Volume 89 Number 1
Coombs negative after receiving 6,000 U during six days in two lots; the saline anti-A titer was as high as 1:8, and the immune titer was 1:16. On the basis of clinical experience and titer analysis, the risk of hemolysis with the PTC preparations appears minimal. REFERENCES 1. Ashenhurst JB, Langehennig PL, Seeler RA, and Teller MC: Hemolytic anemia due to anti-B in antihemophiliac factor concentrates, J PEn~ATR88:257, 1976. 2. Seeler RA: Hemolysis due to anti-A and anti-B in factor VIII preparations, Arch Intern Med 130:101, 1972. 3. Tamagnini GP, Dormandy KM, Ellis D, and Maycock WdA: Factor VIII concentrate in haemophilia, Lancet 2:188, 1975.
Urinary protein excretion in early infancy F. Anders Karlsson, M.D.,* and Kristoffer Hellsing, M.D., Uppsala, Sweden
IN REC~YT "tEARS much progress concerning kidney function has been made by detailed studies of urinary proteins? In particular, measurements of the relative amounts of high and low molecular weight proteins have given valuable information concerning glomerular and tubular function in kidney diseaseY' :' In this study the urinary excretion of a high molecular weight protein, albumin (molecular weight 69,000) and a low molecular weight B~-microglobulin (molecular weight 11,800) has been investigated in 138 healthy infants varying in age from full-term birth to one year. In addition, the urinary protein patterns during this period have been analyzed by electrophoresis in polyacrylamide gel containing sodium dodecylsulfate, a technique that separates proteins according to their molecular weight. Quantitative data on albumin excretion in infants have not been reported earlier. A mean value for/3~-microglobulin in urine from nine newborn infants of 0.51 mg/1 was recently published. 4 From the Institute of Medical and Physiological Chemistry and Department of Clinical Chemistry, University of Uppsala. Supported by Tore Nilson Foundation, Konung Gustaf V 80-hrsfond, and the Swedish Medical Research Council (Project No. 13X-3144). *Reprint address: Institute of Medical and Physiological Chemistry, Box 575, S-751 23 Uppsala 1, Sweden.
Brief clinical and laboratory observations
89
4. Rosati LA, Barnes B, Oberman HA, and Penner JA: Hemolytic anemia due to anti-A in concentrated antihemophilic factor preparations, Transfusion 10:139, 1970. 5. Oberman HA, Barnes BA, and Ginther PL: Erythrocyte sensitization and anemia due to isoantibodies in lyophilized pooled plasma, JAMA 198:233, 1966. 6. Schwartz JM, Cohn BD, Ritz ND, and Levy T: Appendectomy in hemophilia with the use of cryoprecipitated human factor VIII to control bleeding, JAMA 198:1175, 1966. 7. Marder VJ, and Shulman NR: Major surgery in classic hemophilia using fraction 1, Am J Med 41:56, 1966. 8. Schorr JB, Ballard S, and Radel E: Hemolysis associated with "Fibro-AHF" therapy of hemophilia, read before the Thirty-sixth Annual Meeting of the Society for Pediatric Research, Atlantic City, 1966, p 132. 9. Cronin CA: Factor VIII concentrate in haemophilia, Lancet 1:1303, 1975.
Table I. Urinary excretion of albumin and B2-microglobulin in early infancy; the mean and the 95% confidence limits were calculated from the logarithm of the observed values Age (mo)
n
Albumin (mg/l)
fiz-Microglobulin (rag~l)
0- 1 1- 2 2- 4 4- 6 6-12
42 22 30 23 21
10.83 (1.20-98.06) 2.89 (0.25-34.03) 2.40 (0.48-11.93) 3.00 (0.48-18.73) 3.03 (0.70-13.11)
0.640 (0.036-11.135) 0.170 (0.004- 7.221) 0.030 (0.001- 0.782) 0.040 (0.005- 0.283) 0.050 (0.010- 0.246)
MATERIALS
AND METHODS
Urine specimens were collected from healthy infants remaining at the hospital after birth or during routine postnatal examinations. After spontaneous micturition into plastic bags, small aliquots of neutral phosphatebuffer and sodium azide were added to a final concentration of 0.05M and 0.002% (w/v), respectively, and the samples were stored at + 4~ for one to seven days, then a t - 2 0 ~ until analysis. Albumin values were determined with a polymerenhanced immunonephelometric method)/32-Microglobulin measurements were carried out with the Phadebas fl~micro test (Pharmacia Diagnostics, Uppsala, Sweden). Urine specimens were concentrated with Diaflo ultrafiltration equipment, membrane UM 2 (Amicon Corp., Lexington, Mass.). Samples of concentrated urinary proteins (50-200 /~g) were electrophoretically separated in a polyacrylamide gel containing sodium dodecylsulfate (0.5%). The gel concentrations in spacer and running gel were 7 and 11%, respectively, and the buffer system was designed according to Neville. 6 Reference proteins were purchased from Serva, Heidelberg, Germany.
