Relationship of milk intake and vitamin K supplementation to vitamin K status in newborns.

Vitamin K status was evaluated by measuring blood acarboxyprothrombin (PIVKA-II) levels on the fifth day of life. The incidence of PIVKA-II-positive infants was higher in breast-fed babies than in those given supplementary (mixed) feeding. The median of total amount of milk intake during the first 3 days was significantly lower in PIVKA-II-positive infants than in PIVKA-II-negative infants among infants given both types of feedings. In addition, there was a significant negative correlation between a positive PIVKA-II proportion and the amount of milk intake in the breast-fed babies. The minimum dose of vitamin K2 necessary to prevent a positive PIVKA-II reading was 15 micrograms among babies with a normal absorption potential.

Vitamin K effect in low birth weight infants.

Factor II coagulant antigen (FII-AG), the protein induced by vitamin K absence or antagonist II (PIVKA-II), and coagulant activity (normotest) were measured in low birth weight infants. The factor II coagulant antigen and normotest levels in one-day-old babies were lower than those of full-term infants (P less than .005, P less than .01, respectively). Infants whose normotest levels were less than 30% at one day (group A) received vitamin K2, and the others whose normotest levels were greater than 30% at one day (group B) were not treated. At this time, the mean factor II coagulant antigen level was significantly lower in group A than in group B (P less than .05). During the first seven days of life, in 65.2% of the infants in group B the PIVKA-II level became positive, but this did not occur in any infant in group A. After vitamin K treatment, there was greater improvement in the normotest level in infants with positive PIVKA-II levels than in those with negative PIVKA-II levels. This observation indicates that the hypoprothrombinemia found in low birth weight infants at one day of age is attributable to reduced synthesis of factor II coagulant antigen in the liver at this stage, but the prophylactic administration of vitamin K seemed to be effective even in such infants, probably because of the increase in factor II coagulant antigen synthesis (P less than .001) during the first seven days of life.

Des-gamma-carboxyprothrombin (PIVKA II) and plasma vitamin K1 in newborns and their mothers.

Assessments of the vitamin K status in newborns and their mothers by means of des-gamma-carboxy-prothrombin (PIVKA II) measurement have given equivocal results. Part of the variability could be attributed to differences in sensitivity (i.e. the ability to detect small concentrations) and validity (i.e. ability to detect vitamin K deficiency) of the methods applied. None of these methods have yet been validated with respect to plasma vitamin K1. In 22 healthy mother/infant pairs PIVKA II was determined using three different assays including ratio Xa/ecarin (Xa/ec), crossed immunoelectrophoresis (CIE), and an ELISA with a monoclonal antibody (MAB). The results were compared with conventional clotting tests and plasma vitamin K1. The following results were obtained: Cord blood: Clotting tests within age-related normal ranges; PIVKA II detection rates: 0/22 (Xa/ec), 1/22 (CIE), 4/22 (MAB); plasma vitamin K1: undetectable in 20/22. Mothers: Clotting tests all within normal range; PIVKA II detection rates: 1/22 (Xa/ec), 0/22 (CIE), 5/22 (MAB); plasma vitamin K1 (pg/ml) for all mothers (median; range): 186; 55-833; for PIVKA II positive mothers: 213; 59-699. PIVKA II detectability in newborns and mothers was not correlated. The results show an increase in sensitivity for PIVKA II detection in the order of MAB >> CIE > Xa/ec. Due to the very low plasma vitamin K1 at birth, no correlation was possible between cord PIVKA II detectability and plasma vitamin K1. However, in mothers at term PIVKA II MAB appears to be unrelated to the vitamin K status.

Immunoaffinity purification of human erythrocyte prolidase.

A procedure including immunoaffinity gel chromatography of an immobilized monoclonal antibody was used to isolate human erythrocyte prolidase (EC 3.4.13.9). The monoclonal antibody was developed against liver prolidase and the antibody recognized the erythrocyte enzyme. The purification procedure included three steps of DEAE cellulose (batcher), immunoaffinity gel chromatography and gel filtration column chromatography. The overall recovery was approximately 20% and the specific activity of the purified preparation was approximately 260 U/mg of protein, a value exceeding that obtained using conventional procedures.

Clinical evaluation of plasma abnormal prothrombin (PIVKA-II) in patients with hepatocellular carcinoma.

