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Abstract
Background Vitamin K deficiency bleeding (VKDB) in infants is a rare but serious worldwide problem, particularly in Southeast Asia. Apart from exclusive breast feeding, little is known of the maternofetal risk factors that predispose infants to VKDB.
Objectives To assess (a) the relationships between functional vitamin K insufficiency in a large cohort of Thai mothers to that of their newborn infants and (b) the importance of delivery risk factors and maternal intakes of vitamin K as determinants of neonatal vitamin K status.
Methods Vitamin K status was assessed by measuring undercarboxylated prothrombin (protein induced by vitamin K absence/antagonist-II (PIVKA-II)) in 683 mothers and in the cord blood of their babies by sensitive immunoassay. Dietary phylloquinone (vitamin K1; K1) intakes were assessed in 106 of these mothers by food frequency questionnaire.
Results Babies were categorised as ‘normal’ (n=590) or ‘high risk’ (n=93) according to birth weight and delivery type. PIVKA-II was detectable (>0.15 arbitrary units (AU)/ml) in 85 mothers (12.4%) and 109 babies (16.0%) with median levels of 0.78 and 1.04 AU/ml in mothers and babies, respectively. ‘High-risk’ babies had a higher median detectable PIVKA-II concentration than ‘normal-risk’ babies (3.1 vs 1.0 AU/ml, p=0.02) and a higher prevalence of clinically relevant (>5.0 AU/ml) concentrations (p=0.006). Mothers with K1 intakes below the US recommended ‘adequate intake’ for pregnancy (<90 µg/day) had a higher prevalence of detectable PIVKA-II (18.8%) than those with adequate intakes (3.3%) (p=0.01).
Conclusions Functional, clinically relevant, vitamin K insufficiency was more common in ‘high-risk’ than ‘normal-risk’ newborns. Vitamin K insufficiency in mothers was linked to lower dietary K1 intakes during pregnancy.
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Introduction
Vitamin K is an antihaemorrhagic factor needed for the γ carboxylation of prothrombin (factor II) and factors VII, IX and X in the liver. The consequences of vitamin K deficiency for haemostasis are an inability to synthesise functional molecules of factors II, VII, IX and X, resulting in a hypocoagulable state and, with progressive deficiency, a bleeding tendency that is individually unpredictable. Vitamin K deficiency bleeding (VKDB) in infants is a rare but serious worldwide problem with a high risk of mortality and permanent disability resulting from intracranial haemorrhage.1,–,6 Three forms of VKDB have been described according to the age of the infant at presentation.1 They are early (0–24 h), classical (1–7 days) and late (8 days to 6 months but typically 2–12 weeks). Surveillance data show that the introduction of nationwide vitamin K prophylaxis regimens for newborns leads to a dramatic decrease in the incidence of classical and late VKDB.2,–,7
What is already known on this topic
Without adequate vitamin K prophylaxis, the incidence of vitamin K deficiency bleeding (VKDB) in Southeast Asia is up to 10-fold higher than in Europe.
Measurements of undercarboxylated prothrombin (protein induced by vitamin K absence/antagonist-II (PIVKA-II)) offer a sensitive method of assessing functional vitamin K status that can be used to study candidate risk factors for maternal and neonatal vitamin K insufficiency.
What this study adds
This is the largest population study of the prevalence of vitamin K insufficiency in mothers and infants at the time of delivery and the first evaluation of maternal dietary vitamin K intakes during pregnancy.
The findings suggest that vitamin K insufficiency in a minority of newborns may be exacerbated by prematurity, stressful deliveries or intrauterine growth retardation.
The findings show the importance of maintaining adequate dietary intakes of vitamin K throughout pregnancy.
