Vitamin D and mineral metabolism in ' i very low birth weight infant recelv ng

400 IU of vitamin D

the

Richard C o o k e , MD, Bruce Hollis, MD, Cynth ia Conner , RN, Donna Watson, RN, Susan Werkman, RN, and Russell Chesney, MD

From the Departments of Pediatrics and Obstetrics and Gynecology, University of Tennessee, Memphis, and the Department of Pediatrics, Medical University of South Carolina, Charleston

Study objective: To examine (I) the ef fect of vitamin D intake (380 to 480 IU dai ly) on plasma 25-hydroxyvitamin D (25-0HD) and 1,25-dihydroxyvitamin D (I,25- [OH]2D) concentrat ions and (2) the relationship of 1,25-(OH)2D to calc ium and phosphorus absorpt ion and retention in the very low birth weight infant receiv- ing a preterm infant formula. Subjects: Eleven "we l l " infants with a birth weight and gestat ional age (mean _+ SD) of 1,078 _+ 128 gm and 29 • 1.9 weeks, respectively, were studied for a 3-week period. Weight and postnatal age (mean _+ SD) at the beginning of the study were 1132 • 56 gm and 16 • 6 days, respectively. All infants were fed a preterm infant formula and tolerated a full enteral intake (120 kca l / kg /day ) for the duration of the study. Interventions: Plasma 25-0HD and 1,25-(OH)2D concentrat ions were measured at the beginning of the study and at the beginning of each 48-hour ba lance period. Calcium and phosphorus ba lance studies (n = 33) were performed weekly. Main results: Plasma 25-0HD (30 _+ 10 ng/ml) and 1,25-(OH)2D (54 _+ 14 pg/ml) concentrat ions were normal at the beginning of the study. Plasma 25-0HD val- ues did not change, but 1,25-(OH)2D values increased (p <0.001) throughout the study. Plasma 1,25-(OH)2D concentrat ions were not related to calc ium or phos- phorus absorpt ion and retention, but were a l inear function of postconcept iona l age. Conclusions: Normal vitamin D status and act ivi ty are maintained in the very low birth weight infant fed a high calc ium formula (380 to 480 IU of vitamin D daily). Plasma 1,25-(OH)2D concentrat ions are not related to calc ium absorpt ion but are l inearly related to maturity. (J PEDIATR 1990;116:423-8)

The daily requirements of vitamin D for the very low birth weight infant are not completely established, and the effect of vitamin D supplementation on vitamin D status is com- plex. The ultimate action of vitamin D is to increase intes- tinal calcium absorption, 1 but the relationship between plasma 1,25-dihydroxyvitamin D concentrations and cal- cium absorption has not been described in the VLBW infant

Submitted for publication May 17, 1989; accepted Sept. 18, t989. Richard J. Cooke, MD, Newborn Center, 853 Jefferson Ave., Suite 201, Memphis, TN 38163. 9/23/16794

receiving recommended intakes of vitamin D, calcium, and phosphorus. The purpose of this study was to explore the influence of vitamin D on plasma 25-hydroxyvitamin D and 1,25-(OH)2D concentrations, and to study further the rela-

I 1,25-(OH)2D 1,25-Dihydroxyvitamin D I 25-OHD 25-Hydroxyvitamin D I VLBW Very low birth weight

tionship of 1,25-(OH)2D to calcium and phosphorus ab- sorption and retention in the VLBW infant receiving pre- term formula.

423

4 2 4 Cooke et al. The Journal of Pediatrics March 1990

Table I. Anthropometr ic and biochemical results

A g e (days

15 • 6 20 • 6 27 _+ 6 33 • 6 Weight (gm) 1132 + 56 1245 _+ 62 1420 _+ 81 1607 _+ 89 Length (cm) 37.9 • 1.2 38.5 _+ 1.1 39.5 _+ 0.9 40.7 _+ 0.9 Calcium (mg/dl) 9.4 • 0.7 9.8 +_ 0.3* 9.8 _+ 0.4 9.3 _+ 0.8

(mmol/kg) (2.4 + .18) (2.5 +_ .08) (2.5 _+ .10) (2.3 _+ .20) Phosphorus (mg/dl) 5.3 + 0.8 5.9 + 0.4* 5.8 • 0.3* 5.8 _+ 0.8*

(mmol/kg) (1.7 _+ .26) (1.9 + 1.3) (1.9 • 1.0) (1.9 +_ .26) Alkaline phosphatase (IU) 356 +_ 96 408 + 120 387 _+ 130 371 _+ 84

(~kat/L) (5.9 _+ 1.6) (6.8 • 2.0) (6.5 + 2.2) (6.2 • 1.4)

Values are expressed as mean + SD. *p <0.05 as determined by repeated measures analysis of variance.