The clinical usefulness of plasma abnormal prothrombin, defined as a protein induced by vitamin K absence or antagonist-II: PIVKA-II, as a tumor marker for hepatocellular carcinoma (HCC), was evaluated. Plasma PIVKA-II concentration was determined by an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specific for PIVKA-II. Forty-one (65%) out of 63 patients with HCC had an abnormal PIVKA-II level above 0.13 arbitrary units (AU)/ml; the level was above 0.3 AU/ml in 33 patients (52%) and above 0.5 AU/ml in 27 patients (43%). On the other hand, most of the 282 patients with various liver diseases other than HCC had normal or slightly elevated levels of PIVKA-II. Their values were all below 0.5 AU/ml, with the exception of 2 patients with decompensated liver cirrhosis. The patients with PIVKA-II values above 0.5 AU/ml were strongly suspected of having HCC. Plasma PIVKA-II levels were not related to serum alpha-fetoprotein (AFP) levels, but were above 0.5 AU/ml in 14 (44%) out of the 32 patients whose serum AFP levels were below 400 ng/ml. In some patients with HCC, PIVKA-II was increased throughout the course of the disease, and in others it normalized after surgical resection of the tumor. We conclude that the plasma PIVKA-II assay by the ELISA method using a monoclonal antibody is a useful diagnostic tool for monitoring HCC, particularly in HCC patients with low AFP levels.

Assimilatory nitrate reductase in a chlorate-resistant mutant of Escherichia coli.

Nitrate reductase was investigated in extracts from cells of a chlorate-resistant mutant strain of E. coli which grew anaerobically on nitrate as the sole source of nitrogen. The nitrate reductase was of particulate nature and reduced chlorate like the nitrate reductase from the wild strain, but in contrast was inhibited only weakly by azide or cyanide. Nitrate reductase activity was found in extracts from the mutant cells grown on nitrate as the sole source of nitrogen, but not in extracts from cells grown in complex nutrient medium. Addition of ammonia also caused a decrease in activity. Accordingly, the nitrate reductase in the chlorate-resistant mutant is of the assimilatory type.

Effect of vitamin K administration on acarboxy prothrombin (PIVKA-II) levels in newborns.

PIVKA-II (protein induced by vitamin K absence or antagonist-II) was measured in two groups of newborns, one group being given 5 mg vitamin K at birth and the other untreated. The untreated group had a significantly higher proportion of PIVKA-II positive babies at 3 and 5 days of age than did the treated group. When vitamin K was administered to newborn babies whose normotest levels were less than 30%, it was found that the higher the pre-treatment PIVKA-II levels the greater the response to vitamin K, as monitored by the normotest. Thus PIVKA-II levels might be more useful than a coagulation test, since the low activity of vitamin K dependent coagulation factors sometimes reflects not vitamin K deficiency but impaired production of these factors because of immaturity. The findings support the view that vitamin K given prophylactically at birth will help to prevent neonatal bleeding.

Vitamin K deficiency in breast-fed infants at one month of age.

PIVKA-II (protein induced by vitamin K absence or antagonist-II) was measured, as an indicator of vitamin K deficiency, in breast-fed infants of approximately 1 month of age. The infants consisted of three different groups: untreated (group 1); those given 5 mg vitamin K once at birth (group 2); and those given 5 mg twice, at birth and at 14 days after birth (group 3). At 1 month of age, the rate of PIVKA-II-positive infants and their PIVKA-II levels were significantly reduced in group 3 as compared with the levels of the other two groups, whereas these parameters were similar between groups 1 and 2. This observation suggested that vitamin K administration once at birth may be unsafe by 1 month of age. An additional administration of vitamin K seemed to be necessary for complete prevention of vitamin K deficiency, causing severe bleeding in breast-fed infants of approximately 1 month of age.

Screening for late neonatal vitamin K deficiency by acarboxyprothrombin in dried blood spots.

Acarboxyprothrombin (protein induced by vitamin K absence or antagonist-II (PIVKA-II] concentrations in dried blood spots were determined in 19,029 infants at about 1 month of age as an indicator of vitamin K deficiency. We observed 51 cases with raised blood concentrations of PIVKA-II (greater than 4 AU/ml), nine of whom showed very high concentrations (greater than 20 AU/ml). For infants who did not receive vitamin K prophylaxis at birth, the incidence of the PIVKA-II test yielding positive results was significantly higher in those solely breast fed (0.51%) compared with those fed formula milk (0.18%). Among solely breast fed infants, the incidence of a very high result of the PIVKA-II test was 0.14% in those who had not received vitamin K prophylaxis at birth, 0.04% in those who received 2 mg orally, and 0.03% in those who received 2 mg orally plus a further dose of 2-4 mg orally at 7 days. Thus vitamin K prophylaxis at birth did not completely prevent vitamin K deficiency at 1 month. We administered vitamin K therapeutically to all infants whose PIVKA-II test yielded a positive result at 1 month. Only one infant with a positive result developed late neonatal intracranial haemorrhage.