Exclusive breast feeding is a major pathophysiological risk factor in the development of classical and late VKDB6 In the first week of life, there is evidence that vitamin K status (as measured by coagulation factors and protein induced by vitamin K absence/antagonist-II (PIVKA-II) prevalence) is strongly related to the volumes of breast milk that the mother is able to provide to her infant.6 However, little is known about vitamin K requirements during pregnancy and how maternal dietary intakes impact on the vitamin K status of mother and newborn infant. Because of this lack of data, the US recommendations for an ‘adequate intake’ of vitamin K during pregnancy are currently the same as for non-pregnant women, namely 90 µg/day.8 This study reports on the vitamin K status of Thai mothers and their newborn babies and the role of maternal dietary phylloquinone (vitamin K1; K1) intakes as determinants of status.
Methods
Participants
A total of 683 mothers and their newborn babies from Ramathibodi Hospital, Bangkok, Thailand (n=323) and the Mother and Child Health Regional Hospital, Khonkaen, Thailand (n=360) were enrolled in the study. The Apgar scores were 9 to 10 at 1 min and 10 at 5 min. Birth characteristics and delivery risk classification are shown in table 1. Newborns were categorised as ‘normal risk’ if born with birth weights ≥2500 g and by vaginal delivery (n=590) or ‘high risk’ (n=93) if born either <37 weeks' gestational age (n=16), small for gestational age (SGA; n=23), by caesarean section (n=20), forceps extraction (n=16) or vacuum extraction (n=18). Venous blood was drawn from the mothers during the first stage of labour, approximately 6 h before the delivery and from the umbilical vein of the cord after the cord was cut, immediately before the separation of the placenta. After centrifugation at 3000 rpm for 15 min, serum was separated and stored at −70°C until assayed. The Faculty Committee on Medical Research Ethics approved the study, and informed consent was obtained from all participating mothers.
Birth characteristics and delivery risk classification of 683 newborns with distribution of protein induced by vitamin K absence/antagonist-II (PIVKA-II) concentrations in those 109 newborns with detectable PIVKA-II (>0.15 AU/ml) indicative of functional vitamin K insufficiency
Determination of K1 intakes in mothers
None of the mothers had been taking any drugs, for example, oral anticoagulants, phenytoin or rifampicin, known to interfere with vitamin K metabolism. A food frequency questionnaire was designed to estimate K1 consumption from the Thai diet. During the immediate post partum period, the mothers were interviewed by a dietician who recorded the frequency and amount of each type of food eaten in the 3-month period before delivery. The types of food were categorised into seven groups, namely: (1) milk and dairy products; (2) meat and meat products; (3) rice and cereals; (4) legumes, nut, seed and products; (5) fruit; (6) vegetables; and (7) fats and oils. Special attention was paid to vegetables as the major source of K1. Commonly eaten vegetables in Thailand include cucumber, sweet pepper, lettuce, bean sprouts, tomato, asparagus, broccoli, cabbage, carrot, cauliflower, fresh beans, okra, spinach, kale, swamp cabbage, water mimosa, ivy gourd, cassia leaves, pumpkin and potato. Where possible, intakes of fat and oil were obtained from the type and estimated amounts of fat and oil used during food preparation such as stir-frying. In some cases information of the content and type of fat and oil in some cooked and commercial dishes was incomplete. The average frequency of consumption of each food item was recorded. Nine frequency categories ranging from ‘never or less than once a month’ to ‘equal to or more than four times/day’ were indicated in the questionnaire. The average consumed portion sizes were described either in multiples or submultiples of the food item (for example, one egg, half an apple), or in standard weight and volume by using standard measuring cups and spoons. The recorded frequency categories for each food item were converted to daily K1 intakes using the median values of the K1 content of foods obtained from Booth et al,9,–,12 Koivu et al,13 Piironen et al14 and Bolton-Smith et al.15 Certain Thai foods (swamp cabbage, water mimosa) for which K1 contents were not available from the literature were assigned K1 values from ‘like foods’ for which values were available.