90
Briefclinical and laboratory observations
The Journal ofPediatric~ Ju~ 1976
=
:~ ~;,i!iiiii,!iiiiii~i>;~::i~?: ~ 84184184
-A
,0
"" 1
2
3
4
5
---M
6
Fig. 1. Electrophoresis in polyacrylamide,gel containing sodium dodecylsulphate of concentrated urines from (i) a normal adult, (2) a pool of eight equal specimens from children at the age of 0 to l'month, and (3) at the age of 4 to 6 months, (4) a patient with renal tubular disease (chronic cadmium poisoning), (5) a patient with nephrotic syndrome, (6) standard solution containing albumin (A), molecular weight 67,000; ovalbumin (0) tool weight 45,000, and myoglobin (M), mol weight 17,800. The individual proteins responsible for the bands seen in the urine from the newborn infants have not been identified. However, the strongest band most likely represents albumin and the sharp fast-moving one represents fl~-microglobulin. RESULTS
AND DISCUSSION
The urinary concentrations of a l b u m i n and /?~-microglobulin obtained in this study when plotted on a linear scale were found to be asymmetrically distributed. The logarithmic values were more normally distributed and were thus preferred for the calculation of m e a n and confidence limits. The highest concentrations of fl~microglobulin were found after birth (Table i), followed by a gradual decrease during the first four months to an almost constant level of about one-twentieth of the inital value. The a l b u m i n excretion demonstrated a similar tendency, however, the decrease was less pronounced, only about four-fold. During the first months large differences were noticed in protein excretion among individuals. After a few months these differences diminished and the m e a n excretion reached a stable level. These rapid changes are of importance when proteinuria during the first year of life is evaluated. Pooled urine from children at the age of 0 to 1 month and 4 to 6 months, respectively, was subjected to polyacrylamide gel electrophoresis in a buffer system containing sodium dodecylsulphate. As shown in Fig. 1, urine from newborn infants has a higher proportion of low molecular weight proteins than urine from older children, where the electrophoretic pattern is similar to that found in the examination of urine from normal adults. It is generally accepted that in the adult kidney, the tubular reabsorption of low molecular weight protein is more affected than is the reabsorption of high molecular weight proteins by tubular dysfunction. 2-~ Accordingly, a high
proportion of low molecular weight proteins is often found in the urine of patients with renal tubular disease, whereas patients with glomerular damage exhibit a pattern of marked contrast. The quantitative data and the results of the electrophoretic analysis in this study are interpreted as reflecting changes in the renal tubular protein reabsorbing function, which occur after birth. The skillful technical assistance of Mrs. C. Trossvik and Mr. H. Engstr6m and the kin4 cooperation of Dr. L-I Hardell, Dr. G. Sundelin and the staff'~t the Children's clinic at Bandstolsvfigen, Uppsala, are gratefully acknowledged. REFERENCES
1. Hardwicke J: Laboratory aspects of proteinuria in human disease, Clin Nephrol 3:37, 1975. 2. Peterson PA, Evrin P-E, and Berggard I: Differentiation of glomerular, tubular and normal proteinuria: determinations of urinary excretion of beta~2-microglobulin, albumin, and total protein, J Clin Invest 48:1189, 1969. 3. Hardwicke J, Cameron JS, Harrison JR, Hulme B, and Sooothil JF: Proteinuria in kidney disease, in Manuel Proteins in normal and pathological urine, Revitlard JP, and Betuel H, editors: Basel, 1970, S Karger AG, p 111. 4. Jonasson L-E, Evrin P-E, and Wibell L: Content of fi~microglobulin and albumin in human amniotic fluid, Acta Obstet Gynecol Scand 53:49, 1974. 5. Lizana J, and Hellsing K: Polymer enhancement of automated immunological nephelometric analysis, as illustrated by determination of urinary albumin, Clin Chem 20:415, 1974. 6. Neville DM Jr: Molecular weight determination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J Biol Chem 246:6328, 1971.
