BrifishJournd of Urology (1 992), 70, 603409
01992 British Journal of Urology
Proteinuria and Enzymuria in Vesicoureteric Reflux D.C. HANBURY and J. CALVIN Departments of Urology and Clinical Biochemistry, Addenbrooke‘s Hospital, Cambridge
Summary-Vesicoureteric reflux is a common abnormality of the urinary tract leading to significant renal morbidity and premature mortality. No reliable non-invasive method exists for its diagnosis. This study investigated the presence of urinary proteins and enzymes in healthy children and those with reflux. A log normal distribution was found for all analyte/creatinine ratios. Significantly higher tubular protein/creatinine ratios were found in patients with reflux nephropathy. Three enzyme/ creatinine ratios (n-acetyl- B- D-glucosaminidase, gamma-glutamyl transferase and lactate dehydrogenase) were higher in children with reflux who had no renal scarring, but the degree of overlap with the normal range was s u c h that it is doubtful whether any will be of use as a urinary marker.
Vesicoureteric reflux (reflux) is a common abnormality of the urinary tract and occurs in approximately 1 in 250 live births (Hodson, 1978). The renal damage (reflux nephropathy) which may result from a combination of reflux and infection accounts for 24% of all cases of end-stage renal failure in children and young adults (Broyer et al., 1985). Nine of 10 cases of severe hypertension in this age group have a renal cause (De Swiet and Dillon, 1989) and in over 50% this is found to be reflux nephropathy (Londe, 1978). The current method of diagnosis (micturating cystourethrography (MCU))is invasiveand an unsuitable screening method for this common disorder. Although there have been considerable advances in ultrasound and radioisotope techniques, a reliable non-invasive method of diagnosis is still elusive. Heavy proteinuria implies advanced glomerular damage and is a sinister indication of worsening renal function (Kincaid-Smith and Becker, 1978). In a follow-up series (Bailey, 1984), all children deteriorating to end-stage renal failure excreted more than 0.5 g urinary albumin per day at first presentation. The presence of a small loss of albumin (“microalbuminuria”) is a reliable indicator of progression of renal disease in diabetes (Gibb et al., 1989) and hypertension (Losito et al., 1988). Accepted for publication 20 November 1991
Although it is now well established that heavy albuminuria means a poor prognosis in advanced reflux nephropathy, little information is available on its significance in the early stages of the disease. Histological evidence suggests that a marker of tubular damage or dysfunction should be more sensitive in the diagnosis of early nephropathy (Heptinstalland Hodson, 1984).There is additional physiological evidence, in studies of concentrating ability, that reflux causes a reversible tubular injury (Uehling and Wear, 1976; Mundy et al., 1981).I n tubular disease the protein present in urine is a smaller size than albumin (Hardwicke et al., 1970) and could therefore provide clues to the site of damage. Urinary beta, microglobulin (B,M) has been used as a marker of damage in tubular disorders, and heavy metal or aminoglycosidetoxicity (Schardijn and Van Eps, 1987; Karlsson et al., 1980), but it has the particular problem of deteriorating in acid urine (Hardwicke, 1984). Alternatives include alpha, microglobulin (A,M) and retinol binding protein (RBP). Their sensitivity is similar to that of B2Mand they are more stable in acid urine (Bernard et al., 1982; Yu et al., 1983). Over 30 enzymes have been found in the urine (Mattenheimer, 1971). Most urinary enzymes originate from various parts of the renal tubular cell and its luminal brush border (Grotsch and Matten-
603
604 heimer, 1983). A selection has been chosen since reflux may damage particular parts of the renal N-acetyl-B-D-glucosaminidase tubular cells. (NAG) is predominantly lysosomal, while lactate dehydrogenase (LDH) is found in the cytoplasm. Alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and alanine aminopeptidase (AAP) are bound to the brush border membrane and are thought to have reabsorptive functions (Guder and Ross, 1984). High urinary enzyme activity is usually indicative of parenchymal damage, although micro-organisms and white blood cells can increase the apparent activity. Urinary enzymes have been studied particularly in drug and heavy metal toxicity (Price, 1982) and renal transplant rejection (Wellwood et al., 1975). The purpose of this study was to document concentrations of a range of urinary proteins and enzymes, from both healthy and affected children, in order to establish whether any may be useful in the early diagnosis of reflux.
