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Creatine phosphokinase BB isoenzyme in very-low-birth-weight infants: Relationship with mortality and intraventricular hemorrhage
790
Clinical and laboratory observations
Although the means by which heparin causes inhibition of the classical complement pathway has not been determined, there are several proposed sites of inhibition, including direct inhibition of Clq binding to immune complexes, inhibition of the interaction of C]~ with C4 and C2, and potentiation of the effect of C]- inhibitor function? Surface-associated heparin inhibits activation of the alternative pathway of complement by augmenting the regulatory action of the control proteins H and I in such a way that the formation and function of the amplification convertase C3bBb is prevented.4,5 Inhibitory activity of the effector sequence of complement by heparin also has been described. 4 The inhibitory effect of heparin on neutrophilmediated bactericidal activity is dose dependent and appears to be attributable primarily to the inhibition of ingestion.6 Because levels of both classical and alternative complement pathway components are lower in neonates than in older children and adults, ~2 the anticomplementary effect of heparin potentially could have caused significant inhibition of complement-dependent bactericidal activity in neonatal sera. Our data indicate that at the levels required for maintaining catheter patency and for achieving anticoagulation in neonates, the anticomplementary effect of heparin, as assessed by bactericidal activity of sera for type III, group B streptococcus, is not significant.
REFERENCES 1. McDonald MM, Hathaway WE: Anticoagulant therapy by continuous heparinization in newborn and older infants. J PEDIATR101:451, 1982.
The Journal of Pediatrics November 1983 2. McDonald MM, Jacobson LJ, Hay WW Jr, et al: Heparin clearance in the newborn, i'ediatr Res 15:1015, 1981. 3. Caughman GB, Boackle RJ, Vesely J: A postulated mechanism for heparin's potentiation of C1 inhibitor function. Mol Immunol 19:287, 1982. 4. Weiler JM, Yurt RW, Fearon DT, et al: Modulation of the formation of the amplification convertase of complement, C3bBb, by native and commercial heparin. J Exp Med 147:409, 1978. 5. Kazatchkine MD,Fearon DT, Silbert JE, et al: Surfaceassociated heparin inhibits zymosan-inducedactivation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b. J Exp Med 150:1202, 1979. 6. Victor M, Weiss J, Elsbach P: Heparin inhibits phagocytosis by polymorphonuclear leukocytes. Infect Immun 32:295, 1981. 7. Edwards MS, Baker CJ, Kasper DL: Opsonic specificity of human antibody to the type 111 polysaccharide of group B Streptococcus. J Infect Dis 140:1004, 1979. 8. Edwards MS, Nicholson-WellerA, Baker CJ, et al: The role of the alternative complement pathway in opsonophagocytosis of type III, group B Streptococcus. J Exp Med 151:1275, 1980. 9. Baker CJ, Kasper DL, Tager IB, et al: Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B Streptococcus. J Clin Invest 59:810, 1977. 10. Teien AN, Lie M: Evaluation of an amidolytic heparin assay method: Increased sensitivityby adding purified antithrombin III. Thromb Res 10:399, 1977. 11. Estes JW, Poulin PF: Pharmokinetics of heparin. Thromb Haemost 33:26, 1974. 12. Davis CA, Vallota EH, Forristal J: Serum complement levels in infancy: Age related changes. Pediatr Res 13:1043, 1979.
Creatine phosphokinase BB isoenzyme in very-low-birth-weight infants." Relationship with mortality and intraventricular hemorrhage Michael E. Speer, M.D., Ching-Nan Ou, Ph.D., Gregory J. Buffone, Ph.D., and Vickie L. Frawley, M.S. Houston, T e x a s From the Departments of Pediatrics and Pathology, Baylor Cgllege of Medicine, and the Section of Neonatology, Texas Children's Hospital. Presented in part at the Western Society for Pediatric Research, Carmel, California, February 1982, and at the American Society of Pediatrics and Society for Pediat~c~Research, Washington, D.C., May 1982. Reprint requests: Michael E. Speer, M.D., Department of Pediatrics, Baylor College of Medicine, 1200 Morsund Ave., Houston, TX 77030.
