Eur J Pediatr (1990) 149 : 640-644
European Journal of
Pediatrics
9 Springer-Verlag1990
Vitamin K1 levels and Kl-dependent coagulation factors II and X in preterm and small-for-date neonates A. L. J. M. Pietersma-de Bruyn s, P . J . C . van der Straaten 2, P . M . M . van Haard 3, J. C. Kuijpers s, K. Hamulyfik 4, and J. H. Ruys 5 Departments of 1Obstetrics and Gynaecology, 2Paediatrics, Reinier de Graaf Hospital, P.O. Box 5011, 2600 GA Delft, The Netherlands 3Department of Clinical Chemistry, Stichting Samenwerking Delftse Ziekenhuizen (SSDZ), Delft, The Netherlands 4Department of Haematology, University Hospital Maastricht, Maastricht, The Netherlands 5Department of Pediatrics, University Hospital Leiden, Leiden, The Netherlands Received July 14, 1989 / Accepted December 15, 1989
Abstract. In 17 preterm neonates and 7 small-for-date neonates, all formula-fed, vitamin K-dependent coagulation factors II and X remained near 45% of adult values from the m o m e n t of birth until 28 days postnatally. Vitamin Ks levels, however, showed a remarkable rise from below the detection limit of 0.022 ng/ml in umbilical cord blood, to serum levels with a range of 0.99-7.29 ng/ml vitamin Kt on day 3, with a further rise on days 7 and 28 postnatally. Vitamin K1 (Konakion) parenterally given to a third group of four preterm neonates as a i mg dose resulted in very high serum levels of vitamin Ks (64.08-157.10ng/ml), but without any significant increase in plasma levels of vitamin K-dependent coagulation factors II and X, compared to the group without any extra vitamin Ks. It is concluded that in healthy preterm and small-for-date neonates no correlation is seen between serum levels of vitamin Ks and plasma levels of coagulation factors II and X. After administration of i mg Konakion no accelerated increase is seen in coagulatin factor activities.
Key words: Vitamin K1 - Small-for-date - Preterm Neonate - Coagulation
Introduction Preterm and small-for-date neonates are at risk for haemostatic disorders caused by a variety of factors, such as decreased synthesis of coagulation factors, plaOffprint requests to: A. L. J. M. Pietersma-de Bruyn Abbreviations: AT III = antithrombin III; FDP = fibrinogen
degradation products; FII = coagulation factor II (total of non- and carboxylated); F IIC = coagulation factor II-activity; FX = coagulation factor X; HDN = haemorrhagic disease of the newborn; HPLC = high performance liquid chromatography; PIVKA II = descarboxylated coagulation factor II
telet consumption and platelet function abnormalities [3, 9, 11, 14, 23, 36]. Abnormal bleeding tendencies are seen primarily in sick preterm infants with multiple problems, like vomiting and jaundice [23], alpha-l-antitrypsin deficiency [15, 28, 29], need for antibiotic therapy [27], hepatic dysfunction [21] and other complications [19, 24]. Because vitamin K-dependent coagulation factors were found to be 3 0 % - 5 0 % of adult values in preterm as well as in term neonates, most physicians assume a vitamin K deficiency and try to prevent haemostatic disorders in newborns by vitamin K prophylaxis [4, 6, 9, 14, 38]. However, the response to vitamin K administration may be limited due to incapability of the immature liver to synthesize more precursor proteins and carboxylate them [4, 8]. Moreover many other factors may be involved within haemorrhagic disease of the newborn (HDN). This explains why infants may still have haemorrhagic problems despite prophylactic administration of vitamin K [5, 7, 14, 20, 33-35, 40], so the role of vitamin K prophylaxis in this respect is still uncertain [25, 26]. In order to prove a real vitamin K deficiency, measurement of decarboxylated coagulation factor II (PIVKA II) has been propagated, but presence of P I V K A II activity in plasma depends very much on the method being used [26, 41, 42]. To investigate the correlation of serum levels of vitamin Ks and plasma levels of vitamin Kdependent coagulation factors in preterm and small-fordate neonates, levels of vitamin Ks and plasma levels of coagulation (F) factor II, IIc and X were determined.
Materials and methods
Subjects We investigated 28 neonates, divided into the following groups: 1. Group A consisted of 17 preterms, born 32-36 weeks of gestation. The neonates had a mean birth weight of 2480 g, range 18253150 g.
641 2. Group B consisted of seven small-for-dates (< 10th percentile of Kloosterman's birth weight curves, [17]. The neonates were born at 35-41 weeks of gestation, with a mean birth weight of 2200g, range 1330-2760 g. 3. Group C consisted of four preterms receiving 1 mg vitamin Ks (Konakion, Hoffmann-LaRoche, Nijdrecht, The Netherlands) parenterally shortly after birth, born between 29 and 34 weeks of gestation, with a mean birth weight of 1705 g, range 1360-2215 g. Children in group A and B did not receive extra amounts of vitamin Ks. All children were nursed at the neonatal department for some time and were fed formula milk with a high amount of vitamin K1 (Almiron M2 or Nenatal, Nutricia, Zoetermeer, The Netherlands). Apart from being preterm or too small, all children appeared to be healthy at careful physical examination and no signs of abnormal bleeding tendency were seen during the period of investigation. After arrival at the neonatal department, all children started with formula milk 2-4 h after delivery with a dosage of 10 ml milk everey 2-3 h. If necessary the patient was fed by gastric tube. The study was approved by the Medical Ethical Committee of the hospital. Informed consent from the parents was obtained.
Methods The blood sampling protocoll was the same as described previously for term neonates [32]. After clamping the umbilical cord both 5 ml coagulated blood and 5 ml EDTA-blood were obtained by puncturing the umbilical vein. From neonates 3 ml coagulated blood and 2 ml EDTA-blood were taken from a hand vein on days 3, 7 and 28. From their mothers 10 ml of coagulated blood and 5 ml of EDTA-blood were obtained once shortly after delivery. All samples for the vitamin Ks determination were protected from light at all subsequent stages. Immediately after arrival of the blood samples in the laboratory haemoglobin, haematocrit and platelets were determined in EDTA-blood on a Coulter Counter model-S plus (Coulter Electronics, Hileah, Fla., USA) before EDTA-plasma and serum were prepared by centrifugation (2,000g). Remaining EDTA-plasma and serum were stored at -70~ in aliquots of 0.5 ml until needed. Samples from a mother and respective child were assayed in one run. Vitamin K1 was measured by reversed-phase liquid chromatography in combination with electrofluorometry as described by Van Haard [39]. Detection limit using this method was 0.022 ng/ml (= 0.05 nmol/1) requiring a minimum of 0.2ml of serum. FsII, IIc and X were measured with chromogenic substrates in microtitre trays. The extinction (405 nm) was measured in the Titertek Twinreader (Flow General, Flow Laboratories, Irvine, Scotland, UK). Coagulation factors were expressed as percentages of pooled adult normal plasma. Total F I I was assayed as described by Bertina et al. [1] and Peters et al. [30]; the method was adapted for micro-analysis using microtitre trays. The determination of F IIc was based on the F II assay described by Kirchof et al. [16]. Difference between total F II and carboxylated F IIc was used as an indirect method for measuring levels of PIVKA II activity.