A daily K1 intake of 90 µg/day was defined as adequate based on the US recommended ‘adequate intake’ (AI) for pregnancy.8
Determination of undercarboxylated prothrombin
Undercarboxylated prothrombin (PIVKA-II) was assayed by ELISA using a conformation specific monoclonal antibody (C4B6) that selectively binds undercarboxylated species of prothrombin in the presence of calcium ions and does not crossreact with native fully carboxylated prothrombin.16,–,18 Serum standards containing varying concentrations of PIVKA-II were prepared by diluting a stock serum pool obtained from participants receiving warfarin anticoagulant therapy (PIVKA-II positive) with a serum pool from healthy adults (PIVKA-II negative). The concentration of PIVKA-II in the stock warfarin anticoagulated serum was measured by a commercial ELISA PIVKA-II kit (EITEST from Eisai, Tokyo, Japan). PIVKA-II values by our C4B6 assay were reported in arbitrary units (AU) in the same way as those reported by the EITEST assay. This enables the PIVKA-II values of the C4B6 assay to be calibrated against values obtained by the EITEST assay such that 1 AU/ml is equivalent to 1 µg PIVKA-II purified by electrophoresis.16,–,19 The use of arbitrary units instead of weight/molar units is recommended in such assays because even electrophoretically pure PIVKA-II comprises multiple species of partially carboxylated prothrombin, and neither their relative abundance in plasma nor their relative affinity for the antibody is known.16 19 In healthy, vitamin K-replete adult participants, the concentration of PIVKA-II was <0.15 AU/ml; therefore values above this cut-off were reported as ‘detectable’ and indicative of functional vitamin K insufficiency.18 In overt vitamin K deficiency, PIVKA-II circulates at high levels: values were 6.9–99.5 AU/ml (mean 40.0) in 43 adults on warfarin therapy (International Normalized Ratio ≥1.5)18 and 67.9 AU/ml in an infant with fatal late VKDB.20 PIVKA-II values of ≤1.0 AU/ml are considered insignificant to coagulation16 while values ≥5.0 AU/ml are indicative of overt vitamin K deficiency and clinically relevant.
Statistics
χ2 or Fisher exact tests were used for discrete data, where appropriate. Mann–Whitney U or Wilcoxon signed rank tests were used for continuous data. A p value <0.05 was considered statistically significant.
Results
Participant characteristics
All mothers were Thai, with a median age of 27 years (interquartile range 23.0–31.0). They delivered 351 male and 329 female newborn babies with data lacking in 3 babies (tables 1 and 2). No significant differences were found between the data obtained from the two participating hospitals in Bangkok and Khonkaen with respect to the mothers, delivery type, birth weight and gender of the babies, as well as the concentrations of PIVKA-II in mothers and their infants. Bangkok and Khonkaen are cities located in the central and north-eastern parts of Thailand, respectively, separated by 450 km, with a similar lifestyle and Thai food intake.
Protein induced by vitamin K absence/antagonist-II (PIVKA-II) measurements in cord samples categorised according to gender and to maternal factors (gravidity, delivery type, delivery risk, adequacy of vitamin K1 intakes, detectable PIVKA-II concentrations) and showing the proportion of cord samples in each category with a detectable PIVKA-II (>0.15 AU/ml) indicative of functional vitamin K insufficiency
PIVKA-II concentrations in mothers and infants
PIVKA-II was detectable (>0.15 AU/ml) in 85 of 683 mothers (12.4%) (40 from Bangkok) and 109 of 683 babies (16.0%) (56 from Bangkok) with median concentrations of 0.78 AU/ml (interquartile range 0.35–1.40) and 1.04 AU/ml (interquartile range 1.04–2.43), in mothers and babies, respectively. The distribution of detectable PIVKA-II in 85 mothers and 109 babies is shown in figure 1. All eight mothers with PIVKA-II ≥5.0 AU/ml delivered normal babies with undetectable PIVKA-II.
The distribution of detectable protein induced by vitamin K absence/antagonist-II (PIVKA-II) (>0.15 AU/ml) indicative of functional vitamin K insuffi ciency among (A) 85 mothers and (B) 109 babies.