ELEVATED LEVELS OF CPK-BB have been demonstrated in serum after ischemic or traumatic brain injury in adult~ ~-3and after asphyxic insult in neonates?-7 Recently, Shields and Feldman8 reported significant elevations in serum CPK-BB in neonates with periventricular/intravenI
CPK PIVH
Creatinine phosphokinase Periventricular/intraventricular hemorrhage
[
Volume 103 Number 5
Clinical and laboratory observations
79 1
Table. CPK-BB activity With PIVH (U/L)
Sample period
Mean + S E M
1 2 3 4 5
41.44 + 8.41" 1.92 + 0.54 5.63 + 2.40 1.74 + 0.76 7.30 _+ 5.49
Without PIVH (U/L) Range
2.69 to 0.00 to 0.00 to 0.00 to 0.00 to
Mean + S E M
206.72 14.10 50.74 15.02 83.50
9.55 _+ 1.63 1.51 _+ 0.56 2.00 + 0.75 0.81 + 0.25 0.27 _+ 0.19
Range
0.00 to 0.00 to 0.00 to 0.00 to 0.00 to
18.01 6.93 6.29 2.27 1.41
*P < 0.002. tricular hemorrhage, as compared with infants without PIVH. Our study was undertaken to determine whether serum CPK-BB levels could be predictive of short-term outcome in a series of very-low-birth-weight neonates with and without PIVH. METHODS All newborn infants -<32 weeks' gestation, as determined b y maternal dates and Ballard scores, 9 who were admitted to the neonatal intensive 'care unit at Texas Children's Hospital, Houston, and who underwent arterial catheterization were considered for enrollment in the study. If maternal dates and Ballard Scores differed by more than two weeks, the gestational'age assigned was that of the Ballard score. Informed parental consent was obtained for the sequential blood sampling. In all infants --<32 weeks requiring care in the neonatal ICU, one or more ultrasound studies were performed routinely as part of the medical management On or before the sixth day of life. The timing of the ultrasound evaluation(s) was determined bY the attending neonatologisL who was unaware of the CPK-BB test results. Ultrasound imaging was done with a n ATL Mark 3 real-time digital sector scanner (Advanced Technical Laboratory, Bellevue, WA) using a 5 MHz transducer directed in the' sagittal and Coronal planes through the anterior fontanelle, as described by Bejar et al. 1~ The radiologist who performed and interpreted the ultrasound examinations Was unaware of the serum CPK values or the clinical condition of the infant. Autopsies were performed in all patients who died without ultrasound evidence of PIVH. URrasound or autopsy data were used to determine the presence or absence of PIVH. The extent Of PIVH was graded according to the method of Papile et al. l~ The grade of hemorrhage assigned was the maximum extent of bleeding present at 1 week of age. I f death occurred prior to 1 week of age, the grade' of hemorrhage found at autopsy or at the time of the last ultrasound study performed prior to death was assigned. Blood was obtained from the arterial catheter within the
first 12 hours after birth and serially every 24 hours thereafter for five days, or until death if the infant survived less than five days. A 0.7 ml sample was placed in ice and immediately centrifuged at 4 ~ C. The serum was then separated and stored at - 7 0 ~ C until analysis. Total CPK activity was measured, in duplicate, by a modified kinetic procedure after Rosalski, ~zusing a Worthington Statzyme Kit (Worthington Diagnostics, Freehold, N J) and a COBAS-BIO Centrifugal Analyzer (Roche Analytical Instruments, Nutley, N J). The portion of CPK activity contributed by each isoenzyme was assessed by electrophoresis,' followed b y fluorescent detection of enzyme activity, using a Beckman Paragon kit for CK isoenzyme measurement and a Beckman CDS 200 densitometer (Beckman Instruments, Fullerton, CA). Because of the low total activity in most of the sera tested, 6 #l'samples were used. When CPK activity exceeded 300 IU/L, a smaller sample volume was applied in the sample well. A control with known amounts of the three isoenzymes was run with each group of specimens. The data were analyzed by the Studeiit t test, chi-square analysis, or the Mann-Whitney U test, where appropriate. RESULTS Forty-eight infants were entered in the study. All infants weighed <1400 gm and were of --<32 weeks gestation. Thirty-three babies had PIVH and 15 did not, for a PIVH incidence of 69%. Two infants had grade 1 PIVH, eight grade 2, 11 grade 3, and 12 grad e 4. When the CPK-BB levels were examined, an extremely large range of values for each sample period was found (Table). When the first sample values were analyzed, there was a Significant difference in CPK-BB levels between infants who subsequently did or did not develop PIVH (P < 0.002 by Mann-Whitney U test). No differences were found between the two groups when the data from the second, third, fourth, or fifth samples (corrected for multiple comparison) were analyzed. No differences were found between the two groups when gestational age, mode
792
Clinical and laboratory observations