Total F X was assayed as described by Van Wijk et al. [43] and adapted for microtitre trays using reagents of F X Diacrom Kit (Wellcome Diagnostics, Darfford, UK). Fibrinogen degradation products (FDP) were determined in serum using ThromboWellcotest (Wellcome Diagnostics). Antithrombin III (AT III) was assayed as described by Van Wijk et al. [43] after adaption for microtitre trays.
Results T w e n t y - e i g h t m o t h e r s of n e o n a t e s f r o m g r o u p s A , B a n d C s h o w e d s e r u m v i t a m i n Ks levels v a r y i n g f r o m 0.04 to 1.1 ng/ml ( m e a n 0.45 + 0.18 ng/ml). Fs I I , I I c a n d X, A T I I I a n d p l a t e l e t s w e r e within n o r m a l r a n g e for h e a l t h y adults. C o r d b l o o d s a m p l e s of all c h i l d r e n f r o m g r o u p s A , B a n d C s h o w e d v i t a m i n Ks levels b e l o w t h e d e t e c t i o n limit of 0.022 ng/ml. T h e v i t a m i n Ks level o f A l m i r o n M2 was 6 7 . 5 0 n g / m l , t h a t o f N e n a t a l was 9 0 . 5 0 n g / m l , a n d t h e v a r i a t i o n coefficient was 3 % . T h e i n d i v i d u a l r a t i o b e t w e e n F I I a n d I I c was b e t w e e n 0.9 a n d 1.1 in all cases.
Group A A l l 17 p r e t e r m n e o n a t e s s h o w e d d e t e c t a b l e levels o f vit a m i n K1 at d a y 3, v a r y i n g f r o m 0.99 to 7.29 ng/ml ( T a b l e 1). V i t a m i n Ks levels o n days 7 a n d 28 w e r e a l m o s t t h e s a m e as o n d a y 3. F s I I , I I c a n d X s h o w e d m e a n levels o f 3 3 % , 32% a n d 49% r e s p e c t i v e l y in c o r d b l o o d , i n c r e a s e d slightly b u t n o t significantly to 4 5 % , 43% a n d 53% res p e c t i v e l y o n d a y 7, a n d r e m a i n e d n e a r l y t h e s a m e t h e r e a f t e r ( T a b l e 1). A T I I I s h o w e d a m e a n v a l u e o f 40% in c o r d b l o o d , i n c r e a s e d g r a d u a l l y to 60% o n d a y 7 a n d r e m a i n e d t h e s a m e until d a y 28. T h e c o n c e n t r a t i o n o f F D P s was less t h a n 10 ~tg/1 at b i r t h a n d o n all s u b s e q u e n t days.
Group B Seven small-for-date n e o n a t e s showed vitamin K1 levels on d a y 3 c o m p a r a b l e to t h o s e in g r o u p A (2.70 + 1.48 ng/ml, T a b l e 2). V i t a m i n K1 levels i n c r e a s e d to 3.10 + 1.53 ng/ml o n d a y 28. P l a s m a levels o f F s I I a n d X in c o r d b l o o d a n d in t h e n e o n a t a l p e r i o d w e r e c o m p a r a b l e to t h o s e in g r o u p A . D u r i n g 4 w e e k s A T I I I r e m a i n e d at a m e a n of 50%. Platelets in c o r d b l o o d w e r e at a m e a n of 154 • 109/1 i n c r e a s i n g to a m e a n o f 371 • 109/1 on d a y 28. T h e conc e n t r a t i o n o f F D P s was less t h a n 10 gg/1 at b i r t h a n d o n all s u b s e q u e n t days.
Table 1. Preterm neonates (n = 17) 32-36 weeks gestational age. Coagulation parameters and vitamin Ks on days 0, 3, 7 and 28
Ks (ng/ml) FII (%) FIIc (%) FX(%) A T I I I activity (%) Platelets (x 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.50 _+ 0.27 116 + 15 98 + 30 157 + 39 94 + 15 236 + 87 0.39 + 0.04
< 0.02 33 + 6 32 _+ 10 49 + 9 40 + 5 235 + 64 0.49 + 0.08
2.61 + 1.62 39 _ 6 37 _+ 6 44 + 9 48 + 7 222 + 78 0.55 + 0.10
1.89 _+ 2.11 45 _+ 8 43 + 7 53 + 12 60 _+ 9 312 _+ 90 0.48 + 0.07
2.34 _+ 2.38 42 _+ 7 42 _ 8 54 + 21 60 + 13 344 + 78 0.37 + 0.07
642 Table 2. Small-for-date neonates (n = 7) 35-41 weeks gestational age. Coagulation parameters and vitamin K1 on days 0, 3, 7 and 28
K1 (ng/ml) FII (%) FIIc (%) FX (%) AT III activity (%) Platelets (• 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.39 -+ 0.19 102 _+33 129 _+39 179 + 40 91 + 25 206 + 48 0.42 + 0.04
< 0.02 34 + 6 33 + 8 49 + 5 43 + 2 154 + 54 0.54 + 0.07
2.70 _ 1.48 37 _+8 31 _+11 38 _+10 45 _+9 171 + 80 0.57 + 0.08
1.66 + 0.67 44 + 10 38 + 13 48 _+9 55 + 13 274 + 93 0.54 + 0.06
3.10 _+1.53 37 + 6 36 + 4 59 + 7 52 + 12 371 + 73 0.37 + 0.06
Table 3. Preterm neonates (n = 4) 29-34 weeks gestational age on days 0, 3, 7 and 28. Coagulation parameters and vitamin K1. All children
received 1 mg Konakion parenterally after birth
Kz (ng/ml) FII (%) FIIc (%) FX (%) ATIII (%) Platelets (• 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.40 + 99 + 102 + 140 + 72 + 164 + 0.33 +
< 0.02 27 + 4 29 + 5 39 + 6 30 + 3 237 + 75 0.45 + 0.01
124.20 + 42.97 33 + 4 32 + 4 40 _+4 46 + 3 265 + 58 0.47 + 0.04
31.41+ 24.98 40 + 2 36 + 3 45 + 7 54 + 3 400 + 101 0.46 + 0.07
9.99 + 41 + 39 + 62 + 43 + 360 + 0.33 +
0.45 25 31 28 22 82 0.03
Group C The four preterm neonates in group C had received I mg Konakion parenterally within i h after birth and showed very high vitamin K~ levels on day 3, varying from 64.08 to 157.10ng/ml. On day 28, vitamin K~ levels varied from 0.54 to 31.14 ng/ml. Despite the high levels of vitamin K1, mean values of Fs II, IIc and X on day 3 were not significantly higher in comparison with those found in group A and B, namely 33%, 32% and 40% respectively. Only F X increased markedly to 62% on day 28, again approximately the same as in group A and B (Table 3). A T III, platelets and haematocrit did not differ significantly between groups A, B and C. The concentration of FDPs was less than 10 gg/1 at birth and on all subsequent days.