M E T H O D S

Infants weighing <1250 gm at birth and-born at <32 weeks of gestation were considered eligible for the study. Those with evidence of respiratory, neurologic, renal, car- diovascular, hepatic, or gastrointestinal disease or of a con- genital anomaly were excluded. Gestation was determined by means of maternal dates and the Dubowitz examination. If gestational age as determined by maternal dates differed by more than 2 weeks from that obtained by the Dubowitz examination, the latter was used. The study was approved by the University of Tennessee, Memphis, Institutional Review Board. Informed consent was obtained from the parents or guardian. None of the infants received parenteral nutrition or diuretics during the study.

Infants received Similac Special Care formula (Ross Laboratories, Columbus, Ohio) throughout the study. For- mula intake was advanced in a stepwise fashion. Infants who tolerated a minimal enteral intake of 100 kcal/kg/day with an appropriate weight gain (>10 gm/day) by 21 days of age were studied. Enteral intake was usually maintained at 110 to 120 kcal/kg/day. Vitamin D2 supplementation (200 IU daily) was begun at the same time as enteral feed- ing. At full enteral intake (150 ml/kg/day), infants re-

ceived 380 to 480 IU of vitamin D2 daily. The study lasted 3 weeks. During each week, nutrient in-

take and growth were monitored, a blood sample was drawn, and a nutrient balance study was performed. Weight, length, and head circumference were measured immedi- ately before the study began and at the beginning and end of each balance study with methods previously described} Nutrient balance studies were performed with the method previously described. 2 Nutrient intake was calculated from the volume of formula ingested and the concentration of nutrient in the formula. Absorption was defined as the dif- ference between intake and fecal excretion. Retention was defined as the difference between intake and fecal plus uri- nary excretion. Formula, stool, and urine were analyzed for

calcium and phosphorus as previously described} Analyses were done in duplicate and the average value was taken. Formula for the study was manufactured in a single batch. Bottles were chosen at random for nutrient analysis.

Blood sampling was performed immediately before the study began and at the beginning of each balance study. Plasma was separated immediately. Calcium and phospho- rus analyses were performed colorimetrically on an Ektachem 400 analyzer (Eastman Kodak Co., Rochester, N.Y.). Plasma for vitamin D analysis was stored immedi- ately at - 70 ~ C until analysis. Vitamin D assays were per- formed as previously described. 3,4 To determine 1,25-

(OH)2D, we extracted serum samples with acetonitrile. After centrifugation, the supernatant was decanted into a tube containing 0.4 mol/L potassium phosphate (pH = 10.6) and mixed by vortex. The extract was applied to a silica cartridge for final purification of 1,25-(OH)zD, which was measured by means of a radioreeeptor assay. 4 This method is sensitive to 1.0 pg/tube. To determine lev- els of 25-OHD, we also extracted serum with acetonitrile. The sample was then subjected to direct radioimmunoassay using an antibody generated against 23,24,25,26,27-pen- tanor-(22)carboxylic acid. 3 The detection limit of this assay is 3 ng/ml serum.

Nutrient balance results were expressed as milligrams per kilogram per day and as the percentage of intake. Changes in balance variables (e.g., intake, urine, stool), plasma 25-OHD, plasma 1,25-(OHhD, and calcium and phosphorus levels were analyzed by repeated measures analysis of variance. The relationship of mineral absorption and retention to vitamin D levels was analyzed by simple linear regression. The relationship of birth weight, gesta- tion, postnatal age, balance weight, balance length, plasma calcium levels, and plasma phosphorus level to mineral ab- sorption and retention and to vitamin D levels was analyzed by a stepwise regression procedure. All results were consid- ered significant at p < 0.05.

Volume 1l 6 Vi tamin D and mineral metabol i sm in VLB W infants 4 2 5 Number 3

r

o) r

0 0

C

E

>

r

E

Or.

