analyzed. At this time, each subject ingested 10 ml water/kg body weight. After 1 h, an intravenous infusion of 5% (w/v) dextrose was begun with a sustaining infusion of inufin. A primary dose of inufin designed to achieve a plasma inufin concentration of approximately 300 mg/f was given by slow infusion over a 5-lomin period. The purpose of the sustaining infusion of inufin was to maintain the plasma inufin concentration at 300 mg/f. The infusion was continued for 225 min at 2 mf/min (approx. 450 ml solution). Urine was collected at 30-min intervals after the 45-min period which allowed for equilibration of the priming dose of inufin. Blood
TABLE I Summary of the characteristics of the young and elderly subjects
Age (years) (range) Male/Female GFR (ml/s) (range) Serum creatinine (mmolil) Urine creatinine (mmoW
Elderly (n = 17)
Young controk (n = 7)
77.6 f 3.3 71-84 8/9 I.12 f 0.298 0.740-1.88 0.082 zt 0.025
34.4 f 4.9 27-40 413 1.56 L+:0.224* 1.24-1.83 0.067 f 0.008
4.97 f 4.31
6.52 2t: 5.41
Results are given as the mean f 1 S.D. *P = 0.003.
50
samples were drawn before the inulin prime and at the midpoints of the 30-min urine collection periods. Inulin concentrations in plasma and urine were determined using the method of Heyrovsky [7]. Determination of creatinine
The plasma and urine samples were analyzed for creatinine using a manual colorimetric determination (Kit no. 555: Sigma Diagnostics, St. Louis, MO). Measurement of lysosomal enzyme activities
The four lysosomal enzymes were assayed by the fluorometric measurement of 4-methyl-umbelliferone liberated from the appropriate glycoside. The /3-hexosaminidase incubation medium contained 2.0 mM 4-methyl-umbelliferyl-~-D-glucosaminide in 0.1 M sodium citrate buffer (pH 4.6) with bovine serum albumin (1 mg/ml). The a-galactosidase activity was determined using an incubation medium which contained 5.0 mM 4-methyl-umbelliferyl-o-galactopyranoside in 0.1 M sodium acetate buffer (pH 4.5). The fl-galactosidase activity was determined using an incubation medium which contained 3.0 mM 4-methyl-umbelliferyl-/3-D-galactopyranoside in 0.06 M sodium citrate-phosphate buffer with 0.06 M sodium chloride (pH 4.8). The B-glucuronidase activity was determined in a reaction medium that contained 4.0 mM 4-methyl-umbelliferyl-O-D-glucuronide in 0.02 M sodium acetate buffer (pH 4.6). Plasma or urine (0.01 ml) was added to 0.09 ml of incubation mixture for each enzyme assay. All assays were performed at 37°C for 30 min and the reactions were terminated by adding 0.3 M glycine-ammonium hydroxide buffer (pH 10.5) to a final volume of 3.0 ml. The 4-methyl-umbelliferone liberated was measured fluorometrically (Turner Model III fluorometer; Sequoia-Turner Corp., Mountain View, CA) as described elsewhere [6] with excitation at 360 nm and emission being measured at 520-580 nm. The four hydrolase assays are highly reproducible and have a standard error of less than 4%. Plasma and urine activities are expressed as units/mm01 creatinine (U/mmol Cr). One unit of enzyme activity equals 1 pmol of substrate hydrolyzed per min at 37°C. The FE ENZ is calculated as the ratio of enzyme clearance to creatinine clearance: (U/P) enzyme activity
x loo
(U/P) creatinine where U = urine and P = plasma. Results for both the elderly population and the control population are expressed as the mean f 1 standard deviation (SD.). Statistical analyses
Statistical analyses were performed with the aid of the Number Cruncher Statistical System software program (version 5; Kaysville, UT). Group comparisons were made using the Mann-Whitney two sample test. A significance level of P C 0.05 was used for all analyses.
51 TABLE II Comparison of the serum lysosomal hydrolase levels in the young and elderly populations with activities expressed on the basis of serum volume. Enzyme
Activity/l serum Elderly
NAG c&al &Gal &Glu
17.9 0.139 0.229 1.15
Young controls f f f f
4.32 0.043 0.180 0.528
13.0 f 1.74* 0.133 f 0.020 0.207 l 0.106 1.18 f 1.04
Enzyme activities are expressed as pmollmin per I serum. NAG = fl-hexosaminidase; o-Gal = ogalactosidase; @-Gal = fl-galactosidase; @-Glu = /%glucuronidase. Results are given as the mean f 1 SD. *I’ = 0.006.