Patients and Methods Urine was collected from babies and children using the Hollister U-bag or clean catch techniques. In the control group were children of colleagues, those attending toddler and play groups, and some having minor surgical operations. None had a history of urinary symptoms. Babies were recruited from the local maternity hospital and a “well baby” clinic; all had a normal prenatal ultrasound scan. Children in the study groups presented with either urinary tract infection or an abnormal prenatal ultrasound scan. Reflux was shown on subsequent MCU and renal scarring documented by 9 9 m Tdimercaptosuccinic ~ acid (DMSA) scan.ning or intravenous urography. Children with significant renal impairment (serum creatinine > 125 pmol/L) were excluded from the study. The 3 groups studied were controls, children with reflux whose kidneys were normal and children with reflux and renal scarring. Urine was collected into a sterile universal container and processed within a few hours of collection. An aliquot was sent for microscopy and culture and the specimen was rejected if > 50 white cells were seen or > lo5 organisms per ml of urine were cultured. Samples for albumin were mixed with azide (0.1% w/v) and stored at 4°C. Samples for tubular proteins and NAG were neutralised and stored at - 20°C ;those to be analysed for the other enzymes
BRITISH JOURNAL OF UROLOGY
were neutralised and mixed with glycerol before storage at - 20°C (Maruhn er al., 1986). Creatinine was assayed by the Jaffk method (Henry, 1974) on a Monarch centrifugal analyser (Instrumentation Laboratory, Lexington, USA). Albumin was measured by an immunoturbidimetric method on a Monarch centrifugal analyser, with antiserum obtained from Atlantic Antibodies (Incstar Ltd, Winnersh). A range of standards was prepared from human serum albumin (Behring Diagnostics, Hounslow). AIM was assayed by rocket immunoelectropho. resis (Laurell, 1975) with antiserum obtained from Dako Ltd, High Wycombe. Standards were kindly provided by Dr R. Chambers, Bristol. Retinol binding protein (RBP) was measured by radioimmunoassay, according to the method described by Beetham et al. (1985), using standards and reagents purchased from St Bartholomew’s Hospital, London. Urinary enzymes were measured on a Monarch centrifugal analyser at 37°C. N-acetyl-B-D-glucosaminidase (NAG, E.C. 3.2.1.30)was determined by means of a colorimetric procedure using 2-methoxy-4-(2’-nitrovinyl)phenyl-2-acetamido-2-deoxy-B-D-glucopyranoside as substrate, previously described by Yuen et a/. (1984). NAG test kits and calibrators were PUP chased from Cortecs Ltd, Deeside. Aliquots for other enzyme analyses were applied to columns pre-packed with Sephadex G25 (Pharmacia Ltd, Milton Keynes) to remove small molecularweight inhibitors as described by Grotsch and Mattenheimer (1983). Lactate dehydrogenase (LDH, E.C. 1.1.1.27)was measured using the reverse reaction of pyruvate to lactate under Scandinavian conditions (Keiding et al., 1974); NADH was purchased from Sigma Chemical Company, Poole, and pyruvate from BDH Chemicals Ltd, Poole. Alanine aminopeptidase (AAP, E.C. 3.4.11.2) was measured using an optimised kinetic assay with alanine-4-nitroanilide as substrate (Sigma Chemical Company), as described by Jung and Scholz (1980). Alkaline phosphatase (ALP, E.C. 3.1.3.1) was assayed using p-nitrophenylphosphate substrate in 2-amino-methyl-propan-1-01 buffer, pH 10.3, as described by Tietz et al. (1980). ALP kits were purchased from Boehringer Mannheim (Mannheim, Germany). Gamma-glutamyl transferase (GGT, E.C. 2.3.2.2) wasmeasuredusing the substrate L-gammaglutamyl-3-carboxyl-4-nitroanilide,according to
605
PROTEINURIA AND ENZYMURIA IN VESICOURETERIC REFLUX
the method of Persijn and van der Slik (1976), and reagents were purchased from Boehringer Mannheim. Results were expressed as the concentration of protein or activity of enzyme divided by the urinary creatinine. Enzyme activity was expressed as IU/ mmol creatinine where a unit is equivalent to 1 pmol chromophore released per min.