of delivery, Apgar scores at 1 and 5 minutes, and place of delivery (inborn vs outborn) were examined. The CPK-BB values were also examined in relationship to mode and place of delivery, and no differences were found in any of the sample periods. The infants with PIVH were significantly smaller than the infants without PIVH (mean _+ SEM 909.09 _+ 28.47 gm vs 1056 ___53.65 gm, P < 0.01 by Student t test). When the birth weights for each group were plotted on standard curves for gestational age, the weights of the infants with PIVH were significantly lower for gestational age. Second peak CPK-BB levels, defined as the highest serum value attained after the first 12 hours, were analyzed, and no significant differences between the two groups were found. Evidence of PIVH was found by ultrasound or autopsy in 18.2% (6/33) of the patients prior to 12 hours of life, 33.3% (11/33) by 24 hours of age, 54.5% (18/33) by 48 hours, 72.7% (24/33) by 72 hours, and 90.9% (30/33) by 96 hours of age. All 19 patients with first sample values >~19 U / L had PIVH. Thus, this level was 100% predictive of hemorrhage, although the obverse was not true. 'There was a significantly higher mortality during the first two weeks of life in the patients with PIVH with CPK-BB levels _>_19 U / L (13/19), as compared with the patients with PIVH with levels <19 U / L (3/14) and the 15 patients without PIVH, none of whom died during the study period (P < 0.001 by chi-square analysis). No significant differences were found in birth weight, gestational age, Apgar scores, and mode or place of delivery among the three groups. Further, no difference in the amount of hemorrhage could be found between infants with PIVH with first sample CPK-BB values ~ 1 9 or ~<19 U/L. All infants who died within two weeks of birth did so as a consequence of PIVH, with one exception; that infant had a first sample CPK-BB value of 2.69 U / L and grade 2 PIVH, and died secondary to necrotizing enterocolitis on day 12 of life. DISCUSSION Elevation of serum CPK-BB activity has been reported after perinatal asphyxia in both term 5,7 and preterm infants.6 In these same studies, particularly that of Walsh et al., 7 a strong association was noted between the level of CPK-BB activity and the degree of subsequent neurologic impairment. Our data extend the above observations to the VLBW premature infant who dies within two weeks after birth. As no differences could be found in the extent of hemorrhage between the infants wfi~hfirst sample CPK-BB values >~19 or <19 U/L, we believe that the level of CPK-BB may serve as a predictor of early mortality in these infants. Although other organs contain CPK-BB, L3the brain was
The Journal of Pediatrics November 1983
the most likely source of the elevated CPK-BB activity found in these premature infants. It is doubtful that the serum activity came from the lung, gastrointestinal tract, or placenta, as Cuestas 6 could not demonstrate elevation of this enzyme in neonates with purely pulmonary or gastrointestinal tract disease, and the placenta contains CPK-BB in such small amounts that its contribution to the newborn infant's blood would be negligible.~4 The incidence of PIVH in our patients was higher than that generally reported, ~j, ~5and was related most likely to the study design. We only studied infants of --<32 weeks gestation (mean _+ SD 27.9 + 1.65 weeks) and required that an arterial catheter be in place during the study. Thus, only the most seriously ill patients were examined. The infants with PIVH were significantly smaller than those without hemorrhage, even though other factors, including gestational age, were similar. Several explanations for this finding include the possibility that the same intrauterine mechanisms that lead to growth retardation, such as chronic hypoxia, might predispose this group of small premature infants to PIVH; that errors in the assessment of gestational age occurred in the infants who developed PIVH because alterations in the neurologic state resulting from the same insult that caused increased release of CPK-BB caused consistently higher estimates in Ballard scores; or that the infant who is small for gestational age has different problems in the immediate postnatal period, which predispose PIVH: Bejar et al. t~ found that 90% of PIVH occurred on the first day of life, usually in the first hours. Other authors suggest that PIVH occurs later? 5,16 Although We did not find as high an incidence of PIVH on the first day of life as did Bejar et al., ~~our data did demonstrate that more than 50% of the study patients had evidence of hemorrhage before 48 hours of life, and 90% have PIVH prior to 96 hours of age. Because the ultrasound examinations were performed on clinical grounds, it is possible that PIVH actually occura-ed earlier than was documented. However, our findings are in agreement with the data recently reported by Partridge et al. ~5 in a comparable series of VLBW infants. We hypothesize, therefore, that the elevated first sample CPK-BB levels reported here usually precede the appearance of PIVH, and reflect the occurrence of a perinatal event that results in cellular injury and release of CPK-BB into the circulation. This perinatai eyent is then followed by the appearance of PIVH without necessarily a further significant elevation of CPK-BB. Shields and Feldman8 also have reported elevated levels of CPK-BB in patients who later develop PIVH. However, they did not attempt to correlate mortality with the degree of serum elevation of the enzyme. They suggested that a second peak of CPK-BB activity follows the initial eleva-
Volume 103 Number 5
tion a n d t h a t this second peak occurs 12 to 36 hours a f t e r the clinical symptoms of P I V H . U n f o r t u n a t e l y , timing of P I V H could not be ascertained from the data presented in their study to substantiate this concept. Regardless of w h e t h e r a second peak of C P K - B B activity occurs, it appears clear that high levels of this isoenzyme are associated with a significant increase in mortality d u r i n g the first two weeks of life, and t h a t C P K - B B is a useful test to predict both the occurrence of P I V H and s h o r t - t e r m mortality in V L B W infants. We thank Drs. Arnold J. Rudolph and Marvin Fishman for their thoughtful review of the manuscript.
