- Email: [email protected]
Serial measurements of aminolevulinic acid dehydratase in children with lead toxicity
9 16
Clinical and laboratory observations
and mothers have invariably been seropositive for HIV in cases of transplacentally acquired disease. 2The only bloodderived product given that was anti-HIV positive was the HBIg, which is derived from pooled human plasma. There have been no reported cases of A I D S / A R C in recipients of HBIg. Samples of immune serum globulin have been positive for anti-HIV in most cases. 4-6 HIV is inactivated by heating, low and high pH, and chemical disinfectants. The HBIg that we used is prepared by cold alcohol fractionation and therefore probably does not transmit HIV. 4 Current recommendations by the Canadian Pediatric Society and the American Academy of Pediatrics are to give HBIg to the neonate as soon as possible after birth if the mother has HBsAg. In our patient, the HBIg was given before testing for HIV status. Because the baby's serum was anti-HIV positive, he had to be treated as a potentially infected patient, initially causing much concern for the mother and the hospital staff. Subsequently, when the probable cause of the positive study results was thought to be the HBIg, the baby still required special attention until the 3-month follow-up confirmed our suspicions. The manufacturer's package insert makes no reference to this potential clinical complication.
The Journal of Pediatrics June 1988
Although HBIg is considered to be free of infectious HIV, we recommend that when investigation of HIV status is indicated, it be performed before administration of any immun0globulin preparation. We thank Dr. Donald E. Hill, Head, Department of Pediatrics for review of the manuscript. REFERENCES
1. Provisional Public Health Service inter-agency recommendations for screening donated blood and plasma for antibody to the virus causing acquired immunodeficiency syndrome. MMWR 1985;34:1-5. 2. Rubinstein A. Pediatric AIDS. Curr Probl Pediatr 1986;16: 361-409. 3. Th~ryL, Sprecher-GoldbergerS, Jonckheer T, et al. Isolation of AIDS virus from cell-free breast milk of three healthy virus carriers. Lancet 1985;1:891-2. 4. Gocke DJ, Raska K, Pollack W, et al. HTLV-III antibody in commercial immunoglobulin.Lancet 1986;1:37-8. 5. Lai-Goldman M, McBride JH, Howanitz PJ. Presence of HTLV-III antibodiesin immune serum globulin preparations. Am J Clin Pathol 1987;87:635-9. 6. Buskard N, Ballem P, Adatia A, et al. Evaluation of recipients of hepatitis B immunoglobulin (HBIG) for anti-HIV before and after HBIG injection. Sixth National Conference CHICA, Vancouver, British Columbia, Canada, 1987.
Serial measurements of aminolevulinic acid dehydratase in children with lead toxicity V a l e r y Morris, MD, Morri E, M a r k o w i t z , MD, a n d John F, Rosen, MD From the Department of Pediatrics, ALbert Einstein Coilege of Medicine, Montefiore Medical Center, Bronx, New York
Because of the high prevalence of lead poisoning in children 1 to 6 years of age (Centers for Disease Control, January 1985), further characterization of the biochemical toxicity of lead is potentially useful, particularly at blood Pb concentrations most commonly found (25 to 55 #g/ dL)? Several enzymatic steps in heine synthesis are disturbed by Pb. Inhibition of ferrochelatase by Pb concentrations as low as 17 #g/dL results in the accumulation of Supported by NIH Grant ES04039 and NIH Human Developmental Biology Grant 6962-63. Submitted for publication June 26, 1987; accepted Dec. 3l, 1987. Reprint requests: John F. Rosen, MD, Department of Pediatrics, Montefiore Medical Center, 111 E. 210th St., Bronx, NY 10467.