Discussion
Vitamin K1 concentrations in cord blood samples of preterm and small-for-date neonates were consistently below 0.022ng/ml, the detection limit of our assay. In our former study with term neonates [32] we had the same results, comparable with those of McCarthy et al. [23] and Shearer et al. [37]. These authors detected extremely low vitamin K1 concentrations in cord blood samples using a high performance liquid chromatography ( H P L C ) - m e t h o d with dual-electrode electrochemical detection, apparently with an even lower detection limit. Recently Greer et al. [12] also described detectable levels of vitamin K1 in cord blood samples of term neonates but at a five-fold higher level by H P L C involving
12.38 7 5 34 14 31 0.04
electrochemical reduction and fluorescence spectrophotometry. After 3 days all children in group A and B had detectable vitamin K1 levels, that remained nearly constant thereafter. Vitamin Kz levels were comparable to those found in formula-fed term neonates [32]. Vitamin K-dependent Fs II and X did not decrease on day 3 and only slightly increased after 1 and 4 weeks (Table 1, 2). The individual ratio FIIc/total F I I (0.9-1.1), found in all cases of our investigation, is also indicative for sufficient vitamin K1 activity in the neonatal liver. Concentration of vitamin K-dependent coagulation factors depends on gestational age of the fetus [10]. Forestier et al. [10] determined coagulation factors from the 19th to the 28th week of pregnancy by fetal blood sampling from the umbilical vein at the placental cord insertion and found a progressive increase of coagulation factor activities from 19 weeks of gestation onward. Apparently synthesis of vitamin K-dependent coagulation factors in the growing liver is increasing more rapidly than the increase of the total blood volume of fetus, umbilical cord and fetal part of the placenta. Identical levels of Fs II and X in preterm neonates have been described by others [2, 4, 8, 14, 18, 20, 31, 38]. Levels of vitamin K-dependent coagulation factors are often described to be decreased most of all on the 3rd day postpartum. However, these data are almost exclusively obtained from in this respect unreliable thrombotest determinations [13]. Many authors find minimal response to vitamin K1 administration especially in preterm neonates [38]. In four preterm neonates receiving i mg Konakion parenterally serum levels of vitamin K1 were extremely high after 3 days, but plasma levels of Fs II and X did not rise more than in group A.
643 Identical high s e r u m levels of vitamin K1 were described by W i d d e r s h o v e n et al. after administration of K o n a k i o n [42]. T h e limited capacity of the i m m a t u r e liver of p r e t e r m n e o n a t e s to synthesize coagulation factors is also reflected by low levels of vitamin K - i n d e p e n d e n t A T III. O f course, during pathological conditions of the newb o r n o n e should also calculate for intravascular consumption of these factors. In our study groups ( p r e t e r m and small-for-date neonates) all children were formula-fed during the first days postnatally. F o r m u l a e like A l m i r o n M2 and N e n a t a l have a high concentrations of vitamin K1 (-+ 67.50 ng/ml, _+ 90.50 ng/ml respectively), as c o m p a r e d to breast milk (vitamin K1 c o n t e n t 2.29 +_ 1.71ng/ml [32]. T h e r e f o r e formula-fed infants have higher vitamin Kl-levels on day 3 postnatally in c o m p a r i s o n with term breast-fed neonates [32]. F r o m our results and those of others [3, 10, 31] we have reason to believe that the vitamin K1 status in fetal and early n e o n a t a l life is not the limiting factor in the synthesis of vitamin K l - d e p e n d e n t coagulation factors; this applies to term, p r e t e r m and small-for-date neonates. In healthy well-drinking n e o n a t e s the hepatic synthesis of vitamin K~-dependent coagulation factors is not only limited b y p r e s e n c e of sufficient vitamin K, but also by capability of liver tissue to p r o d u c e these (coagulation) proteins. This m a y have c o n s e q u e n c e s in the p r e v e n t i o n of H D N . Sick p r e t e r m or small-for-date n e o n a t e s with disturbed milk intake and/or underlying diseases like malabsorption or liver diseases, do n e e d extra vitamin K administration, besides o t h e r nutritional factors, e.g. amino acids, necessary for protein synthesis. T h e optimal r o u t e to administer vitamin K in those cases seems to be parenteral (for instance subcutaneously). Optimal dosage for vitamin K administration is not yet k n o w n , but to p r e v e n t liver disturbances (like hyperbilirubinaemia) small amunts of vitamin K1 (about 100 Ixg vitamin K1 once or twice a week) might be preferable. A n optimal d o c u m e n t a t i o n of all relevant data concerning n e w b o r n s with bleeding incidences are necessary for a b e t t e r u n d e r s t a n d i n g of w h a t m a y be linked with vitamin K deficiency. A s h a e m o r r h a g e s in n e w b o r n s do not occur very often, a multicentre trial will be the p r o p e r way to clarify the multifactorial causes in H D N . In T h e Netherlands such a co-operative study is in process.