8 0

75 �84

c- O 70

65

60

5 5 {

50

45

40

35

30

25

20 12

t. }. �9 1 , 2 5 ( O H ) 2 D ( p g / m l )

o "25 (OH) D ( n g / m l )

{ {

1 '6 2"0 24 2'8 3'2 36

Postnatal Age (days)

Fig. t. Plasma vitamin D concentrations as a function of postna- tal age in VLBW infants. Asterisk indicates increase of plasma 1,25-(OH)2D values with postnatal age (p <0.001 ).

R E S U L T S

Eleven infants (eight black, three white) were studied. Birth weight and gestational age (mean + SD) were 1078 _+ 128 gm and 29.2 _+ 1.7 weeks, respectively. Post- natal age and weight at the beginning of the study were 15 _+ 6 days and 1132 + 56 gin, respectively. Anthropo- metric and biochemical results are presented in Table I. The average daily increase in weight was 25 _+ 5 gm and in length was 0.t5 +_ .05 cm. Plasma calcium concentration increased (p <0.05) between 15 and 20 days but returned to baseline by 33 days of age, Plasma phosphorus values in- creased (p <0.001) during the study; the increase occurred between 15 and 20 days of age and remained at the higher level until the end of the study. Plasma alkaline phosphatase activity did not change during the study.

Plasma levels of 25-OHD tended to increase (p = 0.10) daring the study (Fig. 1); the increase occurred between 15 and 21 days but returned to baseline by 33 days of age. Plasma 1,25-(OH)2D concentrations increased (p <0.001) during the study; the increase occurred between 15 and 20 days of age and continued throughout the study. Plasma 25-OHD concentrations were not significantly related to 1,25-(OH)2D concentrations, birth weight, gestation, post- natal age, or plasma calcium or plasma phosphorus values. Plasma 1,25-(OH)~D concentrations were not related to

birth weight, plasma calcium levels, plasma phosphorus levels, calcium intake, or phosphorus intake. However, plasma t,25-(OH)2D concentration was related to gestation (r = 0.46; p <0.005) and postnatal age (r = 0.32; p <0.05)

120

o ,.---, 110

o

t 0 0

o

90 o o

8o ~ o o o o n-

O ' 70 o oO o

~ " 60 o o o ~

,,i,,,- o

OF) 40 0~

~ . y = - 154 + 0 . 9 6 x

30 o R 2 = 0 .55 , p = .001

20 �9 J �9 t �9 i . I . i . i . i . i �9 E �9 i �9 i �9 i 200 205 210 215 220 225 230 235 240 245 250 255 260

Postconceptional Age (days)

Fig. 2. Relationship of plasma 1,25-(OH)2D to postconceptional age in VLBW infants.

and was a linear function of postconceptional age (p <0.001; R 2 = 0.55). This relationship was described by the equation y = -154 + 0.96x, where y = 1,25(OH)2D con- centration and x = postconceptional age (Fig. 2). Fifty-five percent of the variation in plasma 1,25-(OH)2D concentra, tions was explained by variation in postconceptional age.

Calcium and phosphorus intake (expressed as milligrams per kilogram per day) increased (p <0.05) during the study (Table I1). Stool and urinary calcium and phosphorus ex- cretion did not change, and no significant changes were noted in calcium and phosphorus absorption and retention. Plasma 25-OHD and 1,25-(OH)2D concentrations were not related to stool calcium, absorption of calcium (r = 0.14; p = NS) and phosphorus, or retention of calcium and phos-

phorus.

D I S C U S S I O N

The relationship between vitamin D intake and plasma 25-OHD concentration is controversial. Early studies sug- gested that plasma 25-OHD concentrations are low (<15 ng/ml) 5'6 and do not increase with 400 IU vitamin D supplementation.6,7 Other studies suggested that plasma

25-OHD increases to normal concentrations (20 to 40 ng/ ml) with vitamin D supplementation. 8q4 However, tlie level of vitamin D supplementation varied (400 to 2000 IU daily), and conclusions regarding optimum vitamin D intake differed. Thus the American Academy of Pediatrics 15 recommends a daily vitamin D intake of 400 IU, Whereas the European Society for Paediatric Gastroen- terology and Nutrition 16 recommends 800 to 1600 IU.