REFERENCES 1. Somer H, Kaste M, Troupp H, Kohttinen A: Brain creatine kinase in blood after acute brain injury. J Neurol Neurosurg Psychiatr 38:572, 1975. 2. Kaste M, Somer H, Konttinen A: Brain-type creatine kinase isoenzyme: Occurrence in acute cerebral disorders. Arch Neurol 34:142, 1977. 3. Phillips JP, Jones HM, Hitchcock R, Adams H, Thompson R J: Radioimmunoassay of serum creatine kinase BB as index of brain damage after head injury. Br Med J 281:777, !980. 4. Belton NR: Creatine phosphokinase: Blood and CSF levels in newborn infants and children (abst). Arch Dis Child 45:600, 1970. 5. Becker M, Menzel K: Brain-typical creatine kinase in the serum of newborn infants with perinatal brain damage. Acta Paediatr Scand 67:177, 1978. 6. Cuestas RA: Creatine kinase isoenzymes in high-risk infants. Pediatr Res 14:935, 1980.
Clinical and laboratory observations
793
7. Walsh P, Jedeikin R, Ellis G, Primhak R, Makela SK: Assessment of neurologic outcome in asphyxiated term infants by use of serial CK-BB isoenzyme measurement. J PED~ATR 101:988, 1982. 8. Shields WD, Feldman RD: Serum CK-BB isoenzyme in preterm infants with periventricular hemorrhage. J PEDIATR 100:464, 1982. 9. Ballard J, Kazmaier K, Driver M: A simplified assessment of gestational age. Pediatr Res 11:374, 1977. 10. Bejar R, Curbelo V, Coen RW, Leopold G, James H, Gluck L: Diagnosis and follow-up of intraventricular hemorrhage by ultrasound studies of infant's brain through the fontanelles and sutures. Pediatrics 66:661, 1980. 1 l. Papile L-A, Burstein J, Burstein R, Koffler H: Incidence and evolution hemorrhage in the high risk preterm infant: A study of infants with birth weights less than 1500 grams. J PED1ATR 92:529, 1978. 12. Rosalski SB: An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med 69:696, 1967. 13. Tsung SH: Creatine kinase isoenzyme patterns in human tissue obtained at surgery. Clin Chem 22:173, 1976. 14. Chemnitz G, Nevermann L, Schmidt E, Schmidt FW, Lorbers J: Creatine Kinase (EC 2.7.3.2) and creatine kinase isoenzymes during pregnancy and labor and in the cord blood. Clin Biochem 12:277, 1979. 15. Partridge JC, Babcock DS, Steichen J J, Han BK: Optimal timing for diagnostic cranial ultrasound in low birth weight infants: Detection of intracranial hemorrhage and ventricular dilation. J PEDIAq'R 102:281, 1983. 16. Tsiantos A, Victorin L, Relier JP, Dyer N, Sundell H, Brill AB, Stahlman M: Intracranial hemorrhage in the prematurely born infant: Timing of clots and evaluation of clinical signs and symptoms. J PEDIATR 85:854, 1974.
Longitudinal study of free thyroxine in low-birth-weight infants by paper disk method Masao Sakaguchi, M.D., Shigeyoshi Suzuki, M.D., Kanji Nagashima, M.D., Satoshi Shimano, M.D., Satoshi Uchida, M.D., and Takayoshi Kuroume, M.D. M a e b a s h i , Gunma, J a p a n
THE HIGH INCIDENCE of transiently low values for thyroid-stimulating h o r m o n e or thyroxine observed in low-birth-weight infants represents a potential pitfall in
From the Department of Pediatrics, Gunma University School of Medicine. Reprint requests: Shigeyoshi Suzuki, M.D., Department of Pediatrics, Gunma University School of Medicine, MaebashL Gunma, Japan
screening programs for congenital hypothyroidism t h a t are based on m e a s u r e m e n t of T 4 or T S H . Previous investigations have indicated t h a t free T4, the biologically active fraction of T4, provides a more accurate assessment of thyroid function t h a n does total T 4 and t h a t it is maintained at a slightly elevated level or within the n o r m a l r a n g e in L B W infants despite the low T4 concentrations. ~'2 T h e r e q u i r e m e n t for a r a t h e r large a m o u n t of blood has
Clinical and laboratory observations
Although the means by which heparin causes inhibition of the classical complement pathway has not been determined, there are several proposed sites of inhibition, including direct inhibition of Clq binding to immune complexes, inhibition of the interaction of C]~ with C4 and C2, and potentiation of the effect of C]- inhibitor function? Surface-associated heparin inhibits activation of the alternative pathway of complement by augmenting the regulatory action of the control proteins H and I in such a way that the formation and function of the amplification convertase C3bBb is prevented.4,5 Inhibitory activity of the effector sequence of complement by heparin also has been described. 4 The inhibitory effect of heparin on neutrophilmediated bactericidal activity is dose dependent and appears to be attributable primarily to the inhibition of ingestion.6 Because levels of both classical and alternative complement pathway components are lower in neonates than in older children and adults, ~2 the anticomplementary effect of heparin potentially could have caused significant inhibition of complement-dependent bactericidal activity in neonatal sera. Our data indicate that at the levels required for maintaining catheter patency and for achieving anticoagulation in neonates, the anticomplementary effect of heparin, as assessed by bactericidal activity of sera for type III, group B streptococcus, is not significant.