Control children
25
.-c 0
P
2 0 p
m
boys girls
Results
Normal ranges were calculated from urine protein and enzyme/creatinine ratios found in healthy children (Table 1); 193 control specimens were analysed but several dilute specimens with low creatinine values (< 1 mmol/L) and/or undetectable analyte concentration were omitted from the statistical analysis. Correlation between urinary creatinine concentration and urine flow rate is known to be poor when the urinary output is high (Price, 1978) and studies have shown that urinary enzyme output is related to diuresis (Raab, 1972; Jung and Schulze, 1986). The results were initially divided into 2 age groups (0-30 and 30-120 months) and the distributions statistically examined using the chi-squared normality test. The distributions were found to be log-normal for all analytes and not Gaussian. Student’s t test, after log transformation of the results, was used for all of the following statistical analyses. There was no statistical difference between the sexes for any of the analytes measured; for example, Figure 1 shows the variation of the AAP/creatinine ratio against age for boys and girls. Normal ranges for the analyte/creatinine ratios are shown in Table2. Variation with age was examined by dividing the control group into 6 cohorts (0-2.9, 3-11.9, 12-23.9, 2435.9, 36-59.9 and 60-120 months). In general, all analyte ratios decreased with the increasing age of the child (for example AAP in Fig. 1). The rate at which this occurred enabled varying age groups to be combined for each analyte. Patient and control groups
0
20
60
40
d=day. m=month.
100
120
Fig. 1 Influence of age and sex on urinary alanine aminopeptidase/creatinine ratio.
were compared where sufficient patient numbers existed within any particular age group. Significant differences between the groups for all analytes studied are shown in Table 3. Few babies and very young children with reflux were seen during the study, but sufficient patients in the older age groups were available for analysis. Children with reflux but no scarring had higher LDH, GGT and NAG ratios in certain age groups, but there was considerable overlap for all analytes such that conclusions could not easily be drawn from any individual result of analyte/creatinine ratio. In children with reflux and scarring, all analyte ratios apart from ALP, LDH and GGT were significantly higher in certain age groups. The most marked differences in the mean values were seen with the tubular proteins A, M and RBP, but Figure 2 (RBP) shows the considerable overlap with controls.
Discussion We found a log distribution for all analyte ratios. This has been described for NAG by Osborne (1980) and Watts et al. (1988), for ALP and LDH
Table 1 Details of Patients
Controls Reflux Reflux with scarring
80
Age (months)
Boys
Girls
Total
Age range
Mean age (months)
101 23 20
92 41 24
193 64 44
ld-120m 3d-120m 5d-120m
34 48 57
606
BRITISH JOURNAL OF UROLOGY
Table 2 Urinary Analyte/Creatinine Ratios in Normal Children. (The mean and 95% confidence limits were calculated from the logarithm of the observed values)
a. Albumin/creatinine ratios
e. Alkaline phosphatase/creatinine ratios
Age (months)
No.