the heme precursor protoporphyrin. 2 The sensitivity of this enzyme to Pb effects, coupled with the relative ease with which erythrocyte protoporphyrin can be measured, has ALAD CaNaEEDTA EP PbPT
~-Aminolevulinicacid dehydratase Calcium disodium ethylenediamine tetraacetic acid Erythrocyte protoporphyrin CaNa2EDTA provocative test
established EP measurements as the current method of choice in screening the pediatric population for Pb toxicity. ~ The activity of fi-aminolevulinic acid dehydratase, an enzyme active earlier in the heme pathway, has been
Volume 112 Number 6
shown to be significantly depressed by Pb. 3"8 Hernberg et al. 6 found 50% of A L A D activity inhibited at a blood Pb concentration of 16 #g/dL. Nieberg et al.7 showed a high correlation between red cell ALAD with chelatable Pb. Therefore, these authors suggested that ALAD is a useful index of the body burden of Pb and may be a useful index of toxicity to determine the need for treatment. Previous studies of A L A D activity in children have generally reported measurements obtained at a single time. 8,9 In Pb-poisoned adults, A L A D activity increases after treatment with CaNaEEDTA? ~ The aim of our study was to assess A L A D activity repetitively in a group of children with Pb toxicity undergoing chelation therapy. By so doing, the clinical utility of this presumably sensitive index of Pb toxicity in pediatric populations could be evaluated, both as a determinant for the need of chelation therapy and as a predictor of the long-term management needs of individual Pb-poisoned children. METHODS Thirty-seven children (ages 12 months to 6 years) without overt clinical symptoms of Pb poisoning were studied. All had Pb levels in whole blood >__23 t~g/dL (range 23 to 116 #g/dL) at the time of initial study. Concurrently, the EP concentration in whole blood was >35 # g / d L (range 39 to 899 #g/dL). None had been given chelating agents in the preceding 2 months. Study design. Seven children with initial blood Pb levels >55 ~zg/dL immediately underwent 5 days of CaNaEEDTA treatment with or without dimercaprol (BAL). The remaining 30 children, who had blood Pb concentrations between 23 and 55 ug/dL, underwent a CaNa/EDTA provocative test: lzq4 500 mg/m 2 of CaNaEEDTA was administered intramuscularly, followed by an 8-hour urine collection. The ratio of urinary Pb excreted (in micrograms) to CaNazEDTA administered (in milligrams) was then calculated. For children ~ 3 years of age a ratio of _>_0.60, and for children >3 years of age a ratio of >--0.70, indicated a positive test result, l' Children with positive provocative test results then received 5 days of CaNaEEDTA (1000 mg/m2/d) in the hospital. Blood samples were obtained at four times: before treatment or before PbPT, 12 hours after completion of chelation treatment, 4 weeks after the initial time, and 7 weeks after the initial time point. At week 7, those children with persistent elevations in blood Pb concentrations (Pb > 25 #g/dL) underwent a second CaNa2EDTA provocative test (PbPT-2). Laboratory studies on the blood samples included measurements of blood Pb, EPI and ALAD, and routine chemistry determinations.
Clinical and laboratory observations
91 7
Laboratory methods. Red blood cell A L A D activity was assayed using the method of Granick et al. ~5 using 20 #L whole blood. Using this assay, a good correlation has been shown between blood Pb levels and the ratio of fully activated ALAD (with dithiothreitol) to the nonactivated enzyme) 5 Expressing the measurement as the ratio of activated to total ALAD circumvents the complication of the genetic heterogeneity of this enzyme. ~5 Pb concentrations in urine and in whole blood and EP in whole blood were measured by methods previously published, n' 13Urine and blood Pb determinations wre carried out on a Spectra 30P atomic absorption spectrophotometer (Varian Associates Inc., Palo Alto, Calif.) with a carbon rod atomizer (Model GTA-96). Recoveries of added 2t~ or 2~ to working standards averaged 97% to 101%. The 99% confidence limits of this method, as previously reported, is 1 # g / d L for Pb in whole bloodJ 2,13 For blood Pb values, spiked whole blood was used for standards. This laboratory participates in the Centers for Disease Control and New York City proficiency testing programs. For measurements of Pb in urine, the method of Kubasik and Volosin was used, as reported previously? z, 13 Spiked urine samples were used as standards. The 99% confidence limit of this method was 5 gg Pb/vol urine) 2 EP was determined fluorometrically by the ethylacetate acetic acid extraction method, t6 The 99% confidence limit of this method is <1 ug/dL. Statistical tests. For each variable, comparison of multiple groups was made using analyses of variance. Two group comparisons between time points for variables with s!gnificant ANOVAs were made using paired t tests. Pearson product moment correlations between A L A D and either blood Pb concentrations, total urinary Pb excretion post-PbPT, or the urinary Pb/CaNazEDTA-dose ratio, at concurrent time points, were calculated based on all available data. Multiple regression analyses were performed using ALAD, EP, and blood Pb values from concurrent time points as predictors of total urine Pb excretion post-PbPT. These independent variables from the pretreatment time point were also used to predict blood Pb or urine Pb excretion at week 7 post-PbPT-2. Analyses were performed on a personal computer ( A T & T PC 6300, American Telephone & Telegraph, New York) using the SYSTAT statistics package version 2.1. RESULTS The data from 20 children who received chelation therapy and had complete data available at all four time points are shown in Table I. As expected, blood Pb concentrations fell with treatment, then rebounded by week 4, but to levels below pretreatment values. In contrast, mean A L A D ratios followed an inverse pattern.