REFERENCES 1. McDonald MM, Hathaway WE: Anticoagulant therapy by continuous heparinization in newborn and older infants. J PEDIATR101:451, 1982.
The Journal of Pediatrics November 1983 2. McDonald MM, Jacobson LJ, Hay WW Jr, et al: Heparin clearance in the newborn, i'ediatr Res 15:1015, 1981. 3. Caughman GB, Boackle RJ, Vesely J: A postulated mechanism for heparin's potentiation of C1 inhibitor function. Mol Immunol 19:287, 1982. 4. Weiler JM, Yurt RW, Fearon DT, et al: Modulation of the formation of the amplification convertase of complement, C3bBb, by native and commercial heparin. J Exp Med 147:409, 1978. 5. Kazatchkine MD,Fearon DT, Silbert JE, et al: Surfaceassociated heparin inhibits zymosan-inducedactivation of the human alternative complement pathway by augmenting the regulatory action of the control proteins on particle-bound C3b. J Exp Med 150:1202, 1979. 6. Victor M, Weiss J, Elsbach P: Heparin inhibits phagocytosis by polymorphonuclear leukocytes. Infect Immun 32:295, 1981. 7. Edwards MS, Baker CJ, Kasper DL: Opsonic specificity of human antibody to the type 111 polysaccharide of group B Streptococcus. J Infect Dis 140:1004, 1979. 8. Edwards MS, Nicholson-WellerA, Baker CJ, et al: The role of the alternative complement pathway in opsonophagocytosis of type III, group B Streptococcus. J Exp Med 151:1275, 1980. 9. Baker CJ, Kasper DL, Tager IB, et al: Quantitative determination of antibody to capsular polysaccharide in infection with type III strains of group B Streptococcus. J Clin Invest 59:810, 1977. 10. Teien AN, Lie M: Evaluation of an amidolytic heparin assay method: Increased sensitivityby adding purified antithrombin III. Thromb Res 10:399, 1977. 11. Estes JW, Poulin PF: Pharmokinetics of heparin. Thromb Haemost 33:26, 1974. 12. Davis CA, Vallota EH, Forristal J: Serum complement levels in infancy: Age related changes. Pediatr Res 13:1043, 1979.
Creatine phosphokinase BB isoenzyme in very-low-birth-weight infants." Relationship with mortality and intraventricular hemorrhage Michael E. Speer, M.D., Ching-Nan Ou, Ph.D., Gregory J. Buffone, Ph.D., and Vickie L. Frawley, M.S. Houston, T e x a s From the Departments of Pediatrics and Pathology, Baylor Cgllege of Medicine, and the Section of Neonatology, Texas Children's Hospital. Presented in part at the Western Society for Pediatric Research, Carmel, California, February 1982, and at the American Society of Pediatrics and Society for Pediat~c~Research, Washington, D.C., May 1982. Reprint requests: Michael E. Speer, M.D., Department of Pediatrics, Baylor College of Medicine, 1200 Morsund Ave., Houston, TX 77030.