Mean 95% confidence (mg/mmol) limits (mglmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IU/mmol)
0-2.9 3-11.9 12-23.9 24-35.9 3659.9 60-120
19 19 21 20 32 33
6.4 2.4 2.4 1.3 0.9 1.0
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 16 20 20 30 35
3.3 2.4 1.3 1.0 0.5 0.4
1.0-10.7 0.2-25.4 0.3-6.5 0.2-3.9 0.3-1.1 0.2-1.1
0.1-33.6 0.7-9.0 0.5-12.5 0.3-6.3 0.3-3.1 0.2-4.3
b. Alpha, microglobulin/creatinineratios
f. Gamma glutamyl transferase/creatinineratios
Age (months)
No.
Mean (mglmmol)
95% confidence limits (mglmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IUlmmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 18 19 26 35 37
4.9 0.9 0.8 0.6 0.4 0.4
1.0-25.2 0.4-2.0 0.2-3.6 0.2-1.5 0.1-1.3 0.1-1.0
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 21 27 26 36 37
10.5 10.7 6.9 6.2 5.8 4.4
4.624.0 4.4-28.3 3.1-15.7 3.2-12.1 3.0-1 1.0 2.4-8.4
c. Retinol binding proteinlcreatinineratios
g. Lactate dehydrogenase/creatinineratios
Age (months)
No.
Mean (pg/mmol)
95% confidence limits (pglrnmol)
Age (months)
No.
Mean (IU/mmol)
95% confidence limits (IUlmmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
16 11 16 21 33 36
46.4 5.9 12.0 8.2 7.5 6.8
3.8-570 3.2-11.0 2.0-70.2 3.1-22.1 3.0-19.2 2.4-19.4
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 20 26 26 36 37
11.4 6.6 2.6 2.0 1.4 1.2
1.6-79.3 1.2-35.4 0.3-21.0 0.5-7.4 0.3-7.4 0.4-4.3
d. Alanine aminopeptidaselcreatinineratios
h. N-acetyl-B-D-glucosaminidase/creatinine ratios
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IUlmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IU/mmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 21 27 26 36 37
9.6 4.7 2.7 2.1 1.6 1.3
3.6-25.2 1.1-19.6 0.8-9.3 0.7-2.2 0.8-3.2 0.7-2.5
0-2.9 3-11.9 12-23.9 24-35.9 3659.9 60-120
19 21 27 27 36 37
1.65 0.87 0.46 0.36 0.28 0.25
0.594. 12 0.49-2.8 1 0.18-1.14 0.14492 0.134.62 0.13-0.50
by Werner et al. (1970) and for AAP by Jung et al. (1986). Bell et al. (1986) and Davies et al. (1984) found log distributions for albumin, whilst Gibb et al. (1989) found the same for RBP. A majority opinion supports a log normal distribution for urinary enzymes and proteins in early childhood. The influence of sex was negligible. Differences
have been reported for ALP, GGT, LDH and NAG by Maruhn et al. (1976), for LDH by Matsukura et al. (1987) and for GGT and NAG by Jungetal. (1986). Other studies found no difference, e.g. NAG by Price (1978), AAP by Jung et ul. (1986) and ALP and GGT by Matsukura et ul. (1987). The rapid fall in output of urinary proteins
PROTEINURIA AND ENZYMURIA IN VESICOURETERICREFLUX
Table 3 Summary of Analytes found to be Significantly Elevated in Patient versus Control Groups Analyte
Patient (mean)
Age (months)
Control (mean)
P
~
Children with reflux NAG 36-120 LDH 36-120 GGT 12-35.9
0.32 2.0 8.3
= 0.03
0.27IU/mmol 1.3 IU/mmol 6.6 IU/mmol
=0.015 =0.018
Children with reflux and renal scarring Albumin 24-120 1.6 1 .O mg/mmol AIM 36-120 0.63 0.38mg/mmol RBP 36-120 13.5 7.1 pg/mmol AAP 36-59.9 2.1 1.6 IU/mmol NAG 36120 0.33 0.27IU/mmol
=0.01 =
01992 British Journal of Urology
Proteinuria and Enzymuria in Vesicoureteric Reflux D.C. HANBURY and J. CALVIN Departments of Urology and Clinical Biochemistry, Addenbrooke‘s Hospital, Cambridge
Summary-Vesicoureteric reflux is a common abnormality of the urinary tract leading to significant renal morbidity and premature mortality. No reliable non-invasive method exists for its diagnosis. This study investigated the presence of urinary proteins and enzymes in healthy children and those with reflux. A log normal distribution was found for all analyte/creatinine ratios. Significantly higher tubular protein/creatinine ratios were found in patients with reflux nephropathy. Three enzyme/ creatinine ratios (n-acetyl- B- D-glucosaminidase, gamma-glutamyl transferase and lactate dehydrogenase) were higher in children with reflux who had no renal scarring, but the degree of overlap with the normal range was s u c h that it is doubtful whether any will be of use as a urinary marker.