9 18
Clinical and laboratory observations
The Journal of Pediatrics June 1988
Table I. Blood laboratory values in 20 CaNaEEDTA-treated children
Blood Pb (t~g/dL) Erythrocyte protoporphyrin (/~g/dL) ALAD
Pretreatment
Trough
Week 4
Week 7
38 _ 12 112 + 71 0.28 _+ 0.06
18 _+ 6* 109 _+ 72 0.41 + 0.09*
29 +_ 8* 96 _+ 57 0.33 _+ 0.09"~
32 _+ 9* 91 _+ 63 0.32 + 0.12
Values are mean +- 1 SD. ALAD, 6-Aminolevulinic acid dehydratase, expressed as ratio of enzyme activity with and without dithiothreitol (DTT): Ratio = (Units A L A D without DTT)/(Units A L A D with DTT). *P <0.001, paired t test, pretreatment vs subsequent time points. t P <0.01, paired t test, pretl"eatment vs subsequent time points.
Table II. Pearson product moment correlations between ALAD activity and blood or urine lead values
Blood Pb Urine Pb Urine Pb/dose CaNazEDTA
ALAD pretreatment
ALAD trough
ALAD Week 4
ALAD Week 7
-0.612" (n = 36) -0.084 (n = 29) -0,149 (n = 29)
-0.466t (n = 28)
-0.525~:(n = 32)
-0.572:~(n = 26) -0.200 (n = 15) -0,263 (n = 15)
*P <0.00i. l P <0.05. :~P <0.01.
ANOVA for blood Pb and ALAD across the four time points were significant at P <0.05. Subsequent analyses using paired t tests of time point 1 compared with the three other time points found significant differences between pretreatment blood Pb concentrations and all posttreatment time points. The same pattern of relationships was true for ALAD pretreatment and 7 weeks posttreatment (P -- 0.07). The decline in mean EP concentrations across time was not statistically significant. The ALAD activity and blood Pb levels were moderately and inversely correlated at all time points (Table II). There was no statistically significant bivariate relationship between urinary Pb excretion and ALAD activity at either time point. To clarify further whether the measurement of ALAD is useful as a predictor of the chelatable body burden of Pb when combined with EP and blood Pb measurements, we performed a series of multiple regression analyses (table of regression equations available on request). At time points 1 and 4, the addition of either EP or ALAD to blood Pb accounted for only a small, nonsignificant increment in the variance of urinary Pb excretion. The highest r z values were noted when both EP and ALAD were entered in the equation (rZp......tment= 0.554, r2week7 = 0,496, P <0.000 with both EP and ALAD in the regression equations vs r2pretreat. mo,t= 0.432, r2w=k7 = 0.376, P <0.01 with only blood Pb as the independent variable). DISCUSSION The population we examined is representative of Pbburdened children, as evidenced by the distribution of
pretreatment blood Pb concentrations, the appropriate reduction of these blood Pb concentrations by chelation therapy, and the ensuing rebound in blood Pb values to less than pretreatment levels. ALAD catalyzes the self-condensation of two molecules of ALAD to form one molecule of porphobilinogen? The enzyme activity is inhibited by Pb in vitro, and is fully activated by dithiothreitol and zinc, thereby allowing for the use of an activity ratio to measure ALAD in Pbexposed subjects? 5 This observation is significant because the baseline enzyme activity shows a significant amount of genetic variability in humansY. 57 We found a significant increase in the ALAD activity ratio immediately after chelation, which decreased at 4 weeks after chelation as the blood Pb level rebounded. As reported in studies using single time measurements, we found the correlation coefficients between ALAD activity ratios and blood Pb concentrations to be statistically significant at all time points. However, these were of only moderate magnitude in accounting for the variance in blood Pb values. Coupled with the lack of a bivariate association between ALAD activity and chelatable Pb in these children, the proposed sensitivity for using ALAD to predict the need for chelation therapy is severely limited in individual children. We did find significant correlations between measurements of EP concentration and urinary Pb levels on each provocative test day (P <0.01 for each), but EP added little predictive information to measurements of the blood Pb concentration, as indicated by the regression equations. In fact, ALAD in combination with blood Pb was a margin-
