Plasma alkaline phosphatase activity and its relation to rickets in pre-term infants

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1 Ann Clin Biochem 1986; 23: Plasma alkaline phosphatase activity and its relation to rickets in pre-term infants E G WALTERS, J F MURPHY', P HENRYt, a P GRAY' and G HELDER From the Departmentsof Medical Biochemistry and Child Health, University Hospital ofwales, Cardiff and the i Departmentof Medical Biochemistry, Llandough Hospital, Cardiff, UK SUMMARY. Sequential measurements of plasma alkaline phosphatase activity were made in 84 pre-term infants delivered before 38 weeks gestation. n 67% of infants, without evidence of rickets but in whom three or more measurements were made and the peak activity was less than 10 times the adult reference range, the activity rose to a peak and declined to previous levels, or lower. The time taken for these changes varied greatly. The range of values was inspected at each week of age, and in all but six cases the level did not exceed 10 times the upper limit of the adult reference range. Three infants had radiological evidence of rickets, and of the six cases in whom higher levels were found, only one had radiological evidence of rickets; the remainder did not. t is recommended that in the latter, the high plasma alkaline phosphatase activity should be regarded as evidence of subclinical bone disease. Rickets is a well recognised cause of neonatal morbidity in pre-term infants, 1 2 although its pathogenesis remains obscure.? Recently, Kovar, Mayne and Barltrop" concluded from a study of 30 neonates, including four cases of rickets, that in the absence of liver disease a plasma alkaline phosphatase activity of less than six times the upper limit of the adult reference range obviates the need for radiological investigation. This has been disputed by Mcntosh et al. 5 who found radiological evidence of rickets in pre-term infants whose serum alkaline phosphatase levels were well within the suggested non-rachitic range. Kovar et al:" also suggested that a level of seven and a half times the upper limit of the adult reference range is compatible with active disease, and that a level of six times the adult range is an indication for an X-ray. n our experience, more than half the pre-term infants whose development gives no cause for concern have plasma alkaline phosphatase levels of this magnitude. n the newborn pre-term infant several factors complicate the interpretation of plasma Correspondence: Dr E G Walters, Department of Chemical Pathology, Level 9, Bristol Royal nfirmary, Bristol BS2 8HW, UK. 652 alkaline phosphatase activities. There is a lack of published reference data.v " Furthermore, plasma alkaline phosphatase activities change during the first few weeks of extra-uterine life so that infants of different ages, and even infants of the same post-delivery age but with different post-conceptual ages, may not be comparable." We have examined the plasma alkaline phosphatase levels found in 84 pre-term babies, including three cases of rickets, in relation to post-delivery and post-conceptional age. Patients nfants born after less than 38 weeks gestation," who were admitted to the Special Care Baby Unit between February 1982 and July 1983, were studied. All were given daily dietary supplements of vitamins (Abidec drops, 0 6 ml, which includes 400 units of Calciferol), and infants of birth weight less than 1 8 kg received additional daily supplements of calcium (10% calcium gluconate, 5 mukg) and also Calciferol, 800 units; the Calciferol was increased to 0 units daily when the plasma alkaline phosphatase activity exceeded six times the upper limit of the adult normal range. Blood samples were obtained from 84 in-

2 Plasma alkaline phosphatase and rickets 653 fants. Serial samples were obtained from the majority, but in 15 cases only one sample was obtained because they were discharged after the first week; in these, the plasma alkaline phosphatase was less than five times the upper limit of our adult reference range (115 lull). Methods Plasma alkaline phosphatase activity was measured using the method of Bessey, Lowry and Brock," adapted to a centrifugal analyser; the between-batch coefficient of variation was 2%. The upper limit of the adult normal range is 115 lull (95% confidence intervals). Different alkaline phosphatase isoenzymes were assessed by visual inspection of the bands obtained by electrophoretic separation on cellulose acetate strips, after staining with fast violet blue salt. The statistical significances of differences between groups were assessed by the Mann Whitney U test. Results The plasma alkaline phosphatase activities of the 84 infants ranged from 121 lull to 2160 lull. Three infants had radiological evidence of rickets, and five others had peak activities greater than 10 times the upper limit of the adult normal range. Exclusion of these cases gives a range of lull. All of the latter were divided into three groups according to the gestational age of the child at delivery and are displayed according to age post-delivery in Figs 1, 2 and 3. The data for those infants with peak plasma alkaline phosphatase activities less than 10 times the adult normal range, who were delivered before 31 weeks gestation (Fig. 1), are similar to the data for those delivered between 31 and 34 weeks gestation (Fig. 2). n each figure the mean values for the first week of life are significantly lower than those recorded in the second week of life (P<0 05). Also, in Fig. 1 the mean value recorded at the third week of life is significantly lower than that for the second week (P<O 01). None of the means for other ages differed significantly from the mean for the second week of life in either group. Moreover, the mean value at each week of age in Fig. 1 is not significantly different from the mean of the corresponding age group in Fig. 2 (P>O 05), except again for the third week (P<O O). The apparent difference between the values recorded in the third week of life may be due to the much smaller numbers of samples from infants delivered before 31 weeks gestation. For those delivered between 35 and 38 weeks gestation (Fig. 3), the alkaline phosphatase levels lie within the ranges exhibited by the other groups, but the number of values in this category is too small to allow examination of any possible trend with age post-delivery. The sequential changes in plasma alkaline phosphatase were similar to those previously reported." Of those without rickets who had :J.,....~ ~ E.g., ~1l : ; -l. O.s;;;;...L. --!-. J... t 1.L Fill :r s: 0. o c-, 1000 ~ c":;: - r a Post-de 1ivery oge (weeks) FG. 1. Total plasma alkaline phosphatase activities for infants without evidence of rickets, delivered before 31 weeks of gestation, with an activity of less than 10 times the upper limit of the adult normal range. ( ) Mean value; ( ) upper limit of the adult reference range.