Vesicoureteric reflux (reflux) is a common abnormality of the urinary tract and occurs in approximately 1 in 250 live births (Hodson, 1978). The renal damage (reflux nephropathy) which may result from a combination of reflux and infection accounts for 24% of all cases of end-stage renal failure in children and young adults (Broyer et al., 1985). Nine of 10 cases of severe hypertension in this age group have a renal cause (De Swiet and Dillon, 1989) and in over 50% this is found to be reflux nephropathy (Londe, 1978). The current method of diagnosis (micturating cystourethrography (MCU))is invasiveand an unsuitable screening method for this common disorder. Although there have been considerable advances in ultrasound and radioisotope techniques, a reliable non-invasive method of diagnosis is still elusive. Heavy proteinuria implies advanced glomerular damage and is a sinister indication of worsening renal function (Kincaid-Smith and Becker, 1978). In a follow-up series (Bailey, 1984), all children deteriorating to end-stage renal failure excreted more than 0.5 g urinary albumin per day at first presentation. The presence of a small loss of albumin (“microalbuminuria”) is a reliable indicator of progression of renal disease in diabetes (Gibb et al., 1989) and hypertension (Losito et al., 1988). Accepted for publication 20 November 1991
Although it is now well established that heavy albuminuria means a poor prognosis in advanced reflux nephropathy, little information is available on its significance in the early stages of the disease. Histological evidence suggests that a marker of tubular damage or dysfunction should be more sensitive in the diagnosis of early nephropathy (Heptinstalland Hodson, 1984).There is additional physiological evidence, in studies of concentrating ability, that reflux causes a reversible tubular injury (Uehling and Wear, 1976; Mundy et al., 1981).I n tubular disease the protein present in urine is a smaller size than albumin (Hardwicke et al., 1970) and could therefore provide clues to the site of damage. Urinary beta, microglobulin (B,M) has been used as a marker of damage in tubular disorders, and heavy metal or aminoglycosidetoxicity (Schardijn and Van Eps, 1987; Karlsson et al., 1980), but it has the particular problem of deteriorating in acid urine (Hardwicke, 1984). Alternatives include alpha, microglobulin (A,M) and retinol binding protein (RBP). Their sensitivity is similar to that of B2Mand they are more stable in acid urine (Bernard et al., 1982; Yu et al., 1983). Over 30 enzymes have been found in the urine (Mattenheimer, 1971). Most urinary enzymes originate from various parts of the renal tubular cell and its luminal brush border (Grotsch and Matten-
603
604 heimer, 1983). A selection has been chosen since reflux may damage particular parts of the renal N-acetyl-B-D-glucosaminidase tubular cells. (NAG) is predominantly lysosomal, while lactate dehydrogenase (LDH) is found in the cytoplasm. Alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and alanine aminopeptidase (AAP) are bound to the brush border membrane and are thought to have reabsorptive functions (Guder and Ross, 1984). High urinary enzyme activity is usually indicative of parenchymal damage, although micro-organisms and white blood cells can increase the apparent activity. Urinary enzymes have been studied particularly in drug and heavy metal toxicity (Price, 1982) and renal transplant rejection (Wellwood et al., 1975). The purpose of this study was to document concentrations of a range of urinary proteins and enzymes, from both healthy and affected children, in order to establish whether any may be useful in the early diagnosis of reflux.