References 1. Bertina RM, Marel vd-Nieuwkoop WY v, Dubbeldam J, Boekhout-Mussert RJ, Veltkamp JJ (1980) New method for the rapid detection of vitamin K deficiency. Clin Chim Acta 105 : 93-98 2. Bleyer WA, Hakami N, Sheperd TH (1971) Haemostatic disorders in the human fetus and newborn infant. J Pediatr 79: 838-853 3. Bor M van de, BriOt E, Bel F van, Ruys JH (1986) Hemostasis and periventricular-intraventricular hemorrhage of the newborn. Am J Dis Child 140 : 1131-1134
4. Buchanan GR (1978) Neonatal coagulation; normal physiology and pathophysiology. Clin Haematol 7: 85-108 5. Biiller H, Burger B, Nagelkerke N, Cate JW ten, Breederveld C, Heymans H (1986) Vitamin K-status beyond the neonatal period. Eur J Pediatr 145 : 496-499 6. Cade JF, Hirsch J, Martin M (1969) Placental barrier to coagulation factors: its relevance to the coagulation defect at birth and to haemorrhage in the newborn. Br Med J 2: 281-283 7. Chaou WT, Chou ML, Eitzman DV (1984) Intracranial haemorrhage and vitamin K deficiency in early infancy. J Pediatr 105 : 880-884 8. Chessells JM, Wigglesworth JS (1972) Coagulation studies in preterm infants with respiratory distress and intracranial haemorrhage. Arch Dis Child 47 : 564-570 9. Editorial (1978) Vitamin K and the newborn. Lancet I:755757 10. Forestier F, Daffos F, Rainaut M, Sold Y, Amiral J (1985) Vitamin K dependent proteins in fetal hemostasis at mid trimester of pregnancy. Thromb Haemost 53(3) : 401-403 11. Gray OP, Acherman A, Fraser AJ (1968) Intracranial haemorrhage and clotting defects in low-birth weight infants. Lancet I : 545-548 12. Greer FR, Mnmmah-Schendel LL, Marshall S, Suttie JW (1988) Vitamin Ks (phylloquinone) and vitamin K: (menaquinone) status in newborns during first week of life. Pediatrics 81 : 137-140 13. Hamulyfik K, Devilee PP, Nieuwenhuizen W, Hemker HC (1988) The prolongation of the thrombotest clotting time in newborns. Thromb Res 52 : 45-52 14. Hathaway WE (1975) The bleeding newborn. Semin Hematol 12 : 175-188 15. Hope PL, Hall MA, Millward-Sadler GH, Normand ICS (1982) Alpha-l-antitrypsin deficiency presenting as a bleeding diathesis in the newborn. Arch Dis Child 57 : 68-79 16. Kirchhof BRJ, Vermeer C, Hemker HC (1978) The determination of prothrombin using synthetic chromogenic substracts: choice of a suitable activator. Thromb Res 13 : 219-232 17. Kloosterman GJ (1969) Over intrauteriene groei en de intrauteriene groeicurve. Ned T Veflosk 69 : 349-365 (in Dutch) 18. Kolindewala JK, Dube B, Bhargava V, Dube RK, Kota VLN, Das BK (1986) Hemostatic parameters in newborn. II Sequential study during the first four weeks of life. Thromb Haemost 55(1) : 51-53 19. Kries R von, Reifenh~iuser A, G6bel U, Mc Carthy P, Shearer MJ, Berkhan P (1985) Late onset haemorrhagic disease of newborn with temporary malabsorption of vitamin K1. Lancet I: 1035 20. Lane PA, Hathaway WE (1985) Vitamin K in infancy. J Pediatr 106 : 351-359 21. Matsuda J, Nishiyama S, Motohara K, Endo F, Ogata T, Futagoishi Y (1989) Late neonatal vitamin K deficiency associated with subclinical liver dysfunction in human milk-fed infants. J Pediatr 114: 602-605 22. Mc Carthy PT, Gau G, Crampton OE (1988) Plasma and liver levels of vitamin K in the newborn. In: Suttie JW (ed) Current advances in vitamin K research. Elsevier, Amsterdam, pp 437464 23. Mc Donald MM, Johnson ML, Rumack CM, Koops BL, Guggenheim MA, Babb C, Hathaway WE (1984) Role of coagulopathy in newborn intracranial hemorrhage. Pediatrics 74:2631 24. Mc Ninch AW, Orme RL, Tripp JH (1983) Haemorrhagic disease of the newborn returns. Lancet I: 1089-1090 25. Minford AMB, Eden OB (1979) Haemorrhage responsive to vitamin K in a 6-week-old infant. Arch Dis Child 54:310-311 26. Motohara K, Matsukura M, Matsuda I, Iribe K, Ikada T, Kondo Y, Yonekubo A, Yamamoto Y (1970) Severe vitamin K deficiency in breast-fed infants. J Pediatr 105 : 943-945 27. Nammacher MA, Willemin M, Hartmann JR, Gaston LW (1970) Vitamin K deficiency in infants beyond the neonatal period. J Pediatr 76: 549-554
644 28. O'Connor ME, Livingstone DS, Hannah J, Wilkins D (1983) Vitamin K deficiency and breast feeding. Am J Dis Child 137 : 601-602 29. Payne NR, Hasegawa DK (1984) Vitamin K deficiency in newborns. A case report in alphal-antitrypsin deficiency and a review of factors predisposing to hemorrhage. Pediatrics 73: 712-716 30. Peters M, Breederveld C, Kahl6 LH, Cate JW ten (1982) Rapid micro-analysis of coagulation parameters by automated chromogenic substrate methods-application in neonatal patients. Thromb Res 28 : 773-781 31. Peters M, Cate JW ten, Jansen E, Breederveld C (1985) Coagulation and fibrinolytic factors in the first week of life in healthy infants. J Pediatr 106: 292-295 32. Pietersma-de Bruyn ALJM, Haard PMM van, Beunis MH, Hamulyfik K, Kuijpers JC (1990) Vitamin K1 levels and coagulation factors in healthy term newborns till 4 weeks after birth. Haemostasis 20 : 8-14 33. Pomerance JJ, Teal JG, Gogolok JF, Brown S, Stewart ME (1987) Maternally administered antenatal vitamin KI: effect on neonatal prothrombin activity, partial thromboplastin time, and intraventricular hemorrhage. Obstet Gyneco170: 235-241 34. Rouw-Timmer ECJ, Jone GA de (1986) Bloedingen in de eerste levensweken en vitamine K profylaxe. Ned T Geneesk 130: 484-487 (in Dutch) 35. Sann L, Leclercq M, Guillaumont M, Trouyez R, Bethenod M, Bourgeay-Causse M (1985) Serum vitamin K1 concentrations after oral administration of vitamin Ks in low-birth weight infants. J Pediatr 107 : 605-611 36. Setzer ES, Webb IB, Wassenaar JW, Reeder JD, Nehta PS, Eitzman DV (1982) Platelet dysfunction and coagulopathy in
37. 38.
39.
40. 41. 42.
43.