3 654 Waiters et al... lift!- 11 :.. T ~- ~ o 246 Post-del ivery age (weeks) 8 10 FG. 2. Total plasma alkaline phosphatase activities for infants without evidence of rickets delivered after weeks gestation, with an activity of less than 10 times the upper limit of the adult normal range. ( ) Mean value; ( ) upper limit of the adult reference range o Post-del ivery age (weeks) 8 FG. 3. Total plasma alkaline phosphatase activities for infants without evidence of rickets delivered between 35 and 38 weeks of gestation, with an activity of less than 10 times the upper limit of the adult normal range. ( ) Upper limit of the adult reference range. three or more measurements, 31 (67'0%) showed a rise to a peak and then a fall to below the initial level. The height of the peak was variable and occurred anywhere between 2 and 14 weeks of age, although in most cases it was reached by the age of 4 weeks, irrespective of the period of gestation. n six cases (13%), the level rose progressively until the infant was discharged and in six others it did not change. n three cases (7%) the levels fell progressively to the 40th week post-conception. Of the remaimng eight infants, three had radiological evidence of rickets as categorised by Koo et ai. l ll They were delivered at gestation periods of 28, 32 and 34 weeks with birth weights of 1 0,1 4 and 1 7 kg respectively. The sequential changes of their plasma alkaline phosphatase activities are shown in Table 1. The remaining five infants all had plasma alkaline phosphatase activities above times the upper limit of the adult normal range, and none of the X-ray changes of rickets appeared.

4 Plasma alkaline phosphatase and rickets 655 TABLE 1. Peak plasma alkaline phosphatase (PAP) activities of infants either with X-ray changes of rickets or with an activity of greater than 10 times the upper limit of the adult normal range Patient no. 1* 2* 3* "Signifies X-ray changes of rickets. The ages of infants in weeks post-delivery at the time of the measurement are shown in parentheses. Thus, no firm diagnosis of the cause of the elevated levels of alkaline phosphatase can be made, but all were of bone isoenzyme. One of these infants did, however, suffer from chest infections and developed pectus excavatum. The sequential changes in alkaline phosphatase levels of these infants are shown in Table 1. n each of the three infants with rickets, the initial plasma alkaline phosphatase levels measured 1 or 2 weeks after birth were within the ranges shown in Figs 1-3. n two of them, the plasma alkaline phosphatase did not rise from its initial low level until after 5 weeks after delivery and a peak value 1362 lull was not reached until 17 weeks after delivery (Table 1). n the five infants without radiological evidence of rickets but with levels greater than 10 times the adult reference range, the sequential changes are very similar to those shown in Table 1. ndeed, even though the patients appeared well clinically and showed no radiological evidence of rickets, the levels are higher than in some of the patients with radiological signs. n all eight of these infants, the plasma alkaline phosphatase levels remained high, or even continued to rise, despite an oral intake of 0 units of vitamin D 2 (Calciferol) daily and calcium supplements. n all cases it returned to lower, physiological levels after many weeks. Discussion nitial PAP activity 960 (3) 457 (2) 781 (2) 904 (2) 713 (3) 584 (1) 839 () 1146 (2) Highest peak PAP activity 1075 (4) 966 (7) 1362 (17) 1800 (17) 1883 (17) 2133 (12) 1314 (3) 1260 (3) Subsequent PAP activity 1089 (5) 566 (17) 570 (20) 760 (32) 393 (34) 1234 (20) 857 (7) 912 (17) Our results are similar to those of Kovar et al," but there are some quantitative differences. Our ranges are wider, and 31% of our cases, whose development was unremarkable, had a peak level of more than six times the upper limit of the adult range, the level above which Kovar et al. consider X-rays to be indicated. Our findings that the range of plasma alkaline phosphatase activities, at least in those born before 36 weeks gestation, is not influenced either by gestational age at birth or post-delivery age, allows the use of a single reference range in pre-term infants. However, the considerable overlap of the levels found in those with and without radiological evidence of rickets means that a value within the upper part of the reference range does not exclude rickets. The infants with clinical or radiological evidence of rickets all ultimately attained a level of plasma alkaline phosphatase activity more than nine times the upper limit of the adult normal range. However, radiological changes of rickets have been reported in association with alkaline phosphatase levels lower even than the mean for babies without X-ray changes.p Some of our cases of rickets also had alkaline phosphatase levels within the reference range for several weeks before they increased. No comment was made by Mcntosh et al." on the value of serial alkaline phosphatase measurements, the importance of which has been stressed by others" and is well illustrated in Table 1. By far the highest alkaline phosphatase levels we saw were in some of the patients without radiological changes. At present the significance of this is unclear. The changes differ from the rare condition of hyperphosphatasemia of infancy.f 9 The latterhas been described in older children and is characterised by an abnormal isoenzyme resembling the liver isoenzyme. n all five cases, the very high levels of alkaline phosphatase were due to an increased bone isoenzyme. Although no bone biopsies were done, it would seem prudent in the light of present knowledge to regard such persistently high levels of bone alkaline phosphatase as indicating sub-clinical metabolic bone disease. The very high plasma enzyme levels developed, whether or not there was overt evidence of rickets, despite the administration of 1 units of Calciferol daily from birth and remains high after increasing the dose to 0 units daily in all cases. This may have been due to defective hydroxylation of Calciferol, as has been suggested on the basis of measurements of plasma 1,25-dihydroxycholecalciferol in the pre-term infant.!g-13 This enzyme defect may be common but of very short duration in preterm infants, and without serious adverse effect in the majority.