Volume 112 Number 6
ally better predictor of urinary Pb excretion than was EP at either time point. Others have shown that this enzyme is remarkably sensitive to Pb, possibly at concentrations as low as 10 # g / d L . 9 Measuring EP and A L A D concurrently in a large group of black children led Rogan et al. 17 to suggest that there may be a subpopulation inherently more sensitive to the inhibitory effects of Pb on heme pathway enzymes; such a finding might prove useful in identifying children at greater risk. Our data show t h a t A L A D measurements serve to confirm the toxic effects of Pb on heme metabolism and to provide an assessment of the immediate effects of treatment in reducing the body Pb burden. However, the A L A D assay is technically more difficult and more expensive than EP determinations. Moreover, the lack of a bivariate correlation between A L A D and CaNa2EDTA-provoked urinary Pb excretion, the current standard method for assessing Pb stores in children, limits its clinical utility as a predictor for the need of chelation treatment in individual children. We thank Dr. Carol Seaman and Dr. Polly Bijur for assistance. REFERENCES
1. Rosen JF. Metabolic and cellular effects of lead: a guide to low level lead toxicity in children. In: Mahaffey KR,. ed. Dietary and environmental lead: human health effects. New York: Elsevier Science, 1985:157. 2. Piomelli, S, Seaman C, Zullow D, Curran A, Davidow B. Threshold for lead damage to heme synthesis in urban children. Proc Natl Acad Sci USA 1982;79:3335-9. 3. Nakao K, Wada O, Yano Y. Delta-aminolevulinic acid dehydratase activity in erythrocytes for the evaluation of lead poisoning. Clin Chim Acta 1968;19:319-25. 4. Alessio L, Castoldi MR, Odone P, Franchini I. Behaviour of indicators of exposure and effect after cessation of occupational exposure to lead. Br J Ind Med 1981;38:262-7. 5. Haeger-Aronsen B, Abdulla M, Fristedt BI. Effect of lead on
Clinical and laboratory observations
6.
7.
8.
9. 10.
11.
12.
13.
14. 15.
16.
17.
9 19
~-aminolevulinic acid dehydratase activity in red blood cells Arch. Environ Health 1971;23:440-5. Hernberg S, Nikkanen J, Mellin G, Lilius H. ~-aminolevulinic acid dehydratase as a measure of lead exposure. Arch Environ Health 1970;21:140-5. Nieburg PI, Weiner LS, Oski BF, Oski FA. Red blood cell b-aminolevulinic acid dehydrase activity. Am J Dis Child 1974;127:348-50. Wagner V, Wagnerova M, Wokounova D, Kriz J, Madlo Z, Mohyla O. Correlations between blood lead concentrations and some blood protein levels in children residing in leadpolluted and control areas. J Hyg Epidemiol Microbiol Immunol 1981;25:97-112. Hernberg S, Nikkanen J. Enzyme inhibited by lead under natural urban conditions. Lancet 1970;1:63-4. Aono H, Akaki S. The effects of CaEDTA injection on lead, zinc, copper, ALAD in erythrocytes, plasma and urine in lead exposed workers: a 24-hour observation. Int Arch Occup Environ Health 1984;55:13-8. Araki S, Aono M, Fukahori M, Tabuki K. Behavior of lead and zinc in plasma, erythrocytes, and urine, and ALAD in erythrocytes following intravenous infusion of CaEDTA in lead workers. Arch Environ Health 1984;39:364-7. Saenger P, Rosen JF, Markowitz ME. Diagnostic significance of CaNaEDTA testing in children with undue lead absorption. Am J Dis Child 1982;136:312-5. Markowitz ME, Rosen JF. Assessment of lead stores in children: validation of an 8-hour EDTA provocative test. J PEDIATR 1984;104:337-41. Piomelli S, Rosen JF, Chisolm J J, Graef JW. Management of childhood lead poisoning. J PEDIATR 1984;105:523-32. Granick JL, Sassa S. Granick S, Levere RC, Kappas A. Studies in lead poisoning. II. Correlation between the ratio of activated to inactivated ~-aminolevulinic acid dehydratase of whole blood and the blood lead level. Bioehem Med 1973;8:149-59. Piomelli S. Free erythrocyte porphyrins in the detection of undue absorption of lead and iron deficiency. Ctin Chern 1977;23:264-9. Rogan WJ, Reigart JR, Gladen BC. Association of amino levulinate dehydratase levels and ferrochelatase inhibition in childhood lead exposure. J PEDtATR 1986;109:60-4.