Patients and Methods Urine was collected from babies and children using the Hollister U-bag or clean catch techniques. In the control group were children of colleagues, those attending toddler and play groups, and some having minor surgical operations. None had a history of urinary symptoms. Babies were recruited from the local maternity hospital and a “well baby” clinic; all had a normal prenatal ultrasound scan. Children in the study groups presented with either urinary tract infection or an abnormal prenatal ultrasound scan. Reflux was shown on subsequent MCU and renal scarring documented by 9 9 m Tdimercaptosuccinic ~ acid (DMSA) scan.ning or intravenous urography. Children with significant renal impairment (serum creatinine > 125 pmol/L) were excluded from the study. The 3 groups studied were controls, children with reflux whose kidneys were normal and children with reflux and renal scarring. Urine was collected into a sterile universal container and processed within a few hours of collection. An aliquot was sent for microscopy and culture and the specimen was rejected if > 50 white cells were seen or > lo5 organisms per ml of urine were cultured. Samples for albumin were mixed with azide (0.1% w/v) and stored at 4°C. Samples for tubular proteins and NAG were neutralised and stored at - 20°C ;those to be analysed for the other enzymes
BRITISH JOURNAL OF UROLOGY
were neutralised and mixed with glycerol before storage at - 20°C (Maruhn er al., 1986). Creatinine was assayed by the Jaffk method (Henry, 1974) on a Monarch centrifugal analyser (Instrumentation Laboratory, Lexington, USA). Albumin was measured by an immunoturbidimetric method on a Monarch centrifugal analyser, with antiserum obtained from Atlantic Antibodies (Incstar Ltd, Winnersh). A range of standards was prepared from human serum albumin (Behring Diagnostics, Hounslow). AIM was assayed by rocket immunoelectropho. resis (Laurell, 1975) with antiserum obtained from Dako Ltd, High Wycombe. Standards were kindly provided by Dr R. Chambers, Bristol. Retinol binding protein (RBP) was measured by radioimmunoassay, according to the method described by Beetham et al. (1985), using standards and reagents purchased from St Bartholomew’s Hospital, London. Urinary enzymes were measured on a Monarch centrifugal analyser at 37°C. N-acetyl-B-D-glucosaminidase (NAG, E.C. 3.2.1.30)was determined by means of a colorimetric procedure using 2-methoxy-4-(2’-nitrovinyl)phenyl-2-acetamido-2-deoxy-B-D-glucopyranoside as substrate, previously described by Yuen et a/. (1984). NAG test kits and calibrators were PUP chased from Cortecs Ltd, Deeside. Aliquots for other enzyme analyses were applied to columns pre-packed with Sephadex G25 (Pharmacia Ltd, Milton Keynes) to remove small molecularweight inhibitors as described by Grotsch and Mattenheimer (1983). Lactate dehydrogenase (LDH, E.C. 1.1.1.27)was measured using the reverse reaction of pyruvate to lactate under Scandinavian conditions (Keiding et al., 1974); NADH was purchased from Sigma Chemical Company, Poole, and pyruvate from BDH Chemicals Ltd, Poole. Alanine aminopeptidase (AAP, E.C. 3.4.11.2) was measured using an optimised kinetic assay with alanine-4-nitroanilide as substrate (Sigma Chemical Company), as described by Jung and Scholz (1980). Alkaline phosphatase (ALP, E.C. 3.1.3.1) was assayed using p-nitrophenylphosphate substrate in 2-amino-methyl-propan-1-01 buffer, pH 10.3, as described by Tietz et al. (1980). ALP kits were purchased from Boehringer Mannheim (Mannheim, Germany). Gamma-glutamyl transferase (GGT, E.C. 2.3.2.2) wasmeasuredusing the substrate L-gammaglutamyl-3-carboxyl-4-nitroanilide,according to
605
PROTEINURIA AND ENZYMURIA IN VESICOURETERIC REFLUX
the method of Persijn and van der Slik (1976), and reagents were purchased from Boehringer Mannheim. Results were expressed as the concentration of protein or activity of enzyme divided by the urinary creatinine. Enzyme activity was expressed as IU/ mmol creatinine where a unit is equivalent to 1 pmol chromophore released per min.