Coombs negative after receiving 6,000 U during six days in two lots; the saline anti-A titer was as high as 1:8, and the immune titer was 1:16. On the basis of clinical experience and titer analysis, the risk of hemolysis with the PTC preparations appears minimal. REFERENCES 1. Ashenhurst JB, Langehennig PL, Seeler RA, and Teller MC: Hemolytic anemia due to anti-B in antihemophiliac factor concentrates, J PEn~ATR88:257, 1976. 2. Seeler RA: Hemolysis due to anti-A and anti-B in factor VIII preparations, Arch Intern Med 130:101, 1972. 3. Tamagnini GP, Dormandy KM, Ellis D, and Maycock WdA: Factor VIII concentrate in haemophilia, Lancet 2:188, 1975.
Urinary protein excretion in early infancy F. Anders Karlsson, M.D.,* and Kristoffer Hellsing, M.D., Uppsala, Sweden
IN REC~YT "tEARS much progress concerning kidney function has been made by detailed studies of urinary proteins? In particular, measurements of the relative amounts of high and low molecular weight proteins have given valuable information concerning glomerular and tubular function in kidney diseaseY' :' In this study the urinary excretion of a high molecular weight protein, albumin (molecular weight 69,000) and a low molecular weight B~-microglobulin (molecular weight 11,800) has been investigated in 138 healthy infants varying in age from full-term birth to one year. In addition, the urinary protein patterns during this period have been analyzed by electrophoresis in polyacrylamide gel containing sodium dodecylsulfate, a technique that separates proteins according to their molecular weight. Quantitative data on albumin excretion in infants have not been reported earlier. A mean value for/3~-microglobulin in urine from nine newborn infants of 0.51 mg/1 was recently published. 4 From the Institute of Medical and Physiological Chemistry and Department of Clinical Chemistry, University of Uppsala. Supported by Tore Nilson Foundation, Konung Gustaf V 80-hrsfond, and the Swedish Medical Research Council (Project No. 13X-3144). *Reprint address: Institute of Medical and Physiological Chemistry, Box 575, S-751 23 Uppsala 1, Sweden.
Brief clinical and laboratory observations
89
4. Rosati LA, Barnes B, Oberman HA, and Penner JA: Hemolytic anemia due to anti-A in concentrated antihemophilic factor preparations, Transfusion 10:139, 1970. 5. Oberman HA, Barnes BA, and Ginther PL: Erythrocyte sensitization and anemia due to isoantibodies in lyophilized pooled plasma, JAMA 198:233, 1966. 6. Schwartz JM, Cohn BD, Ritz ND, and Levy T: Appendectomy in hemophilia with the use of cryoprecipitated human factor VIII to control bleeding, JAMA 198:1175, 1966. 7. Marder VJ, and Shulman NR: Major surgery in classic hemophilia using fraction 1, Am J Med 41:56, 1966. 8. Schorr JB, Ballard S, and Radel E: Hemolysis associated with "Fibro-AHF" therapy of hemophilia, read before the Thirty-sixth Annual Meeting of the Society for Pediatric Research, Atlantic City, 1966, p 132. 9. Cronin CA: Factor VIII concentrate in haemophilia, Lancet 1:1303, 1975.
Table I. Urinary excretion of albumin and B2-microglobulin in early infancy; the mean and the 95% confidence limits were calculated from the logarithm of the observed values Age (mo)
n
Albumin (mg/l)
fiz-Microglobulin (rag~l)
0- 1 1- 2 2- 4 4- 6 6-12
42 22 30 23 21
10.83 (1.20-98.06) 2.89 (0.25-34.03) 2.40 (0.48-11.93) 3.00 (0.48-18.73) 3.03 (0.70-13.11)
0.640 (0.036-11.135) 0.170 (0.004- 7.221) 0.030 (0.001- 0.782) 0.040 (0.005- 0.283) 0.050 (0.010- 0.246)
MATERIALS
AND METHODS
Urine specimens were collected from healthy infants remaining at the hospital after birth or during routine postnatal examinations. After spontaneous micturition into plastic bags, small aliquots of neutral phosphatebuffer and sodium azide were added to a final concentration of 0.05M and 0.002% (w/v), respectively, and the samples were stored at + 4~ for one to seven days, then a t - 2 0 ~ until analysis. Albumin values were determined with a polymerenhanced immunonephelometric method)/32-Microglobulin measurements were carried out with the Phadebas fl~micro test (Pharmacia Diagnostics, Uppsala, Sweden). Urine specimens were concentrated with Diaflo ultrafiltration equipment, membrane UM 2 (Amicon Corp., Lexington, Mass.). Samples of concentrated urinary proteins (50-200 /~g) were electrophoretically separated in a polyacrylamide gel containing sodium dodecylsulfate (0.5%). The gel concentrations in spacer and running gel were 7 and 11%, respectively, and the buffer system was designed according to Neville. 6 Reference proteins were purchased from Serva, Heidelberg, Germany.