The distribution and concentrations of detectable PIVKA-II (>0.15 AU/ml) in 109 newborns according to delivery type and associated risk factors are shown in tables 1 and 2. No statistically significant difference was found in the prevalence of detectable PIVKA-II between primigravida and multigravida, vaginal and operative delivery, male and female babies and ‘high-risk’ and ‘normal-risk’ babies. However, the median concentration of detectable PIVKA-II of ‘high-risk’ babies was significantly higher than that of ‘normal-risk’ babies (3.1 vs 1.0 AU/ml, p=0.02). Moreover, the prevalence of PIVKA-II concentrations ≥1.0 AU/ml and ≥5.0 AU/ml in ‘high-risk’ babies (8/10 and 4/10, respectively) was higher than that in ‘normal-risk’ babies (51/99 and 6/99, respectively) with corresponding p values of 0.10 and 0.006, respectively. The four ‘high-risk’ infants with PIVKA-II concentrations representing overt vitamin K deficiency (≥5.0 AU/ml) were distributed equally among premature, SGA, caesarean section and vacuum extraction groups (table 1). Their mothers all had an undetectable PIVKA-II. Two mothers in the ‘normal-risk’ group who delivered babies with a PIVKA-II ≥5.0 AU/ml had detectable PIVKA-II of 0.2 and 1.0 AU/ml, respectively. Mothers with detectable PIVKA-II delivered a higher proportion of babies with detectable PIVKA-II compared to mothers with undetectable PIVKA-II (22% vs 15%; table 2) without quite attaining statistical significance (p=0.08).
Maternal dietary K1 intakes and PIVKA-II concentrations
Complete dietary records of K1 intakes in the 3-month period immediately before delivery were obtained for 106 mothers from Ramathibodi Hospital in Bangkok. As previously reported in a UK population,21 the distribution of K1 intakes was positively skewed. The mean intake (SD) of K1 was 306 µg/day (252) and the median intake (interquartile range) was 241 µg/day (122–417). The main sources of dietary K1 were green leafy vegetables with significant amounts from fats and oils including soybean oil, palm oil, safflower oil and sunflower oil. Rice is a staple food in Thailand, but contains very low amounts of K1.15
Of the 106 mothers in whom K1 intakes were determined, 16 had intakes below the US recommended AI of <90 µg/day with a median intake (interquartile range) of 61.6 µg/day (49.7–85.3). The prevalence of detectable PIVKA-II in mothers with ‘inadequate’ intakes was significantly higher than those with ‘adequate intakes’ (3/16 vs 3/90, p=0.01) but the absolute concentrations were all ≤0.5 AU/ml and clinically insignificant. None of these mothers had any signs or symptoms of systemic diseases or malabsorption except for one mother who was diagnosed as having heterozygous haemoglobin E. Their weight gains during pregnancy were within the normal range and all of them delivered neonates with birth weights >2500 g. The only finding in common to all these mothers was that they had an aversion to eat green leafy vegetables.
Discussion
The prevention of VKDB has been a major health concern in Thailand since 1963, when this country was the first to recognise and describe the pathophysiology of the syndrome now known as late VKDB.6 22 Data collected from literature reports and a field survey suggested that until the late 1970s the prevalence of classical and late VKDB were both of the order of 1 per 1000 births but thereafter declined dramatically as the availability of vitamin K prophylaxis has increased.5 When complete national coverage in Thailand was achieved in 1994, the incidence of late VKDB had declined to European levels of 4.2–7.8 per 100 000 births.5 6 As found in many studies worldwide, the two major risk factors associated with idiopathic late VKDB in Thailand are exclusively breast feeding and not receiving vitamin K prophylaxis at birth.5 A similarly high incidence of late VKDB without vitamin K prophylaxis has been reported in several countries in Asia including Japan, Vietnam and China.23,–,25 This high incidence, often 10-fold higher than in Europe, suggests that there are environmental or genetic factors that predispose to VKDB in Asian populations.