Growth hormone assessment and short-term treatment with growth hormone in Turner syndrome Tsu-Hui Lin, MD, John L. Kirkland, MD, a n d R e b e c c a T. Kirkland, MD From the Department of Pediatrics, Baylor College of Medicine, Houston
Children with Turner syndrome have abnormal growth patterns and short stature as adults. ~-6 The cause of these Submitted for publication Oct. 27, 1987; accepted Jan. 22, 1988. Reprint requests: Tsu-Hui Lin, MD, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
abnormalities in growth is unknown but may be related to perturbations of growth hormone secretion as measured by responses to secretagogues and by 24-hour tests of G H secretion. 7-9 Recent data obtained by several investigators indicate that G H administration increases the velocity of linear growth and the predicted adult height in children
Clinical and laboratory observations
and mothers have invariably been seropositive for HIV in cases of transplacentally acquired disease. 2The only bloodderived product given that was anti-HIV positive was the HBIg, which is derived from pooled human plasma. There have been no reported cases of A I D S / A R C in recipients of HBIg. Samples of immune serum globulin have been positive for anti-HIV in most cases. 4-6 HIV is inactivated by heating, low and high pH, and chemical disinfectants. The HBIg that we used is prepared by cold alcohol fractionation and therefore probably does not transmit HIV. 4 Current recommendations by the Canadian Pediatric Society and the American Academy of Pediatrics are to give HBIg to the neonate as soon as possible after birth if the mother has HBsAg. In our patient, the HBIg was given before testing for HIV status. Because the baby's serum was anti-HIV positive, he had to be treated as a potentially infected patient, initially causing much concern for the mother and the hospital staff. Subsequently, when the probable cause of the positive study results was thought to be the HBIg, the baby still required special attention until the 3-month follow-up confirmed our suspicions. The manufacturer's package insert makes no reference to this potential clinical complication.
The Journal of Pediatrics June 1988
Although HBIg is considered to be free of infectious HIV, we recommend that when investigation of HIV status is indicated, it be performed before administration of any immun0globulin preparation. We thank Dr. Donald E. Hill, Head, Department of Pediatrics for review of the manuscript. REFERENCES
1. Provisional Public Health Service inter-agency recommendations for screening donated blood and plasma for antibody to the virus causing acquired immunodeficiency syndrome. MMWR 1985;34:1-5. 2. Rubinstein A. Pediatric AIDS. Curr Probl Pediatr 1986;16: 361-409. 3. Th~ryL, Sprecher-GoldbergerS, Jonckheer T, et al. Isolation of AIDS virus from cell-free breast milk of three healthy virus carriers. Lancet 1985;1:891-2. 4. Gocke DJ, Raska K, Pollack W, et al. HTLV-III antibody in commercial immunoglobulin.Lancet 1986;1:37-8. 5. Lai-Goldman M, McBride JH, Howanitz PJ. Presence of HTLV-III antibodiesin immune serum globulin preparations. Am J Clin Pathol 1987;87:635-9. 6. Buskard N, Ballem P, Adatia A, et al. Evaluation of recipients of hepatitis B immunoglobulin (HBIG) for anti-HIV before and after HBIG injection. Sixth National Conference CHICA, Vancouver, British Columbia, Canada, 1987.
Serial measurements of aminolevulinic acid dehydratase in children with lead toxicity V a l e r y Morris, MD, Morri E, M a r k o w i t z , MD, a n d John F, Rosen, MD From the Department of Pediatrics, ALbert Einstein Coilege of Medicine, Montefiore Medical Center, Bronx, New York
Because of the high prevalence of lead poisoning in children 1 to 6 years of age (Centers for Disease Control, January 1985), further characterization of the biochemical toxicity of lead is potentially useful, particularly at blood Pb concentrations most commonly found (25 to 55 #g/ dL)? Several enzymatic steps in heine synthesis are disturbed by Pb. Inhibition of ferrochelatase by Pb concentrations as low as 17 #g/dL results in the accumulation of Supported by NIH Grant ES04039 and NIH Human Developmental Biology Grant 6962-63. Submitted for publication June 26, 1987; accepted Dec. 3l, 1987. Reprint requests: John F. Rosen, MD, Department of Pediatrics, Montefiore Medical Center, 111 E. 210th St., Bronx, NY 10467.