ELEVATED LEVELS OF CPK-BB have been demonstrated in serum after ischemic or traumatic brain injury in adult~ ~-3and after asphyxic insult in neonates?-7 Recently, Shields and Feldman8 reported significant elevations in serum CPK-BB in neonates with periventricular/intravenI
CPK PIVH
Creatinine phosphokinase Periventricular/intraventricular hemorrhage
[
Volume 103 Number 5
Clinical and laboratory observations
79 1
Table. CPK-BB activity With PIVH (U/L)
Sample period
Mean + S E M
1 2 3 4 5
41.44 + 8.41" 1.92 + 0.54 5.63 + 2.40 1.74 + 0.76 7.30 _+ 5.49
Without PIVH (U/L) Range
2.69 to 0.00 to 0.00 to 0.00 to 0.00 to
Mean + S E M
206.72 14.10 50.74 15.02 83.50
9.55 _+ 1.63 1.51 _+ 0.56 2.00 + 0.75 0.81 + 0.25 0.27 _+ 0.19
Range
0.00 to 0.00 to 0.00 to 0.00 to 0.00 to
18.01 6.93 6.29 2.27 1.41
*P < 0.002. tricular hemorrhage, as compared with infants without PIVH. Our study was undertaken to determine whether serum CPK-BB levels could be predictive of short-term outcome in a series of very-low-birth-weight neonates with and without PIVH. METHODS All newborn infants -<32 weeks' gestation, as determined b y maternal dates and Ballard scores, 9 who were admitted to the neonatal intensive 'care unit at Texas Children's Hospital, Houston, and who underwent arterial catheterization were considered for enrollment in the study. If maternal dates and Ballard Scores differed by more than two weeks, the gestational'age assigned was that of the Ballard score. Informed parental consent was obtained for the sequential blood sampling. In all infants --<32 weeks requiring care in the neonatal ICU, one or more ultrasound studies were performed routinely as part of the medical management On or before the sixth day of life. The timing of the ultrasound evaluation(s) was determined bY the attending neonatologisL who was unaware of the CPK-BB test results. Ultrasound imaging was done with a n ATL Mark 3 real-time digital sector scanner (Advanced Technical Laboratory, Bellevue, WA) using a 5 MHz transducer directed in the' sagittal and Coronal planes through the anterior fontanelle, as described by Bejar et al. 1~ The radiologist who performed and interpreted the ultrasound examinations Was unaware of the serum CPK values or the clinical condition of the infant. Autopsies were performed in all patients who died without ultrasound evidence of PIVH. URrasound or autopsy data were used to determine the presence or absence of PIVH. The extent Of PIVH was graded according to the method of Papile et al. l~ The grade of hemorrhage assigned was the maximum extent of bleeding present at 1 week of age. I f death occurred prior to 1 week of age, the grade' of hemorrhage found at autopsy or at the time of the last ultrasound study performed prior to death was assigned. Blood was obtained from the arterial catheter within the
first 12 hours after birth and serially every 24 hours thereafter for five days, or until death if the infant survived less than five days. A 0.7 ml sample was placed in ice and immediately centrifuged at 4 ~ C. The serum was then separated and stored at - 7 0 ~ C until analysis. Total CPK activity was measured, in duplicate, by a modified kinetic procedure after Rosalski, ~zusing a Worthington Statzyme Kit (Worthington Diagnostics, Freehold, N J) and a COBAS-BIO Centrifugal Analyzer (Roche Analytical Instruments, Nutley, N J). The portion of CPK activity contributed by each isoenzyme was assessed by electrophoresis,' followed b y fluorescent detection of enzyme activity, using a Beckman Paragon kit for CK isoenzyme measurement and a Beckman CDS 200 densitometer (Beckman Instruments, Fullerton, CA). Because of the low total activity in most of the sera tested, 6 #l'samples were used. When CPK activity exceeded 300 IU/L, a smaller sample volume was applied in the sample well. A control with known amounts of the three isoenzymes was run with each group of specimens. The data were analyzed by the Studeiit t test, chi-square analysis, or the Mann-Whitney U test, where appropriate. RESULTS Forty-eight infants were entered in the study. All infants weighed <1400 gm and were of --<32 weeks gestation. Thirty-three babies had PIVH and 15 did not, for a PIVH incidence of 69%. Two infants had grade 1 PIVH, eight grade 2, 11 grade 3, and 12 grad e 4. When the CPK-BB levels were examined, an extremely large range of values for each sample period was found (Table). When the first sample values were analyzed, there was a Significant difference in CPK-BB levels between infants who subsequently did or did not develop PIVH (P < 0.002 by Mann-Whitney U test). No differences were found between the two groups when the data from the second, third, fourth, or fifth samples (corrected for multiple comparison) were analyzed. No differences were found between the two groups when gestational age, mode
792
Clinical and laboratory observations