Results As shown in Table I, the average glomerular filtration rate for the elderly group was 1.12 ml/s, whereas that of the control population was 1.56 ml/s. The difference between the GFR values of the two groups was significant (P = 0.003). The mean plasma creatinine level was 22% higher in the elderly population (0.082 f 0.025 vs. 0.067 f 0.008 mmol/l), but this difference was not significant. When the levels of the four lysosomal enzymes in the plasma of the two groups were compared on a volume basis, the differences were significant only for NAG (P = 0.006) (Table II). Comparison of FE ENZ values for four enzymes in elderly and young populations By measuring the plasma and urine activities of each of the four hydrolases and the corresponding creatinine concentrations in the two fluids, we were able to calculate FE ENZ values for these enzymes. As shown in Table III, if one focuses
TABLE III Comparison of the FE ENZ values for four lysosomal hydrolases in young and elderly subjects Enzyme
NAG o-Gal P-Gal P-Glu
Fractional enzyme’ excretion (FE ENZ) Elderly
Young controls
0.518 0.836 7.97 1.26
0.277 1.50 5.42 1.55
f 0.311 f 0.738 f 12.70 f 0.973
f f f f
0.152* 1.07 7.88 1.75
Results are given as the mean k 1 S.D. Enzyme abbreviations are given in Table II. *P = 0.06.
52
lirst on the values for the young population and regards these data as the reference points, the four lysosomal hydrolases appear to fall into three categories. The first consists of enzymes whose FE EN2 values are close to unity and is exemplified by /3-glucuronidase. The other category is occupied by P-galactosidase for which the mean FE ENZ value is well above 1.00, in this case 5.42. The third category is typified by &hexosaminidase, an enzyme whose mean FE ENZ value is well below unity, for example 0.277 in the young population. When the two study groups were compared in terms of their FE EN2 values, a marginal difference between the groups was observed only for ~-hexosaminidase (P = 0.06), indicating that the elderly excreted 2.2-fold more ~-hexosaminidase in their urine than did the younger subjects. The mean FE ENZ values for /3-glucuronidase or &galactosidase for the two groups were nearly identical, 1.55 vs. 1.26 and 7.97 vs. 5.42, respectively (Table III). Noteworthy, though not attaining statistical significance, is the lower FE ENZ value for a-galactosidase in the elderly group (0.836 vs. 1.50). The increased urinary excretion of &hexosaminidase and the decreased excretion of cr-galactosidase with advancing age that was revealed through comparison of FE EN2 values (Table III) was also evident when urinary enzyme levels were expressed as activity per mmol creatinine (Table IV). Compared to the data for the young population, urinary ar-galactosidase activity was about 50% lower in the elderly but ~-hexo~minidase activity was 220% higher. Both differences were statistically significant (P = 0.03 and P = 0.008, respectively). We found that an even more effective way of distinguishing elderly from young subjects on the basis of the serum and urinary lysosomal hydrolase data was to divide the units/mm01 creatinine enzyme value for fl-hexosaminidase by the corresponding value for a-galactosidase. As shown in the Fig. 1, in which the units P-hexosaminidase per mmol creatinine/units cu-galactosidase per mmol creatinine ratios are plotted on a log scale, the ratios obtained for the elderly subjects are more variable than those obtained for the young controls. Furthermore, the difference between the means of these ratios in the elderly and young subjects was highly significant (P = 0.002).
TABLE IV Comparison of the urinary lysosomal hydrolase activities for young and elderly subjects based on the urine creatinine concentration. Enzyme
Activity, units/mmol creatinine Young controls
Elderly NAG a-Gal @-Gal @-Glu
1.11 0.0144 0.110 0.143
f f rt f
*P = 0 *008.I l*P = 0.03. Enzyme abbreviations are given in Table II.
0.579 0.013 0.123 0.069
0.510 0.0280 0.114 0.140
f f zt f
0.241r 0.016** 0.123 0.095
53 l
1529
.
5 0
400
-
-b ?
300
--
200
-
100
-
: 'C