Control children
25
.-c 0
P
2 0 p
m
boys girls
Results
Normal ranges were calculated from urine protein and enzyme/creatinine ratios found in healthy children (Table 1); 193 control specimens were analysed but several dilute specimens with low creatinine values (< 1 mmol/L) and/or undetectable analyte concentration were omitted from the statistical analysis. Correlation between urinary creatinine concentration and urine flow rate is known to be poor when the urinary output is high (Price, 1978) and studies have shown that urinary enzyme output is related to diuresis (Raab, 1972; Jung and Schulze, 1986). The results were initially divided into 2 age groups (0-30 and 30-120 months) and the distributions statistically examined using the chi-squared normality test. The distributions were found to be log-normal for all analytes and not Gaussian. Student’s t test, after log transformation of the results, was used for all of the following statistical analyses. There was no statistical difference between the sexes for any of the analytes measured; for example, Figure 1 shows the variation of the AAP/creatinine ratio against age for boys and girls. Normal ranges for the analyte/creatinine ratios are shown in Table2. Variation with age was examined by dividing the control group into 6 cohorts (0-2.9, 3-11.9, 12-23.9, 2435.9, 36-59.9 and 60-120 months). In general, all analyte ratios decreased with the increasing age of the child (for example AAP in Fig. 1). The rate at which this occurred enabled varying age groups to be combined for each analyte. Patient and control groups
0
20
60
40
d=day. m=month.
100
120
Fig. 1 Influence of age and sex on urinary alanine aminopeptidase/creatinine ratio.
were compared where sufficient patient numbers existed within any particular age group. Significant differences between the groups for all analytes studied are shown in Table 3. Few babies and very young children with reflux were seen during the study, but sufficient patients in the older age groups were available for analysis. Children with reflux but no scarring had higher LDH, GGT and NAG ratios in certain age groups, but there was considerable overlap for all analytes such that conclusions could not easily be drawn from any individual result of analyte/creatinine ratio. In children with reflux and scarring, all analyte ratios apart from ALP, LDH and GGT were significantly higher in certain age groups. The most marked differences in the mean values were seen with the tubular proteins A, M and RBP, but Figure 2 (RBP) shows the considerable overlap with controls.
Discussion We found a log distribution for all analyte ratios. This has been described for NAG by Osborne (1980) and Watts et al. (1988), for ALP and LDH
Table 1 Details of Patients
Controls Reflux Reflux with scarring
80
Age (months)
Boys
Girls
Total
Age range
Mean age (months)
101 23 20
92 41 24
193 64 44
ld-120m 3d-120m 5d-120m
34 48 57
606
BRITISH JOURNAL OF UROLOGY
Table 2 Urinary Analyte/Creatinine Ratios in Normal Children. (The mean and 95% confidence limits were calculated from the logarithm of the observed values)
a. Albumin/creatinine ratios
e. Alkaline phosphatase/creatinine ratios
Age (months)
No.