the heme precursor protoporphyrin. 2 The sensitivity of this enzyme to Pb effects, coupled with the relative ease with which erythrocyte protoporphyrin can be measured, has ALAD CaNaEEDTA EP PbPT
~-Aminolevulinicacid dehydratase Calcium disodium ethylenediamine tetraacetic acid Erythrocyte protoporphyrin CaNa2EDTA provocative test
established EP measurements as the current method of choice in screening the pediatric population for Pb toxicity. ~ The activity of fi-aminolevulinic acid dehydratase, an enzyme active earlier in the heme pathway, has been
Volume 112 Number 6
shown to be significantly depressed by Pb. 3"8 Hernberg et al. 6 found 50% of A L A D activity inhibited at a blood Pb concentration of 16 #g/dL. Nieberg et al.7 showed a high correlation between red cell ALAD with chelatable Pb. Therefore, these authors suggested that ALAD is a useful index of the body burden of Pb and may be a useful index of toxicity to determine the need for treatment. Previous studies of A L A D activity in children have generally reported measurements obtained at a single time. 8,9 In Pb-poisoned adults, A L A D activity increases after treatment with CaNaEEDTA? ~ The aim of our study was to assess A L A D activity repetitively in a group of children with Pb toxicity undergoing chelation therapy. By so doing, the clinical utility of this presumably sensitive index of Pb toxicity in pediatric populations could be evaluated, both as a determinant for the need of chelation therapy and as a predictor of the long-term management needs of individual Pb-poisoned children. METHODS Thirty-seven children (ages 12 months to 6 years) without overt clinical symptoms of Pb poisoning were studied. All had Pb levels in whole blood >__23 t~g/dL (range 23 to 116 #g/dL) at the time of initial study. Concurrently, the EP concentration in whole blood was >35 # g / d L (range 39 to 899 #g/dL). None had been given chelating agents in the preceding 2 months. Study design. Seven children with initial blood Pb levels >55 ~zg/dL immediately underwent 5 days of CaNaEEDTA treatment with or without dimercaprol (BAL). The remaining 30 children, who had blood Pb concentrations between 23 and 55 ug/dL, underwent a CaNa/EDTA provocative test: lzq4 500 mg/m 2 of CaNaEEDTA was administered intramuscularly, followed by an 8-hour urine collection. The ratio of urinary Pb excreted (in micrograms) to CaNazEDTA administered (in milligrams) was then calculated. For children ~ 3 years of age a ratio of _>_0.60, and for children >3 years of age a ratio of >--0.70, indicated a positive test result, l' Children with positive provocative test results then received 5 days of CaNaEEDTA (1000 mg/m2/d) in the hospital. Blood samples were obtained at four times: before treatment or before PbPT, 12 hours after completion of chelation treatment, 4 weeks after the initial time, and 7 weeks after the initial time point. At week 7, those children with persistent elevations in blood Pb concentrations (Pb > 25 #g/dL) underwent a second CaNa2EDTA provocative test (PbPT-2). Laboratory studies on the blood samples included measurements of blood Pb, EPI and ALAD, and routine chemistry determinations.
Clinical and laboratory observations
91 7
Laboratory methods. Red blood cell A L A D activity was assayed using the method of Granick et al. ~5 using 20 #L whole blood. Using this assay, a good correlation has been shown between blood Pb levels and the ratio of fully activated ALAD (with dithiothreitol) to the nonactivated enzyme) 5 Expressing the measurement as the ratio of activated to total ALAD circumvents the complication of the genetic heterogeneity of this enzyme. ~5 Pb concentrations in urine and in whole blood and EP in whole blood were measured by methods previously published, n' 13Urine and blood Pb determinations wre carried out on a Spectra 30P atomic absorption spectrophotometer (Varian Associates Inc., Palo Alto, Calif.) with a carbon rod atomizer (Model GTA-96). Recoveries of added 2t~ or 2~ to working standards averaged 97% to 101%. The 99% confidence limits of this method, as previously reported, is 1 # g / d L for Pb in whole bloodJ 2,13 For blood Pb values, spiked whole blood was used for standards. This laboratory participates in the Centers for Disease Control and New York City proficiency testing programs. For measurements of Pb in urine, the method of Kubasik and Volosin was used, as reported previously? z, 13 Spiked urine samples were used as standards. The 99% confidence limit of this method was 5 gg Pb/vol urine) 2 EP was determined fluorometrically by the ethylacetate acetic acid extraction method, t6 The 99% confidence limit of this method is <1 ug/dL. Statistical tests. For each variable, comparison of multiple groups was made using analyses of variance. Two group comparisons between time points for variables with s!gnificant ANOVAs were made using paired t tests. Pearson product moment correlations between A L A D and either blood Pb concentrations, total urinary Pb excretion post-PbPT, or the urinary Pb/CaNazEDTA-dose ratio, at concurrent time points, were calculated based on all available data. Multiple regression analyses were performed using ALAD, EP, and blood Pb values from concurrent time points as predictors of total urine Pb excretion post-PbPT. These independent variables from the pretreatment time point were also used to predict blood Pb or urine Pb excretion at week 7 post-PbPT-2. Analyses were performed on a personal computer ( A T & T PC 6300, American Telephone & Telegraph, New York) using the SYSTAT statistics package version 2.1. RESULTS The data from 20 children who received chelation therapy and had complete data available at all four time points are shown in Table I. As expected, blood Pb concentrations fell with treatment, then rebounded by week 4, but to levels below pretreatment values. In contrast, mean A L A D ratios followed an inverse pattern.