intraventricular hemorrhage in the premature infant. J Pediatr 100 : 599-605 Shearer MJ, Crampton OE, McCarthy PT, Mattock MB (1986) Vitamin K1 in plasma: relationship to vitamin K status, age, pregnancy, diet and disease. Haemostasis 16 (5) : 83 Terwiel JP, Veltkamp JJ, Bertina RJ, Linden IK van der, Tilburg NH van (1985) Coagulation factors in the premature infant born after about 32 weeks of gestation. Biol Neonate 47: 9-18 Van Haard PMM, Engel R, Pietersma-de Bruyn ALJM (1986) Quantitation of trans-vitamin K1 in small serum samples by off-line multidimensional liquid chromatography. Clin Chim Acta 157 : 221-230 Verity CM, Carswell F, Scott GL (1983) Vitamin K deficiency causing intracranial haemorrhage after the neonatal period. Lancet I: 1439 Widdershoven JAM, Kollde LAA, Oostrom CG van, Munster P van, Monnens LAH (1986) Vitamine K defici~ntie bij zuigelingen. Ned T Geneesk 130 : 473-476 (in Dutch) Widdershoven J, Lambert W, Motohara K, Monnens L, Leenheer A de, Matsuda I, Endo F (1988) Plasma concentrations of vitamin K1 and PIVKA II in bottle-fed and breast-fed infants with and without vitamin-K prophylaxis at birth. Eur J Paediatr 148 : 139-142 Wijk EM van, Kahl6 LH, Care JW ten (1980) Mechanized amidolytic technique for determination of factor X and factor X-antigen, and its application to patients being treated with oral anticoagulants. Clin Chem 26 : 885-890
European Journal of
Pediatrics
9 Springer-Verlag1990
Vitamin K1 levels and Kl-dependent coagulation factors II and X in preterm and small-for-date neonates A. L. J. M. Pietersma-de Bruyn s, P . J . C . van der Straaten 2, P . M . M . van Haard 3, J. C. Kuijpers s, K. Hamulyfik 4, and J. H. Ruys 5 Departments of 1Obstetrics and Gynaecology, 2Paediatrics, Reinier de Graaf Hospital, P.O. Box 5011, 2600 GA Delft, The Netherlands 3Department of Clinical Chemistry, Stichting Samenwerking Delftse Ziekenhuizen (SSDZ), Delft, The Netherlands 4Department of Haematology, University Hospital Maastricht, Maastricht, The Netherlands 5Department of Pediatrics, University Hospital Leiden, Leiden, The Netherlands Received July 14, 1989 / Accepted December 15, 1989
Abstract. In 17 preterm neonates and 7 small-for-date neonates, all formula-fed, vitamin K-dependent coagulation factors II and X remained near 45% of adult values from the m o m e n t of birth until 28 days postnatally. Vitamin Ks levels, however, showed a remarkable rise from below the detection limit of 0.022 ng/ml in umbilical cord blood, to serum levels with a range of 0.99-7.29 ng/ml vitamin Kt on day 3, with a further rise on days 7 and 28 postnatally. Vitamin K1 (Konakion) parenterally given to a third group of four preterm neonates as a i mg dose resulted in very high serum levels of vitamin Ks (64.08-157.10ng/ml), but without any significant increase in plasma levels of vitamin K-dependent coagulation factors II and X, compared to the group without any extra vitamin Ks. It is concluded that in healthy preterm and small-for-date neonates no correlation is seen between serum levels of vitamin Ks and plasma levels of coagulation factors II and X. After administration of i mg Konakion no accelerated increase is seen in coagulatin factor activities.
Key words: Vitamin K1 - Small-for-date - Preterm Neonate - Coagulation
Introduction Preterm and small-for-date neonates are at risk for haemostatic disorders caused by a variety of factors, such as decreased synthesis of coagulation factors, plaOffprint requests to: A. L. J. M. Pietersma-de Bruyn Abbreviations: AT III = antithrombin III; FDP = fibrinogen
degradation products; FII = coagulation factor II (total of non- and carboxylated); F IIC = coagulation factor II-activity; FX = coagulation factor X; HDN = haemorrhagic disease of the newborn; HPLC = high performance liquid chromatography; PIVKA II = descarboxylated coagulation factor II
telet consumption and platelet function abnormalities [3, 9, 11, 14, 23, 36]. Abnormal bleeding tendencies are seen primarily in sick preterm infants with multiple problems, like vomiting and jaundice [23], alpha-l-antitrypsin deficiency [15, 28, 29], need for antibiotic therapy [27], hepatic dysfunction [21] and other complications [19, 24]. Because vitamin K-dependent coagulation factors were found to be 3 0 % - 5 0 % of adult values in preterm as well as in term neonates, most physicians assume a vitamin K deficiency and try to prevent haemostatic disorders in newborns by vitamin K prophylaxis [4, 6, 9, 14, 38]. However, the response to vitamin K administration may be limited due to incapability of the immature liver to synthesize more precursor proteins and carboxylate them [4, 8]. Moreover many other factors may be involved within haemorrhagic disease of the newborn (HDN). This explains why infants may still have haemorrhagic problems despite prophylactic administration of vitamin K [5, 7, 14, 20, 33-35, 40], so the role of vitamin K prophylaxis in this respect is still uncertain [25, 26]. In order to prove a real vitamin K deficiency, measurement of decarboxylated coagulation factor II (PIVKA II) has been propagated, but presence of P I V K A II activity in plasma depends very much on the method being used [26, 41, 42]. To investigate the correlation of serum levels of vitamin Ks and plasma levels of vitamin Kdependent coagulation factors in preterm and small-fordate neonates, levels of vitamin Ks and plasma levels of coagulation (F) factor II, IIc and X were determined.
Materials and methods
Subjects We investigated 28 neonates, divided into the following groups: 1. Group A consisted of 17 preterms, born 32-36 weeks of gestation. The neonates had a mean birth weight of 2480 g, range 18253150 g.
641 2. Group B consisted of seven small-for-dates (< 10th percentile of Kloosterman's birth weight curves, [17]. The neonates were born at 35-41 weeks of gestation, with a mean birth weight of 2200g, range 1330-2760 g. 3. Group C consisted of four preterms receiving 1 mg vitamin Ks (Konakion, Hoffmann-LaRoche, Nijdrecht, The Netherlands) parenterally shortly after birth, born between 29 and 34 weeks of gestation, with a mean birth weight of 1705 g, range 1360-2215 g. Children in group A and B did not receive extra amounts of vitamin Ks. All children were nursed at the neonatal department for some time and were fed formula milk with a high amount of vitamin K1 (Almiron M2 or Nenatal, Nutricia, Zoetermeer, The Netherlands). Apart from being preterm or too small, all children appeared to be healthy at careful physical examination and no signs of abnormal bleeding tendency were seen during the period of investigation. After arrival at the neonatal department, all children started with formula milk 2-4 h after delivery with a dosage of 10 ml milk everey 2-3 h. If necessary the patient was fed by gastric tube. The study was approved by the Medical Ethical Committee of the hospital. Informed consent from the parents was obtained.
Methods The blood sampling protocoll was the same as described previously for term neonates [32]. After clamping the umbilical cord both 5 ml coagulated blood and 5 ml EDTA-blood were obtained by puncturing the umbilical vein. From neonates 3 ml coagulated blood and 2 ml EDTA-blood were taken from a hand vein on days 3, 7 and 28. From their mothers 10 ml of coagulated blood and 5 ml of EDTA-blood were obtained once shortly after delivery. All samples for the vitamin Ks determination were protected from light at all subsequent stages. Immediately after arrival of the blood samples in the laboratory haemoglobin, haematocrit and platelets were determined in EDTA-blood on a Coulter Counter model-S plus (Coulter Electronics, Hileah, Fla., USA) before EDTA-plasma and serum were prepared by centrifugation (2,000g). Remaining EDTA-plasma and serum were stored at -70~ in aliquots of 0.5 ml until needed. Samples from a mother and respective child were assayed in one run. Vitamin K1 was measured by reversed-phase liquid chromatography in combination with electrofluorometry as described by Van Haard [39]. Detection limit using this method was 0.022 ng/ml (= 0.05 nmol/1) requiring a minimum of 0.2ml of serum. FsII, IIc and X were measured with chromogenic substrates in microtitre trays. The extinction (405 nm) was measured in the Titertek Twinreader (Flow General, Flow Laboratories, Irvine, Scotland, UK). Coagulation factors were expressed as percentages of pooled adult normal plasma. Total F I I was assayed as described by Bertina et al. [1] and Peters et al. [30]; the method was adapted for micro-analysis using microtitre trays. The determination of F IIc was based on the F II assay described by Kirchof et al. [16]. Difference between total F II and carboxylated F IIc was used as an indirect method for measuring levels of PIVKA II activity.