of delivery, Apgar scores at 1 and 5 minutes, and place of delivery (inborn vs outborn) were examined. The CPK-BB values were also examined in relationship to mode and place of delivery, and no differences were found in any of the sample periods. The infants with PIVH were significantly smaller than the infants without PIVH (mean _+ SEM 909.09 _+ 28.47 gm vs 1056 ___53.65 gm, P < 0.01 by Student t test). When the birth weights for each group were plotted on standard curves for gestational age, the weights of the infants with PIVH were significantly lower for gestational age. Second peak CPK-BB levels, defined as the highest serum value attained after the first 12 hours, were analyzed, and no significant differences between the two groups were found. Evidence of PIVH was found by ultrasound or autopsy in 18.2% (6/33) of the patients prior to 12 hours of life, 33.3% (11/33) by 24 hours of age, 54.5% (18/33) by 48 hours, 72.7% (24/33) by 72 hours, and 90.9% (30/33) by 96 hours of age. All 19 patients with first sample values >~19 U / L had PIVH. Thus, this level was 100% predictive of hemorrhage, although the obverse was not true. 'There was a significantly higher mortality during the first two weeks of life in the patients with PIVH with CPK-BB levels _>_19 U / L (13/19), as compared with the patients with PIVH with levels <19 U / L (3/14) and the 15 patients without PIVH, none of whom died during the study period (P < 0.001 by chi-square analysis). No significant differences were found in birth weight, gestational age, Apgar scores, and mode or place of delivery among the three groups. Further, no difference in the amount of hemorrhage could be found between infants with PIVH with first sample CPK-BB values ~ 1 9 or ~<19 U/L. All infants who died within two weeks of birth did so as a consequence of PIVH, with one exception; that infant had a first sample CPK-BB value of 2.69 U / L and grade 2 PIVH, and died secondary to necrotizing enterocolitis on day 12 of life. DISCUSSION Elevation of serum CPK-BB activity has been reported after perinatal asphyxia in both term 5,7 and preterm infants.6 In these same studies, particularly that of Walsh et al., 7 a strong association was noted between the level of CPK-BB activity and the degree of subsequent neurologic impairment. Our data extend the above observations to the VLBW premature infant who dies within two weeks after birth. As no differences could be found in the extent of hemorrhage between the infants wfi~hfirst sample CPK-BB values >~19 or <19 U/L, we believe that the level of CPK-BB may serve as a predictor of early mortality in these infants. Although other organs contain CPK-BB, L3the brain was
The Journal of Pediatrics November 1983
the most likely source of the elevated CPK-BB activity found in these premature infants. It is doubtful that the serum activity came from the lung, gastrointestinal tract, or placenta, as Cuestas 6 could not demonstrate elevation of this enzyme in neonates with purely pulmonary or gastrointestinal tract disease, and the placenta contains CPK-BB in such small amounts that its contribution to the newborn infant's blood would be negligible.~4 The incidence of PIVH in our patients was higher than that generally reported, ~j, ~5and was related most likely to the study design. We only studied infants of --<32 weeks gestation (mean _+ SD 27.9 + 1.65 weeks) and required that an arterial catheter be in place during the study. Thus, only the most seriously ill patients were examined. The infants with PIVH were significantly smaller than those without hemorrhage, even though other factors, including gestational age, were similar. Several explanations for this finding include the possibility that the same intrauterine mechanisms that lead to growth retardation, such as chronic hypoxia, might predispose this group of small premature infants to PIVH; that errors in the assessment of gestational age occurred in the infants who developed PIVH because alterations in the neurologic state resulting from the same insult that caused increased release of CPK-BB caused consistently higher estimates in Ballard scores; or that the infant who is small for gestational age has different problems in the immediate postnatal period, which predispose PIVH: Bejar et al. t~ found that 90% of PIVH occurred on the first day of life, usually in the first hours. Other authors suggest that PIVH occurs later? 5,16 Although We did not find as high an incidence of PIVH on the first day of life as did Bejar et al., ~~our data did demonstrate that more than 50% of the study patients had evidence of hemorrhage before 48 hours of life, and 90% have PIVH prior to 96 hours of age. Because the ultrasound examinations were performed on clinical grounds, it is possible that PIVH actually occura-ed earlier than was documented. However, our findings are in agreement with the data recently reported by Partridge et al. ~5 in a comparable series of VLBW infants. We hypothesize, therefore, that the elevated first sample CPK-BB levels reported here usually precede the appearance of PIVH, and reflect the occurrence of a perinatal event that results in cellular injury and release of CPK-BB into the circulation. This perinatai eyent is then followed by the appearance of PIVH without necessarily a further significant elevation of CPK-BB. Shields and Feldman8 also have reported elevated levels of CPK-BB in patients who later develop PIVH. However, they did not attempt to correlate mortality with the degree of serum elevation of the enzyme. They suggested that a second peak of CPK-BB activity follows the initial eleva-
Volume 103 Number 5
tion a n d t h a t this second peak occurs 12 to 36 hours a f t e r the clinical symptoms of P I V H . U n f o r t u n a t e l y , timing of P I V H could not be ascertained from the data presented in their study to substantiate this concept. Regardless of w h e t h e r a second peak of C P K - B B activity occurs, it appears clear that high levels of this isoenzyme are associated with a significant increase in mortality d u r i n g the first two weeks of life, and t h a t C P K - B B is a useful test to predict both the occurrence of P I V H and s h o r t - t e r m mortality in V L B W infants. We thank Drs. Arnold J. Rudolph and Marvin Fishman for their thoughtful review of the manuscript.