90
Briefclinical and laboratory observations
The Journal ofPediatric~ Ju~ 1976
=
:~ ~;,i!iiiii,!iiiiii~i>;~::i~?: ~ 84184184
-A
,0
"" 1
2
3
4
5
---M
6
Fig. 1. Electrophoresis in polyacrylamide,gel containing sodium dodecylsulphate of concentrated urines from (i) a normal adult, (2) a pool of eight equal specimens from children at the age of 0 to l'month, and (3) at the age of 4 to 6 months, (4) a patient with renal tubular disease (chronic cadmium poisoning), (5) a patient with nephrotic syndrome, (6) standard solution containing albumin (A), molecular weight 67,000; ovalbumin (0) tool weight 45,000, and myoglobin (M), mol weight 17,800. The individual proteins responsible for the bands seen in the urine from the newborn infants have not been identified. However, the strongest band most likely represents albumin and the sharp fast-moving one represents fl~-microglobulin. RESULTS
AND DISCUSSION
The urinary concentrations of a l b u m i n and /?~-microglobulin obtained in this study when plotted on a linear scale were found to be asymmetrically distributed. The logarithmic values were more normally distributed and were thus preferred for the calculation of m e a n and confidence limits. The highest concentrations of fl~microglobulin were found after birth (Table i), followed by a gradual decrease during the first four months to an almost constant level of about one-twentieth of the inital value. The a l b u m i n excretion demonstrated a similar tendency, however, the decrease was less pronounced, only about four-fold. During the first months large differences were noticed in protein excretion among individuals. After a few months these differences diminished and the m e a n excretion reached a stable level. These rapid changes are of importance when proteinuria during the first year of life is evaluated. Pooled urine from children at the age of 0 to 1 month and 4 to 6 months, respectively, was subjected to polyacrylamide gel electrophoresis in a buffer system containing sodium dodecylsulphate. As shown in Fig. 1, urine from newborn infants has a higher proportion of low molecular weight proteins than urine from older children, where the electrophoretic pattern is similar to that found in the examination of urine from normal adults. It is generally accepted that in the adult kidney, the tubular reabsorption of low molecular weight protein is more affected than is the reabsorption of high molecular weight proteins by tubular dysfunction. 2-~ Accordingly, a high
proportion of low molecular weight proteins is often found in the urine of patients with renal tubular disease, whereas patients with glomerular damage exhibit a pattern of marked contrast. The quantitative data and the results of the electrophoretic analysis in this study are interpreted as reflecting changes in the renal tubular protein reabsorbing function, which occur after birth. The skillful technical assistance of Mrs. C. Trossvik and Mr. H. Engstr6m and the kin4 cooperation of Dr. L-I Hardell, Dr. G. Sundelin and the staff'~t the Children's clinic at Bandstolsvfigen, Uppsala, are gratefully acknowledged. REFERENCES
1. Hardwicke J: Laboratory aspects of proteinuria in human disease, Clin Nephrol 3:37, 1975. 2. Peterson PA, Evrin P-E, and Berggard I: Differentiation of glomerular, tubular and normal proteinuria: determinations of urinary excretion of beta~2-microglobulin, albumin, and total protein, J Clin Invest 48:1189, 1969. 3. Hardwicke J, Cameron JS, Harrison JR, Hulme B, and Sooothil JF: Proteinuria in kidney disease, in Manuel Proteins in normal and pathological urine, Revitlard JP, and Betuel H, editors: Basel, 1970, S Karger AG, p 111. 4. Jonasson L-E, Evrin P-E, and Wibell L: Content of fi~microglobulin and albumin in human amniotic fluid, Acta Obstet Gynecol Scand 53:49, 1974. 5. Lizana J, and Hellsing K: Polymer enhancement of automated immunological nephelometric analysis, as illustrated by determination of urinary albumin, Clin Chem 20:415, 1974. 6. Neville DM Jr: Molecular weight determination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system, J Biol Chem 246:6328, 1971.