Total F X was assayed as described by Van Wijk et al. [43] and adapted for microtitre trays using reagents of F X Diacrom Kit (Wellcome Diagnostics, Darfford, UK). Fibrinogen degradation products (FDP) were determined in serum using ThromboWellcotest (Wellcome Diagnostics). Antithrombin III (AT III) was assayed as described by Van Wijk et al. [43] after adaption for microtitre trays.
Results T w e n t y - e i g h t m o t h e r s of n e o n a t e s f r o m g r o u p s A , B a n d C s h o w e d s e r u m v i t a m i n Ks levels v a r y i n g f r o m 0.04 to 1.1 ng/ml ( m e a n 0.45 + 0.18 ng/ml). Fs I I , I I c a n d X, A T I I I a n d p l a t e l e t s w e r e within n o r m a l r a n g e for h e a l t h y adults. C o r d b l o o d s a m p l e s of all c h i l d r e n f r o m g r o u p s A , B a n d C s h o w e d v i t a m i n Ks levels b e l o w t h e d e t e c t i o n limit of 0.022 ng/ml. T h e v i t a m i n Ks level o f A l m i r o n M2 was 6 7 . 5 0 n g / m l , t h a t o f N e n a t a l was 9 0 . 5 0 n g / m l , a n d t h e v a r i a t i o n coefficient was 3 % . T h e i n d i v i d u a l r a t i o b e t w e e n F I I a n d I I c was b e t w e e n 0.9 a n d 1.1 in all cases.
Group A A l l 17 p r e t e r m n e o n a t e s s h o w e d d e t e c t a b l e levels o f vit a m i n K1 at d a y 3, v a r y i n g f r o m 0.99 to 7.29 ng/ml ( T a b l e 1). V i t a m i n Ks levels o n days 7 a n d 28 w e r e a l m o s t t h e s a m e as o n d a y 3. F s I I , I I c a n d X s h o w e d m e a n levels o f 3 3 % , 32% a n d 49% r e s p e c t i v e l y in c o r d b l o o d , i n c r e a s e d slightly b u t n o t significantly to 4 5 % , 43% a n d 53% res p e c t i v e l y o n d a y 7, a n d r e m a i n e d n e a r l y t h e s a m e t h e r e a f t e r ( T a b l e 1). A T I I I s h o w e d a m e a n v a l u e o f 40% in c o r d b l o o d , i n c r e a s e d g r a d u a l l y to 60% o n d a y 7 a n d r e m a i n e d t h e s a m e until d a y 28. T h e c o n c e n t r a t i o n o f F D P s was less t h a n 10 ~tg/1 at b i r t h a n d o n all s u b s e q u e n t days.
Group B Seven small-for-date n e o n a t e s showed vitamin K1 levels on d a y 3 c o m p a r a b l e to t h o s e in g r o u p A (2.70 + 1.48 ng/ml, T a b l e 2). V i t a m i n K1 levels i n c r e a s e d to 3.10 + 1.53 ng/ml o n d a y 28. P l a s m a levels o f F s I I a n d X in c o r d b l o o d a n d in t h e n e o n a t a l p e r i o d w e r e c o m p a r a b l e to t h o s e in g r o u p A . D u r i n g 4 w e e k s A T I I I r e m a i n e d at a m e a n of 50%. Platelets in c o r d b l o o d w e r e at a m e a n of 154 • 109/1 i n c r e a s i n g to a m e a n o f 371 • 109/1 on d a y 28. T h e conc e n t r a t i o n o f F D P s was less t h a n 10 gg/1 at b i r t h a n d o n all s u b s e q u e n t days.
Table 1. Preterm neonates (n = 17) 32-36 weeks gestational age. Coagulation parameters and vitamin Ks on days 0, 3, 7 and 28
Ks (ng/ml) FII (%) FIIc (%) FX(%) A T I I I activity (%) Platelets (x 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.50 _+ 0.27 116 + 15 98 + 30 157 + 39 94 + 15 236 + 87 0.39 + 0.04
< 0.02 33 + 6 32 _+ 10 49 + 9 40 + 5 235 + 64 0.49 + 0.08
2.61 + 1.62 39 _ 6 37 _+ 6 44 + 9 48 + 7 222 + 78 0.55 + 0.10
1.89 _+ 2.11 45 _+ 8 43 + 7 53 + 12 60 _+ 9 312 _+ 90 0.48 + 0.07
2.34 _+ 2.38 42 _+ 7 42 _ 8 54 + 21 60 + 13 344 + 78 0.37 + 0.07
642 Table 2. Small-for-date neonates (n = 7) 35-41 weeks gestational age. Coagulation parameters and vitamin K1 on days 0, 3, 7 and 28
K1 (ng/ml) FII (%) FIIc (%) FX (%) AT III activity (%) Platelets (• 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.39 -+ 0.19 102 _+33 129 _+39 179 + 40 91 + 25 206 + 48 0.42 + 0.04
< 0.02 34 + 6 33 + 8 49 + 5 43 + 2 154 + 54 0.54 + 0.07
2.70 _ 1.48 37 _+8 31 _+11 38 _+10 45 _+9 171 + 80 0.57 + 0.08
1.66 + 0.67 44 + 10 38 + 13 48 _+9 55 + 13 274 + 93 0.54 + 0.06
3.10 _+1.53 37 + 6 36 + 4 59 + 7 52 + 12 371 + 73 0.37 + 0.06
Table 3. Preterm neonates (n = 4) 29-34 weeks gestational age on days 0, 3, 7 and 28. Coagulation parameters and vitamin K1. All children
received 1 mg Konakion parenterally after birth
Kz (ng/ml) FII (%) FIIc (%) FX (%) ATIII (%) Platelets (• 109/1) Haematocrit
Mother
Cord blood
Day 3
Day 7
Day 28
0.40 + 99 + 102 + 140 + 72 + 164 + 0.33 +
< 0.02 27 + 4 29 + 5 39 + 6 30 + 3 237 + 75 0.45 + 0.01
124.20 + 42.97 33 + 4 32 + 4 40 _+4 46 + 3 265 + 58 0.47 + 0.04
31.41+ 24.98 40 + 2 36 + 3 45 + 7 54 + 3 400 + 101 0.46 + 0.07
9.99 + 41 + 39 + 62 + 43 + 360 + 0.33 +
0.45 25 31 28 22 82 0.03
Group C The four preterm neonates in group C had received I mg Konakion parenterally within i h after birth and showed very high vitamin K~ levels on day 3, varying from 64.08 to 157.10ng/ml. On day 28, vitamin K~ levels varied from 0.54 to 31.14 ng/ml. Despite the high levels of vitamin K1, mean values of Fs II, IIc and X on day 3 were not significantly higher in comparison with those found in group A and B, namely 33%, 32% and 40% respectively. Only F X increased markedly to 62% on day 28, again approximately the same as in group A and B (Table 3). A T III, platelets and haematocrit did not differ significantly between groups A, B and C. The concentration of FDPs was less than 10 gg/1 at birth and on all subsequent days.