In the present study, we found that 12.4% of mothers had evidence of a functional vitamin K insufficiency and that the proportion of these mothers with vitamin K intakes below the US recommendation (AI) for pregnancy (90 µg/day) was higher than in those mothers who met this level of adequacy. As far as we are aware this is the first study to measure dietary intakes of vitamin K in pregnancy in any population. The association between dietary intakes and vitamin K status provides some evidence of the value of the food frequency questionnaire that was used to assess K1 intakes. Nevertheless as with all dietary assessments there are limitations that apply to the accuracy of the food composition database for K1 and to the questionnaire instrument. One of these limitations is the wide variability of K1 contents of oils and fats and the problem in finding out the fat composition of different food dishes.15 As found in the National Diet and Nutrition Survey in Thailand,26 some pregnant women habitually ate a diet that was extremely low in green leafy vegetables.
Despite the relationship between vitamin K status and intakes in mothers, no firm relationship was found between the vitamin K status of mothers and that of their infants although there was a trend (p=0.08) for mothers with detectable PIVKA-II to deliver babies with detectable PIVKA-II. A lack of correlation between PIVKA-II detectability in mothers and newborns was previously reported for a small European cohort of 22 maternal/cord samples and may be due to several unknowns which include individual differences in maternal placental transport and the lipoprotein transport system that delivers vitamin K to the placenta.27 The factors that govern the placental transport of vitamin K are poorly understood but K1 does not easily cross the placenta and the maternal/cord concentration gradient is within the range of 20:1 to 40:1.28 For this reason it is not feasible to accurately measure K1 concentrations in cord plasma. The importance of maintaining adequate vitamin K intakes during pregnancy is illustrated by case reports showing that maternal dietary depletion can lead to fetal cerebral haemorrhage29,–,31 or at early stages to fetal skeletal abnormalities, probably due to inactivity of the vitamin K-dependent matrix Gla protein.32 33 In one of these cases of fetal subdural haematoma, the mother's PIVKA-II was only slightly elevated showing that fetal VKDB can develop even when the maternal deficiency is mild.31.
To our knowledge, this is the largest study of vitamin K status of mother–infant pairs at the time of birth. Comparison of PIVKA-II detectability with other studies is difficult because of methodological34 and population differences. With our C4B6 MAb-based assay, a PIVKA-II concentration of ≥1.0 AU/ml represents an unequivocally abnormal undercarboxylated level of prothrombin.17 It is noteworthy that 8.6% (59/683) of Thai newborns in this study had PIVKA-II concentrations ≥1.0 AU/ml, compared to only 1.1% (1/90) of British preterm infants using the same assay.17 A further 1.5% (10/683) of our Thai cord samples had PIVKA-II concentrations ≥5.0 AU/ml, values approaching or within the range (6.9–99.5 AU/ml) found in patients during warfarin therapy. This compares favourably with a previous Thai study by Laosombat et al35 who found that 1.7% (4/230) of cord samples had PIVKA-II values ≥5.0 AU/ml using the commercial EITEST assay against which our assay was calibrated.
Recommendations for groups at risk of VKDB who should always receive vitamin K prophylaxis have traditionally included preterm and low-birthweight infants and those who have had difficult births36 although supportive evidence of increased risk is difficult to find. In 1 small study, 7 of 19 term infants with complications of labour and delivery were shown to have reduced factor II coagulant activity to antigen ratios consistent with vitamin K deficiency.37 In the present study the overall prevalence of vitamin K insufficiency did not differ between ‘normal-risk’ and ‘high-risk’ infants but infants with a more advanced, potentially clinically relevant, vitamin K insufficiency (PIVKA-II >5.0 AU/ml) were significantly more likely to occur in the ‘high-risk’ group (p=0.006). With the median levels of PIVKA-II also being significantly greater in the ‘high-risk’ group these findings provide supportive evidence that vitamin K insufficiency may be exacerbated by certain factors such as prematurity, stressful deliveries or intrauterine growth retardation although this was found only in a minority of babies.
Acknowledgments
The authors would like to thank Miss Pakaimas Pintadit and Miss Saranya Rurgkhum for their laboratory assistance and Thailand Research Fund-Senior Research Scholar 2006 (AC).
References
Footnotes
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Competing interests None.
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Ethics approval This study was conducted with the approval of the Ethical Clearance Committee on Human Rights Related to Researches Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University.
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Provenance and peer review Not commissioned; not externally peer reviewed.
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Patient consent Obtained.