5 656 Walters et al. t seems that both the estimation of total plasma alkaline phosphatase and radiological examination are required to detect early metabolic bone disease in pre-term infants, and both have their limitations. But whereas it is easy to make serial measurements of plasma alkaline phosphatase for screening purposes it is doubtful whether repeated X-ray examination of these children would be acceptable. Frequent measurements of plasma alkaline phosphatase should be made, preferably weekly whilst the baby is in hospital, even in the absence of clinical signs of rickets. Radiological examinations should be reserved for cases in which the condition is suspected clinically because of a high plasma alkaline phosphatase activity. Whether the persistence of very high alkaline phosphatase levels without radiological changes always presages the development of clinical rickets cannot be determined from the information presently available. However, the index of suspicion must be high in neonates delivered before the 30th week of gestation and in those infants with subsequent illness, as these are at the highest risk of developing rickets. Acknowledgements We wish to thank Miss S Muxworthy for collection of the blood samples. Also Dr D Bingham, Department of Chemical Pathology, Bristol Royal nfirmary, for his assistance in the analysis of the data. References 1 Kullarni PB, Hall RT, Rhodes PS et al. Rickets in very low birth weight infants. 1 Pediatr 1980; 96: Callenbach lc, Sheehan MB, Abramson Sl, Hall RT. Etiological factors in rickets of very low birth weight infants. 1 Pediatr 1981; 98: Tsang RC. The quandry of vitamin D in the newborn infant. Lancet 1983; : Kovar, Mayne P, Barltrop D. Plasma alkaline phosphatase activity: a screening test for rickets in preterm neonates. Lancet 1982; i: Mcntosh N, Williams le, Lyon Al, Wheeler KA. Diagnosis of rickets of prematurity. Lancet 1984; ii: Ballard 1, Kazmaier K, Driver M. A simplified assessment of gestational age. Pediatr Res 1977; : Bessey OA, Lowry OH, Brock Ml. A method for rapid determination of alkaline phosphatase with five cubic millimetres of serum. 1 Bio Chern 1946; 164: Steinherz PG, Steinherz U, Nisseibaum rs, Murphy L. Transient, marked, unexplained elevation of serum alkaline phosphatase. lama 1984; 252: Rosalki SB, Foo AY. More on transient hyperphosphatasaemia of infancy. Clin Chern 1983; 29: Koo WWK, Gupta 1M, Nayanar VV et al. Skeletal changes in pre term infants. Arch Dis Child 1982; 57: Walters EG, Murphy lf, Brown RC et al. Vitamin D metabolism in rachitic pre term infants. Lancet 1983; : Hillman LS, Haddad lg. Perinatal vitamin D metabolism. 1 Pediatr 1975; 86: Seino Y, shii T, Shimotsuji T et al. Plasma active vitamin D concentration in low birthweight infants with rickets and its responses to vitamin D. treatment. Arch Dis Child 1981; 56: Accepted for publication 16 April 1986