Discussion
Vitamin K1 concentrations in cord blood samples of preterm and small-for-date neonates were consistently below 0.022ng/ml, the detection limit of our assay. In our former study with term neonates [32] we had the same results, comparable with those of McCarthy et al. [23] and Shearer et al. [37]. These authors detected extremely low vitamin K1 concentrations in cord blood samples using a high performance liquid chromatography ( H P L C ) - m e t h o d with dual-electrode electrochemical detection, apparently with an even lower detection limit. Recently Greer et al. [12] also described detectable levels of vitamin K1 in cord blood samples of term neonates but at a five-fold higher level by H P L C involving
12.38 7 5 34 14 31 0.04
electrochemical reduction and fluorescence spectrophotometry. After 3 days all children in group A and B had detectable vitamin K1 levels, that remained nearly constant thereafter. Vitamin Kz levels were comparable to those found in formula-fed term neonates [32]. Vitamin K-dependent Fs II and X did not decrease on day 3 and only slightly increased after 1 and 4 weeks (Table 1, 2). The individual ratio FIIc/total F I I (0.9-1.1), found in all cases of our investigation, is also indicative for sufficient vitamin K1 activity in the neonatal liver. Concentration of vitamin K-dependent coagulation factors depends on gestational age of the fetus [10]. Forestier et al. [10] determined coagulation factors from the 19th to the 28th week of pregnancy by fetal blood sampling from the umbilical vein at the placental cord insertion and found a progressive increase of coagulation factor activities from 19 weeks of gestation onward. Apparently synthesis of vitamin K-dependent coagulation factors in the growing liver is increasing more rapidly than the increase of the total blood volume of fetus, umbilical cord and fetal part of the placenta. Identical levels of Fs II and X in preterm neonates have been described by others [2, 4, 8, 14, 18, 20, 31, 38]. Levels of vitamin K-dependent coagulation factors are often described to be decreased most of all on the 3rd day postpartum. However, these data are almost exclusively obtained from in this respect unreliable thrombotest determinations [13]. Many authors find minimal response to vitamin K1 administration especially in preterm neonates [38]. In four preterm neonates receiving i mg Konakion parenterally serum levels of vitamin K1 were extremely high after 3 days, but plasma levels of Fs II and X did not rise more than in group A.
643 Identical high s e r u m levels of vitamin K1 were described by W i d d e r s h o v e n et al. after administration of K o n a k i o n [42]. T h e limited capacity of the i m m a t u r e liver of p r e t e r m n e o n a t e s to synthesize coagulation factors is also reflected by low levels of vitamin K - i n d e p e n d e n t A T III. O f course, during pathological conditions of the newb o r n o n e should also calculate for intravascular consumption of these factors. In our study groups ( p r e t e r m and small-for-date neonates) all children were formula-fed during the first days postnatally. F o r m u l a e like A l m i r o n M2 and N e n a t a l have a high concentrations of vitamin K1 (-+ 67.50 ng/ml, _+ 90.50 ng/ml respectively), as c o m p a r e d to breast milk (vitamin K1 c o n t e n t 2.29 +_ 1.71ng/ml [32]. T h e r e f o r e formula-fed infants have higher vitamin Kl-levels on day 3 postnatally in c o m p a r i s o n with term breast-fed neonates [32]. F r o m our results and those of others [3, 10, 31] we have reason to believe that the vitamin K1 status in fetal and early n e o n a t a l life is not the limiting factor in the synthesis of vitamin K l - d e p e n d e n t coagulation factors; this applies to term, p r e t e r m and small-for-date neonates. In healthy well-drinking n e o n a t e s the hepatic synthesis of vitamin K~-dependent coagulation factors is not only limited b y p r e s e n c e of sufficient vitamin K, but also by capability of liver tissue to p r o d u c e these (coagulation) proteins. This m a y have c o n s e q u e n c e s in the p r e v e n t i o n of H D N . Sick p r e t e r m or small-for-date n e o n a t e s with disturbed milk intake and/or underlying diseases like malabsorption or liver diseases, do n e e d extra vitamin K administration, besides o t h e r nutritional factors, e.g. amino acids, necessary for protein synthesis. T h e optimal r o u t e to administer vitamin K in those cases seems to be parenteral (for instance subcutaneously). Optimal dosage for vitamin K administration is not yet k n o w n , but to p r e v e n t liver disturbances (like hyperbilirubinaemia) small amunts of vitamin K1 (about 100 Ixg vitamin K1 once or twice a week) might be preferable. A n optimal d o c u m e n t a t i o n of all relevant data concerning n e w b o r n s with bleeding incidences are necessary for a b e t t e r u n d e r s t a n d i n g of w h a t m a y be linked with vitamin K deficiency. A s h a e m o r r h a g e s in n e w b o r n s do not occur very often, a multicentre trial will be the p r o p e r way to clarify the multifactorial causes in H D N . In T h e Netherlands such a co-operative study is in process.