Mean 95% confidence (mg/mmol) limits (mglmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IU/mmol)
0-2.9 3-11.9 12-23.9 24-35.9 3659.9 60-120
19 19 21 20 32 33
6.4 2.4 2.4 1.3 0.9 1.0
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 16 20 20 30 35
3.3 2.4 1.3 1.0 0.5 0.4
1.0-10.7 0.2-25.4 0.3-6.5 0.2-3.9 0.3-1.1 0.2-1.1
0.1-33.6 0.7-9.0 0.5-12.5 0.3-6.3 0.3-3.1 0.2-4.3
b. Alpha, microglobulin/creatinineratios
f. Gamma glutamyl transferase/creatinineratios
Age (months)
No.
Mean (mglmmol)
95% confidence limits (mglmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IUlmmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 18 19 26 35 37
4.9 0.9 0.8 0.6 0.4 0.4
1.0-25.2 0.4-2.0 0.2-3.6 0.2-1.5 0.1-1.3 0.1-1.0
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 21 27 26 36 37
10.5 10.7 6.9 6.2 5.8 4.4
4.624.0 4.4-28.3 3.1-15.7 3.2-12.1 3.0-1 1.0 2.4-8.4
c. Retinol binding proteinlcreatinineratios
g. Lactate dehydrogenase/creatinineratios
Age (months)
No.
Mean (pg/mmol)
95% confidence limits (pglrnmol)
Age (months)
No.
Mean (IU/mmol)
95% confidence limits (IUlmmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
16 11 16 21 33 36
46.4 5.9 12.0 8.2 7.5 6.8
3.8-570 3.2-11.0 2.0-70.2 3.1-22.1 3.0-19.2 2.4-19.4
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 20 26 26 36 37
11.4 6.6 2.6 2.0 1.4 1.2
1.6-79.3 1.2-35.4 0.3-21.0 0.5-7.4 0.3-7.4 0.4-4.3
d. Alanine aminopeptidaselcreatinineratios
h. N-acetyl-B-D-glucosaminidase/creatinine ratios
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IUlmmol)
Age (months)
No.
Mean (IUlmmol)
95% confidence limits (IU/mmol)
0-2.9 3-11.9 12-23.9 24-35.9 36-59.9 60-120
19 21 27 26 36 37
9.6 4.7 2.7 2.1 1.6 1.3
3.6-25.2 1.1-19.6 0.8-9.3 0.7-2.2 0.8-3.2 0.7-2.5
0-2.9 3-11.9 12-23.9 24-35.9 3659.9 60-120
19 21 27 27 36 37
1.65 0.87 0.46 0.36 0.28 0.25
0.594. 12 0.49-2.8 1 0.18-1.14 0.14492 0.134.62 0.13-0.50
by Werner et al. (1970) and for AAP by Jung et al. (1986). Bell et al. (1986) and Davies et al. (1984) found log distributions for albumin, whilst Gibb et al. (1989) found the same for RBP. A majority opinion supports a log normal distribution for urinary enzymes and proteins in early childhood. The influence of sex was negligible. Differences
have been reported for ALP, GGT, LDH and NAG by Maruhn et al. (1976), for LDH by Matsukura et al. (1987) and for GGT and NAG by Jungetal. (1986). Other studies found no difference, e.g. NAG by Price (1978), AAP by Jung et ul. (1986) and ALP and GGT by Matsukura et ul. (1987). The rapid fall in output of urinary proteins
PROTEINURIA AND ENZYMURIA IN VESICOURETERICREFLUX
Table 3 Summary of Analytes found to be Significantly Elevated in Patient versus Control Groups Analyte
Patient (mean)
Age (months)
Control (mean)
P
~
Children with reflux NAG 36-120 LDH 36-120 GGT 12-35.9
0.32 2.0 8.3
= 0.03
0.27IU/mmol 1.3 IU/mmol 6.6 IU/mmol
=0.015 =0.018
Children with reflux and renal scarring Albumin 24-120 1.6 1 .O mg/mmol AIM 36-120 0.63 0.38mg/mmol RBP 36-120 13.5 7.1 pg/mmol AAP 36-59.9 2.1 1.6 IU/mmol NAG 36120 0.33 0.27IU/mmol
=0.01 =