9 18
Clinical and laboratory observations
The Journal of Pediatrics June 1988
Table I. Blood laboratory values in 20 CaNaEEDTA-treated children
Blood Pb (t~g/dL) Erythrocyte protoporphyrin (/~g/dL) ALAD
Pretreatment
Trough
Week 4
Week 7
38 _ 12 112 + 71 0.28 _+ 0.06
18 _+ 6* 109 _+ 72 0.41 + 0.09*
29 +_ 8* 96 _+ 57 0.33 _+ 0.09"~
32 _+ 9* 91 _+ 63 0.32 + 0.12
Values are mean +- 1 SD. ALAD, 6-Aminolevulinic acid dehydratase, expressed as ratio of enzyme activity with and without dithiothreitol (DTT): Ratio = (Units A L A D without DTT)/(Units A L A D with DTT). *P <0.001, paired t test, pretreatment vs subsequent time points. t P <0.01, paired t test, pretl"eatment vs subsequent time points.
Table II. Pearson product moment correlations between ALAD activity and blood or urine lead values
Blood Pb Urine Pb Urine Pb/dose CaNazEDTA
ALAD pretreatment
ALAD trough
ALAD Week 4
ALAD Week 7
-0.612" (n = 36) -0.084 (n = 29) -0,149 (n = 29)
-0.466t (n = 28)
-0.525~:(n = 32)
-0.572:~(n = 26) -0.200 (n = 15) -0,263 (n = 15)
*P <0.00i. l P <0.05. :~P <0.01.
ANOVA for blood Pb and ALAD across the four time points were significant at P <0.05. Subsequent analyses using paired t tests of time point 1 compared with the three other time points found significant differences between pretreatment blood Pb concentrations and all posttreatment time points. The same pattern of relationships was true for ALAD pretreatment and 7 weeks posttreatment (P -- 0.07). The decline in mean EP concentrations across time was not statistically significant. The ALAD activity and blood Pb levels were moderately and inversely correlated at all time points (Table II). There was no statistically significant bivariate relationship between urinary Pb excretion and ALAD activity at either time point. To clarify further whether the measurement of ALAD is useful as a predictor of the chelatable body burden of Pb when combined with EP and blood Pb measurements, we performed a series of multiple regression analyses (table of regression equations available on request). At time points 1 and 4, the addition of either EP or ALAD to blood Pb accounted for only a small, nonsignificant increment in the variance of urinary Pb excretion. The highest r z values were noted when both EP and ALAD were entered in the equation (rZp......tment= 0.554, r2week7 = 0,496, P <0.000 with both EP and ALAD in the regression equations vs r2pretreat. mo,t= 0.432, r2w=k7 = 0.376, P <0.01 with only blood Pb as the independent variable). DISCUSSION The population we examined is representative of Pbburdened children, as evidenced by the distribution of
pretreatment blood Pb concentrations, the appropriate reduction of these blood Pb concentrations by chelation therapy, and the ensuing rebound in blood Pb values to less than pretreatment levels. ALAD catalyzes the self-condensation of two molecules of ALAD to form one molecule of porphobilinogen? The enzyme activity is inhibited by Pb in vitro, and is fully activated by dithiothreitol and zinc, thereby allowing for the use of an activity ratio to measure ALAD in Pbexposed subjects? 5 This observation is significant because the baseline enzyme activity shows a significant amount of genetic variability in humansY. 57 We found a significant increase in the ALAD activity ratio immediately after chelation, which decreased at 4 weeks after chelation as the blood Pb level rebounded. As reported in studies using single time measurements, we found the correlation coefficients between ALAD activity ratios and blood Pb concentrations to be statistically significant at all time points. However, these were of only moderate magnitude in accounting for the variance in blood Pb values. Coupled with the lack of a bivariate association between ALAD activity and chelatable Pb in these children, the proposed sensitivity for using ALAD to predict the need for chelation therapy is severely limited in individual children. We did find significant correlations between measurements of EP concentration and urinary Pb levels on each provocative test day (P <0.01 for each), but EP added little predictive information to measurements of the blood Pb concentration, as indicated by the regression equations. In fact, ALAD in combination with blood Pb was a margin-
Volume 112 Number 6