REFERENCES 1. Somer H, Kaste M, Troupp H, Kohttinen A: Brain creatine kinase in blood after acute brain injury. J Neurol Neurosurg Psychiatr 38:572, 1975. 2. Kaste M, Somer H, Konttinen A: Brain-type creatine kinase isoenzyme: Occurrence in acute cerebral disorders. Arch Neurol 34:142, 1977. 3. Phillips JP, Jones HM, Hitchcock R, Adams H, Thompson R J: Radioimmunoassay of serum creatine kinase BB as index of brain damage after head injury. Br Med J 281:777, !980. 4. Belton NR: Creatine phosphokinase: Blood and CSF levels in newborn infants and children (abst). Arch Dis Child 45:600, 1970. 5. Becker M, Menzel K: Brain-typical creatine kinase in the serum of newborn infants with perinatal brain damage. Acta Paediatr Scand 67:177, 1978. 6. Cuestas RA: Creatine kinase isoenzymes in high-risk infants. Pediatr Res 14:935, 1980.
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7. Walsh P, Jedeikin R, Ellis G, Primhak R, Makela SK: Assessment of neurologic outcome in asphyxiated term infants by use of serial CK-BB isoenzyme measurement. J PED~ATR 101:988, 1982. 8. Shields WD, Feldman RD: Serum CK-BB isoenzyme in preterm infants with periventricular hemorrhage. J PEDIATR 100:464, 1982. 9. Ballard J, Kazmaier K, Driver M: A simplified assessment of gestational age. Pediatr Res 11:374, 1977. 10. Bejar R, Curbelo V, Coen RW, Leopold G, James H, Gluck L: Diagnosis and follow-up of intraventricular hemorrhage by ultrasound studies of infant's brain through the fontanelles and sutures. Pediatrics 66:661, 1980. 1 l. Papile L-A, Burstein J, Burstein R, Koffler H: Incidence and evolution hemorrhage in the high risk preterm infant: A study of infants with birth weights less than 1500 grams. J PED1ATR 92:529, 1978. 12. Rosalski SB: An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med 69:696, 1967. 13. Tsung SH: Creatine kinase isoenzyme patterns in human tissue obtained at surgery. Clin Chem 22:173, 1976. 14. Chemnitz G, Nevermann L, Schmidt E, Schmidt FW, Lorbers J: Creatine Kinase (EC 2.7.3.2) and creatine kinase isoenzymes during pregnancy and labor and in the cord blood. Clin Biochem 12:277, 1979. 15. Partridge JC, Babcock DS, Steichen J J, Han BK: Optimal timing for diagnostic cranial ultrasound in low birth weight infants: Detection of intracranial hemorrhage and ventricular dilation. J PEDIAq'R 102:281, 1983. 16. Tsiantos A, Victorin L, Relier JP, Dyer N, Sundell H, Brill AB, Stahlman M: Intracranial hemorrhage in the prematurely born infant: Timing of clots and evaluation of clinical signs and symptoms. J PEDIATR 85:854, 1974.
Longitudinal study of free thyroxine in low-birth-weight infants by paper disk method Masao Sakaguchi, M.D., Shigeyoshi Suzuki, M.D., Kanji Nagashima, M.D., Satoshi Shimano, M.D., Satoshi Uchida, M.D., and Takayoshi Kuroume, M.D. M a e b a s h i , Gunma, J a p a n
THE HIGH INCIDENCE of transiently low values for thyroid-stimulating h o r m o n e or thyroxine observed in low-birth-weight infants represents a potential pitfall in
From the Department of Pediatrics, Gunma University School of Medicine. Reprint requests: Shigeyoshi Suzuki, M.D., Department of Pediatrics, Gunma University School of Medicine, MaebashL Gunma, Japan
screening programs for congenital hypothyroidism t h a t are based on m e a s u r e m e n t of T 4 or T S H . Previous investigations have indicated t h a t free T4, the biologically active fraction of T4, provides a more accurate assessment of thyroid function t h a n does total T 4 and t h a t it is maintained at a slightly elevated level or within the n o r m a l r a n g e in L B W infants despite the low T4 concentrations. ~'2 T h e r e q u i r e m e n t for a r a t h e r large a m o u n t of blood has