References 1. Bertina RM, Marel vd-Nieuwkoop WY v, Dubbeldam J, Boekhout-Mussert RJ, Veltkamp JJ (1980) New method for the rapid detection of vitamin K deficiency. Clin Chim Acta 105 : 93-98 2. Bleyer WA, Hakami N, Sheperd TH (1971) Haemostatic disorders in the human fetus and newborn infant. J Pediatr 79: 838-853 3. Bor M van de, BriOt E, Bel F van, Ruys JH (1986) Hemostasis and periventricular-intraventricular hemorrhage of the newborn. Am J Dis Child 140 : 1131-1134
4. Buchanan GR (1978) Neonatal coagulation; normal physiology and pathophysiology. Clin Haematol 7: 85-108 5. Biiller H, Burger B, Nagelkerke N, Cate JW ten, Breederveld C, Heymans H (1986) Vitamin K-status beyond the neonatal period. Eur J Pediatr 145 : 496-499 6. Cade JF, Hirsch J, Martin M (1969) Placental barrier to coagulation factors: its relevance to the coagulation defect at birth and to haemorrhage in the newborn. Br Med J 2: 281-283 7. Chaou WT, Chou ML, Eitzman DV (1984) Intracranial haemorrhage and vitamin K deficiency in early infancy. J Pediatr 105 : 880-884 8. Chessells JM, Wigglesworth JS (1972) Coagulation studies in preterm infants with respiratory distress and intracranial haemorrhage. Arch Dis Child 47 : 564-570 9. Editorial (1978) Vitamin K and the newborn. Lancet I:755757 10. Forestier F, Daffos F, Rainaut M, Sold Y, Amiral J (1985) Vitamin K dependent proteins in fetal hemostasis at mid trimester of pregnancy. Thromb Haemost 53(3) : 401-403 11. Gray OP, Acherman A, Fraser AJ (1968) Intracranial haemorrhage and clotting defects in low-birth weight infants. Lancet I : 545-548 12. Greer FR, Mnmmah-Schendel LL, Marshall S, Suttie JW (1988) Vitamin Ks (phylloquinone) and vitamin K: (menaquinone) status in newborns during first week of life. Pediatrics 81 : 137-140 13. Hamulyfik K, Devilee PP, Nieuwenhuizen W, Hemker HC (1988) The prolongation of the thrombotest clotting time in newborns. Thromb Res 52 : 45-52 14. Hathaway WE (1975) The bleeding newborn. Semin Hematol 12 : 175-188 15. Hope PL, Hall MA, Millward-Sadler GH, Normand ICS (1982) Alpha-l-antitrypsin deficiency presenting as a bleeding diathesis in the newborn. Arch Dis Child 57 : 68-79 16. Kirchhof BRJ, Vermeer C, Hemker HC (1978) The determination of prothrombin using synthetic chromogenic substracts: choice of a suitable activator. Thromb Res 13 : 219-232 17. Kloosterman GJ (1969) Over intrauteriene groei en de intrauteriene groeicurve. Ned T Veflosk 69 : 349-365 (in Dutch) 18. Kolindewala JK, Dube B, Bhargava V, Dube RK, Kota VLN, Das BK (1986) Hemostatic parameters in newborn. II Sequential study during the first four weeks of life. Thromb Haemost 55(1) : 51-53 19. Kries R von, Reifenh~iuser A, G6bel U, Mc Carthy P, Shearer MJ, Berkhan P (1985) Late onset haemorrhagic disease of newborn with temporary malabsorption of vitamin K1. Lancet I: 1035 20. Lane PA, Hathaway WE (1985) Vitamin K in infancy. J Pediatr 106 : 351-359 21. Matsuda J, Nishiyama S, Motohara K, Endo F, Ogata T, Futagoishi Y (1989) Late neonatal vitamin K deficiency associated with subclinical liver dysfunction in human milk-fed infants. J Pediatr 114: 602-605 22. Mc Carthy PT, Gau G, Crampton OE (1988) Plasma and liver levels of vitamin K in the newborn. In: Suttie JW (ed) Current advances in vitamin K research. Elsevier, Amsterdam, pp 437464 23. Mc Donald MM, Johnson ML, Rumack CM, Koops BL, Guggenheim MA, Babb C, Hathaway WE (1984) Role of coagulopathy in newborn intracranial hemorrhage. Pediatrics 74:2631 24. Mc Ninch AW, Orme RL, Tripp JH (1983) Haemorrhagic disease of the newborn returns. Lancet I: 1089-1090 25. Minford AMB, Eden OB (1979) Haemorrhage responsive to vitamin K in a 6-week-old infant. Arch Dis Child 54:310-311 26. Motohara K, Matsukura M, Matsuda I, Iribe K, Ikada T, Kondo Y, Yonekubo A, Yamamoto Y (1970) Severe vitamin K deficiency in breast-fed infants. J Pediatr 105 : 943-945 27. Nammacher MA, Willemin M, Hartmann JR, Gaston LW (1970) Vitamin K deficiency in infants beyond the neonatal period. J Pediatr 76: 549-554
644 28. O'Connor ME, Livingstone DS, Hannah J, Wilkins D (1983) Vitamin K deficiency and breast feeding. Am J Dis Child 137 : 601-602 29. Payne NR, Hasegawa DK (1984) Vitamin K deficiency in newborns. A case report in alphal-antitrypsin deficiency and a review of factors predisposing to hemorrhage. Pediatrics 73: 712-716 30. Peters M, Breederveld C, Kahl6 LH, Cate JW ten (1982) Rapid micro-analysis of coagulation parameters by automated chromogenic substrate methods-application in neonatal patients. Thromb Res 28 : 773-781 31. Peters M, Cate JW ten, Jansen E, Breederveld C (1985) Coagulation and fibrinolytic factors in the first week of life in healthy infants. J Pediatr 106: 292-295 32. Pietersma-de Bruyn ALJM, Haard PMM van, Beunis MH, Hamulyfik K, Kuijpers JC (1990) Vitamin K1 levels and coagulation factors in healthy term newborns till 4 weeks after birth. Haemostasis 20 : 8-14 33. Pomerance JJ, Teal JG, Gogolok JF, Brown S, Stewart ME (1987) Maternally administered antenatal vitamin KI: effect on neonatal prothrombin activity, partial thromboplastin time, and intraventricular hemorrhage. Obstet Gyneco170: 235-241 34. Rouw-Timmer ECJ, Jone GA de (1986) Bloedingen in de eerste levensweken en vitamine K profylaxe. Ned T Geneesk 130: 484-487 (in Dutch) 35. Sann L, Leclercq M, Guillaumont M, Trouyez R, Bethenod M, Bourgeay-Causse M (1985) Serum vitamin K1 concentrations after oral administration of vitamin Ks in low-birth weight infants. J Pediatr 107 : 605-611 36. Setzer ES, Webb IB, Wassenaar JW, Reeder JD, Nehta PS, Eitzman DV (1982) Platelet dysfunction and coagulopathy in
37. 38.
39.
40. 41. 42.
43.
intraventricular hemorrhage in the premature infant. J Pediatr 100 : 599-605 Shearer MJ, Crampton OE, McCarthy PT, Mattock MB (1986) Vitamin K1 in plasma: relationship to vitamin K status, age, pregnancy, diet and disease. Haemostasis 16 (5) : 83 Terwiel JP, Veltkamp JJ, Bertina RJ, Linden IK van der, Tilburg NH van (1985) Coagulation factors in the premature infant born after about 32 weeks of gestation. Biol Neonate 47: 9-18 Van Haard PMM, Engel R, Pietersma-de Bruyn ALJM (1986) Quantitation of trans-vitamin K1 in small serum samples by off-line multidimensional liquid chromatography. Clin Chim Acta 157 : 221-230 Verity CM, Carswell F, Scott GL (1983) Vitamin K deficiency causing intracranial haemorrhage after the neonatal period. Lancet I: 1439 Widdershoven JAM, Kollde LAA, Oostrom CG van, Munster P van, Monnens LAH (1986) Vitamine K defici~ntie bij zuigelingen. Ned T Geneesk 130 : 473-476 (in Dutch) Widdershoven J, Lambert W, Motohara K, Monnens L, Leenheer A de, Matsuda I, Endo F (1988) Plasma concentrations of vitamin K1 and PIVKA II in bottle-fed and breast-fed infants with and without vitamin-K prophylaxis at birth. Eur J Paediatr 148 : 139-142 Wijk EM van, Kahl6 LH, Care JW ten (1980) Mechanized amidolytic technique for determination of factor X and factor X-antigen, and its application to patients being treated with oral anticoagulants. Clin Chem 26 : 885-890