ally better predictor of urinary Pb excretion than was EP at either time point. Others have shown that this enzyme is remarkably sensitive to Pb, possibly at concentrations as low as 10 # g / d L . 9 Measuring EP and A L A D concurrently in a large group of black children led Rogan et al. 17 to suggest that there may be a subpopulation inherently more sensitive to the inhibitory effects of Pb on heme pathway enzymes; such a finding might prove useful in identifying children at greater risk. Our data show t h a t A L A D measurements serve to confirm the toxic effects of Pb on heme metabolism and to provide an assessment of the immediate effects of treatment in reducing the body Pb burden. However, the A L A D assay is technically more difficult and more expensive than EP determinations. Moreover, the lack of a bivariate correlation between A L A D and CaNa2EDTA-provoked urinary Pb excretion, the current standard method for assessing Pb stores in children, limits its clinical utility as a predictor for the need of chelation treatment in individual children. We thank Dr. Carol Seaman and Dr. Polly Bijur for assistance. REFERENCES
1. Rosen JF. Metabolic and cellular effects of lead: a guide to low level lead toxicity in children. In: Mahaffey KR,. ed. Dietary and environmental lead: human health effects. New York: Elsevier Science, 1985:157. 2. Piomelli, S, Seaman C, Zullow D, Curran A, Davidow B. Threshold for lead damage to heme synthesis in urban children. Proc Natl Acad Sci USA 1982;79:3335-9. 3. Nakao K, Wada O, Yano Y. Delta-aminolevulinic acid dehydratase activity in erythrocytes for the evaluation of lead poisoning. Clin Chim Acta 1968;19:319-25. 4. Alessio L, Castoldi MR, Odone P, Franchini I. Behaviour of indicators of exposure and effect after cessation of occupational exposure to lead. Br J Ind Med 1981;38:262-7. 5. Haeger-Aronsen B, Abdulla M, Fristedt BI. Effect of lead on
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~-aminolevulinic acid dehydratase activity in red blood cells Arch. Environ Health 1971;23:440-5. Hernberg S, Nikkanen J, Mellin G, Lilius H. ~-aminolevulinic acid dehydratase as a measure of lead exposure. Arch Environ Health 1970;21:140-5. Nieburg PI, Weiner LS, Oski BF, Oski FA. Red blood cell b-aminolevulinic acid dehydrase activity. Am J Dis Child 1974;127:348-50. Wagner V, Wagnerova M, Wokounova D, Kriz J, Madlo Z, Mohyla O. Correlations between blood lead concentrations and some blood protein levels in children residing in leadpolluted and control areas. J Hyg Epidemiol Microbiol Immunol 1981;25:97-112. Hernberg S, Nikkanen J. Enzyme inhibited by lead under natural urban conditions. Lancet 1970;1:63-4. Aono H, Akaki S. The effects of CaEDTA injection on lead, zinc, copper, ALAD in erythrocytes, plasma and urine in lead exposed workers: a 24-hour observation. Int Arch Occup Environ Health 1984;55:13-8. Araki S, Aono M, Fukahori M, Tabuki K. Behavior of lead and zinc in plasma, erythrocytes, and urine, and ALAD in erythrocytes following intravenous infusion of CaEDTA in lead workers. Arch Environ Health 1984;39:364-7. Saenger P, Rosen JF, Markowitz ME. Diagnostic significance of CaNaEDTA testing in children with undue lead absorption. Am J Dis Child 1982;136:312-5. Markowitz ME, Rosen JF. Assessment of lead stores in children: validation of an 8-hour EDTA provocative test. J PEDIATR 1984;104:337-41. Piomelli S, Rosen JF, Chisolm J J, Graef JW. Management of childhood lead poisoning. J PEDIATR 1984;105:523-32. Granick JL, Sassa S. Granick S, Levere RC, Kappas A. Studies in lead poisoning. II. Correlation between the ratio of activated to inactivated ~-aminolevulinic acid dehydratase of whole blood and the blood lead level. Bioehem Med 1973;8:149-59. Piomelli S. Free erythrocyte porphyrins in the detection of undue absorption of lead and iron deficiency. Ctin Chern 1977;23:264-9. Rogan WJ, Reigart JR, Gladen BC. Association of amino levulinate dehydratase levels and ferrochelatase inhibition in childhood lead exposure. J PEDtATR 1986;109:60-4.
Growth hormone assessment and short-term treatment with growth hormone in Turner syndrome Tsu-Hui Lin, MD, John L. Kirkland, MD, a n d R e b e c c a T. Kirkland, MD From the Department of Pediatrics, Baylor College of Medicine, Houston
Children with Turner syndrome have abnormal growth patterns and short stature as adults. ~-6 The cause of these Submitted for publication Oct. 27, 1987; accepted Jan. 22, 1988. Reprint requests: Tsu-Hui Lin, MD, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.
abnormalities in growth is unknown but may be related to perturbations of growth hormone secretion as measured by responses to secretagogues and by 24-hour tests of G H secretion. 7-9 Recent data obtained by several investigators indicate that G H administration increases the velocity of linear growth and the predicted adult height in children