The results of this study show that a daily vitamin D in-

4 2 6 Cooke et al. The Journal o f Pediatrics March 1990

Table II. Minera l balance results in V L B W infants receiving a p re te rm infant formula

Balance at a g e Balance of a g e Balance at age 20 • 6 days 27 _+ 6 days 33 • 6 days

Calcium Intake (mg/kg/day) Urine (mg/kg/day) Stool (mg/kg/day) Absorption (mg/kg/day) Retention (mg/kg/day) Absorption (%) Retention (%)

Phosphorus Intake (mg/kg/day) Urine (mg/kg/day) Stool (mg/kg/day) Absorption (mg/kg/day) Retention (mg/kg/day) Absorption (%) Retention (%)

219 -+ 6 223 _+ 2 225 _+ 6" 4.4_+ 2 4.5 +_ 2 4.4_+ 2 133 _+ 18 141 _+ 26 145 _+ 12 87 _+ 19 81 ___ 26 81 +_ 14 82 _+ 19 77 _+ 26 76 _+ 13 40_+ 8 37 _+ 12 36 • 6 37_+ 8 35 _+ 11 34 + 5.3

117 _+ 3 118 _+ 1 120 _+ 4* 8_+4 10_+6 1 2 + 8

43 -+ 7 45 -+ 10 44 -+ 6 73 _+ 8 73 _+ 11 76-+ 7 65_+ 5 63-+ 6 64+ 5 63 -+ 6 62-+ 9 64_+ 5 56-+ 4 53 • 6 53-+ 6

Values are expressed as mean _+ SD. *p <0.05 as determined by repeated measures analysis of variance.

take of 380 to 480 IU maintains normal plasma 25-OHD

concentrations (in comparison with values in older children

and adults 17) but should be interpreted with caution. Vita-

min D requirements are a function of fetal stores, which are

dependent on maternal vitamin D status isz~ and are accu-

mulated during the last trimester. If maternal vitamin D

status is poor or infants are delivered very prematurely, then

the infant's vitamin D requirements may be increased.

Study infants received 200 IU of D daily from 4 to 6 days

of age. Normal plasma 25-OHD concentrations at 15 _+ 6

days of age suggest that fetal status was adequate in this

group of infants. Such may not have been the case in other

studies, in which plasma 25-OHD concentrations were low

and increased slowly despite a high level of vitamin D intake (1200 to 2100 IU/day). 8, 10-12

The results also indicate that plasma, 1,25-(OH)2D con-

centrations increase with gestation, postnatal age, and

postconceptional age. Renal 1-hydroxylase activity and

1,25-(OH)zD production are affected by growth, plasma

calcium, and plasma phosphorus. 21 In this study, no rela-

tionship could be detected with growth, plasma calcium, or

plasma phosphorus. Increased plasma 1,25-(OH)zD con-

centrations have also been related to mineral intake. 22 No

relationship could be detected between plasma 1,25-

(OH)2D and calcium-phosphorus intake. Thus, although

the mechanism is unclear, maturation appears to be an im-

portant regulator of renal 1-hydroxylase activity and 1,25-

(OH)zD production. Whether maturation is rate limiting

needs to be examined.

No relationship was noted between plasma 1,25-(OH)2D

concentrations and intestinal calcium absorption. Calcium

absorption is the sum of two mechanisms: "active" (vitamin

D-mediated), which is operative at low calcium intakes, and

"passive," which is operative at high calcium intakes. These

infants received a high calcium intake (220 mg/kg/day), so

the passive pathway may have been the primary mechanism

of absorption. 23 The newborn rat is born with a decreased

number of cell receptors for 1,25-(OH)2D, 24, 25 without

functional intestinal calcium-binding protein 26 and without

an "active" calcium transport mechanism. 27, 28 If such is the

case in the human infant, plasma 1,25-(OH)2D levels may

not be paralleled by changes in "active" calcium transport.

"Net" absorption, as determined by nutrient balances, does

not reflect "true" absorption because fecal calcium is both

dietary and endogenous in origin. Thus plasma 1,25-

(OH)2D levels may have been paralleled by changes in

"true" but not "net" calcium absorption. Additional stud-

ies are needed to examine this possibility.

Previous studies have suggested that VLBW infants fed

Similac Special Care formula accreted calcium at intra- uterine rates (120 to 150 mg/kg/day). 14, 29 Calcium ab-

sorption was less in this study compared with previous

studies, and retention (75 mg/kg/day) was less than that in

utero. The difference in absorption is not explained easily.

One possibility is that calcium absorption was overesti-

mated in the previous studies. Sedimentation of calcium was

noted in the bottle and in the feeding set. 14 This loss varied

from 2.4% to 12.2% 14 and was taken into account during

calculations. However, a subsequent study demonstrated up

to 50% loss of calcium under similar conditions. 3~ If the ef-

fects of sedimentation were underestimated, absorption

would be overestimated. Significant sedimentation was not

Volume 116 Vi tamin D and mineral metabol ism in VLB W infants 4 2 7 Number 3

observed during our study. To prevent any loss of minerals,

we rinsed all bottles and feeding sets with distilled, deion-

ized water and fed the r inse to the infant. 2

Another possibility is tha t calcium absorption was de-

creased in this study. This decrease may reflect a difference

in calcium bioavailabili ty because the calcium salts in Sim-

ilac Special Care infant formula have been changed since

the previous studies. However, a net calcium absorption rate

of 38% is comparable to tha t observed previously in p re te rm

infants fed a pre term formula (42%) 31 and is consistent with

a " t rue" calcium absorption rate of 80%, as has been shown

with stable isotope techniques. 32

Although calcium retent ion was less than tha t in utero,

biochemical evidence of rickets did not develop in any of the

infants. The absence of rickets is consistent with previous

studies tha t have shown normal bone mineral izat ion in

V L B W infants fed Similac Special Care formula. 33 Bio-

chemical indexes are an early indicator of bone

mineralization,34 36 so it appears tha t adequate bone min-

eralization occurred when calcium and phosphorus reten-

tion were 80 and 65 m g / k g / d a y , respectively. However,

fur ther studies are needed to examine mineral requirements

and the relationship of minera'l absorpt ion and retent ion to

bone mineral izat ion in the V L B W infant.

R E F E R E N C E S

1. Nordin BEC, ed. Calcium in human biology. London: Spring- er-Verlag, 1988.

2. Cooke R J, Perrin F, Moore J, Paule C, Ruckman K. Method- ology of nutrient balance studies in the preterm infant. J Pe- diatr Gastroenterol Nutr 1988;7:434-40.

3. Hollis BW, Napoli JL. Improved radioimmunoassay for vita- min D and its use in assessing vitamin D status. Clin Chem 1985;31:1815-9.

4. Hollis BW. Assay of circulating 1,25(OH)2D involving a novel single-cartridge extraction and purification procedure. Clin Chem 1986;32:2060-3.

5. Rosen J, Roginsky M, Nathenson G, Finberg L. 25-Hydrox- yvitamin D: Plasma levels in mothers and their premature in- fants with neonatal hypocalcemia. Am J Dis Child 1974; 127:220-3.

6. Hillman L, Haddad J. Human perinatal vitamin D metabo- lism. 11. Serial 25-hydroxyvitamin D concentration in sera of term and premature infants. J PEDIATR 1975;86:928-35.

7. Hillman LS, HoffN, Salmons S, et al. Mineral homeostasis in very premature infants: serial evaluation of serum 25-hydrox- yvitamin D, serum minerals and bone mineralization. J PEDI- ATR 1985;106:970-80.

8. Wolf H, Graft V, Offerman G. The vitamin D requirements for premature infants. In: Norman A, Schaefer K, Herrath D, Grigoleit H, eds. Vitamin D bone and its clinical application. Berlin: Walter de Gruyter, 1979:349-52.

9. Robinson M, Merrett A,'Tetlow V, Compston J. Plasma 25- hydroxyvitamir/D concentrations in preterm infants receiving oral vitamin D supplements. Arch Dis Child 1981;56:144-5.

10. Glorieux FH, Salle B, Delvin EE, David L. Vitamin D metab- olism in preterm infants: its relation to early neonatal hypo-

calcemia. In: Stern L, Salle B, eds. Intensive care in the new- born; Vol 3. New York: Masson, 1981:127-34.

11. Salle B, David L, Glorieux F, Delvin E, Senterre J, Renaud H. The effect of early oral administration of vitamin D and its metabolites in premature neonates on mineral homeostasis. Pediatr Res 1982;16:75-8.

12. Salle B, Glorieux F, Delvin E, David L; Meunier G. Vitamin D metabolism in preterm infants. Acta Paediatr Scand 1983;72:203-6.

13. Markstad T, Aksnes L, Finne P, Araskog D. Vitamin D nu- tritional status of premature infants supplemented with 500 IU vitamin D2 per day. Acta Paediatr Scand 1983;73:517- 20.

14. Huston RK, Reynolds JW, Jensen C, Buist NRM. Nutrient and mineral retention and vitamin D absorption in low-birth- weight infants: effect of medium-chain triglycerides. Pediatrics 1983;72:44-8.

15. Committee on Nutrition. Pediatric nutrition handbook. Elk Grove Village, Ill.: American Academy of Pediatrics, 1985.

16. European Society of Paediatric Gastroenterology and Nutri- tion. In: Wharton BA, ed. Nutrition and feeding of preterm infants. Oxford, England: Blackwell, 1987.

17. Lentner C, ed. Geigy scientific tables; vol 3. Basel: Ciba-Geigy, 1984:126.

18. Gertner JM, Glassman MS, Coustan DR, Goodman DBP. Fetomaternal vitamin D relationships at term. J PEDIATR 1980;97:637-40.

19. Atkinson SA. Calcium and phosphorus requirements of low birth weight infants: a nutritional and endocrinological per- spective. Nutr Rev 1983;41:69-78.

20. Delvin EE, Glorieux FH, Salle BL, David L, Varenne JP. Control of vitamin D metabolism in preterm infants: feto-ma- ternal relationshsips. Arch Dis Child 1982;57:754-7.

21. Fraser DR. Regulation of the metabolism of vitamin D. Phys- ioI Rev 1980;60:551-613.

22. Markstad T, Aksnes L, Finne-P, Aarskog D. Plasma concen- trations of vitamin D metabolites in premature infants. Pedi- atr Res 1984;18:269-72.

23. Wilkinson R. Absorption of calcium, phosphorus and magne- sium. In: Nordin BEC, ed. Calcium, phosphate, and magne- sium metabolism. Edinburgh: Churchill Livingstone, 1976:36- 112.

24. Halloran BP, DeLuca HF. Calcium transport in small intes- tine during early development: role of vitamin D. Am J Phys- iol 1980;239:G473-9.

25. Halloran BP, DeLuca HF. Appearance of the intestinal cyto- solic receptor for 1,25-dihydroxyvitamin D3 during neonatal development in the rat'. J Biol Chem 1981;256:7338-42.

26. Ueng T-H, Golub EE, Bronner F. The effect of age and 1,25- dihydroxyvitamin D3 treatment on the intestinal calcium- binding protein of suckling rats. Arch Biochem Biophys 1979;196:624-30.

27. Pansu D, Bellaton C, Bronner F. Developmental changes in the mechanisms of duodenal calcium transport in the rat. Am J Physiol 1983;244:G20-6.

28. Dostal LA, Toverud SU. Effect of vitamin D3 on duodenal calcium absorption in vivo during early development. Am J Physiol 1984;246:G528-34.

29. Shenai JP, Reynolds JW, Babson SG. Nutritional balance studies in very-low-birth-weight infants: enhanced nutrient re- tention rates by an experimental formula. Pediatrics 1980;66:233-8.

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30. Bhatia J, Fomon SJ. Formulas for premature infants: fate of the calcium and phosphorus. Pediatrics 1983;72:37-40.

31. Ehrenkranz RA. Session II: discussion. J PEDIATR 1988; 113(suppl):247.

32. Ehrenkranz RA, Ackerman BA, Nelli CM, Janghorbani M. Absorption of calcium in premature infants as measured with a stable isotope 46Ca extrinsic tag. Pediatr Res 1985;19:178- 84.

33. GreerFR. Determination ofradialbonemineralcontentinlow birth weight infants by photon absorptiometry. J PEDIATR 1988;113:213-9.

34. Kovar I, Mayne P, Barltrop D. Plasma alkaline phosphatase activity: a screening test for rickets in preterm neonates. Lan- cet 1982;1:308-10.

35. Glass E J, Hume R, Hendry GMA, Strange RC, Forfar JO. Plasma alkaline phosphatase activity in rickets of prematurity. Arch Dis Child 1982;57:373-6.

36. Waiters EG, Murphy JF, Henry P, Gray OP, Elder GH. plasma alkaline phosphatase activity and its relation to rickets in preterm infants. Ann Clin Biochem 1986;23:652-6.

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