Immunochemical studies of human prolidase with monoclonal and polyclonal antibodies: absence of the subunit of prolidase in erythrocytes from a patient with prolidase deficiency.

Prolidase was highly purified from human liver and erythrocytes. NaDodSO4/acrylamide gel electrophoresis revealed that these preparations contained a major protein with MW = 56,000. The mass of prolidase was estimated on gel filtration to be MW = 97,000, for both enzyme preparations. A monoclonal antibody was raised against the liver enzyme and a specific antiserum against the erythrocyte enzyme. The monoclonal antibody (EP-2) recognized prolidase from erythrocytes and liver, in equal proportions. The antiserum also recognized the enzyme from erythrocytes and liver. Immunoprecipitation studies with these antibodies suggested only a single species of prolidase in erythrocytes and liver. Using an immobilized monoclonal antibody (EP-2) as an immunoadsorbent, prolidase was partially purified from crude extracts, and the protein of the partially purified enzyme was identified by immunoblotting using antiserum. A protein band with a MW = 56,000 was demonstrated specifically when crude extracts from the liver and erythrocytes were examined using NaDodSO4/acrylamide gel electrophoresis. The subunit protein was absent in erythrocytes from a patient with prolidase deficiency. We propose that the absence of the subunit is one cause of the prolidase deficiency.

Acarboxy prothrombin (PIVKA-II) as a marker of hepatoblastoma in infants.

We evaluated plasma PIVKA-II (protein induced by vitamin K absence or antagonist-II, acarboxy prothrombin) levels in three infants with hepatoblastoma as a tumor marker. PIVKA-II levels were highly elevated in all three patients. Vitamin K administration, performed in two patients, resulted in only moderate reduction of PIVKA-II levels. Chemotherapy against tumor cells reduced the PIVKA-II levels without exception. Immunohistochemical study of the liver tissue indicated the presence of PIVKA-II in the hepatoblastoma cell. These findings suggest that elevated PIVKA-II in these patients was not due to nutritional vitamin K deficiency, but to excess production of tumor cells. A measurement of plasma PIVKA-II may be useful as a new marker of hepatoblastoma.

Supplementation of vitamin K in pregnant women receiving anticonvulsant therapy prevents neonatal vitamin K deficiency.

OBJECTIVE: The null hypothesis of this study is that extra vitamin K administered to pregnant women on a regimen of enzyme-inducing anticonvulsant therapy will not decrease the frequency of symptoms of vitamin K deficiency in their neonates. STUDY DESIGN: A multicenter case-control study was performed on 16 pregnant women on anticonvulsant therapy who received 10 mg of vitamin K1 daily from 36 weeks of pregnancy onward. Concentrations of PIVKA-II (protein induced by vitamin K absence for factor II) and of vitamin K1 were determined in cord blood and compared with those in 20 controls. RESULTS: In none of 17 cord samples was PIVKA-II detectable, compared with 13 of 20 in controls (chi 2, p < 0.001). Median cord vitamin K1 level was 530 pg/ml compared with below detection limit in most controls. CONCLUSIONS: Antenatal vitamin K1 treatment decreases the frequency of vitamin K deficiency in neonates of mothers on anticonvulsant therapy.

Vitamin K status in cystic fibrosis.

Appearance of PIVKA-II (protein induced by vitamin K absence-II) in serum is a biochemical sign of insufficient vitamin K-dependent carboxylation of prothrombin. Plasma concentrations of PIVKA-II and vitamin K1 were determined in 24 children with cystic fibrosis. Eight were supplemented with vitamin K1. The purpose of the study was to determine the occurrence of vitamin K deficiency in cystic fibrosis and to evaluate the effect of vitamin K supplementation. PIVKA-II was detectable in only one unsupplemented child. In this patient, the concentration of vitamin K1 was below the limit of detection of 60 ng/l. Vitamin K1 levels in the other unsupplemented children were normal (mean 476 ng/l = 1 mmol/l). The supplemented patients showed extremely high levels of vitamin K1 (mean 22445 ng/l = 50 nmol/l). In conclusion, vitamin K deficiency occurs infrequently in cystic fibrosis. Checking the coagulation system is advised, but routine vitamin K supplementation is not recommended. If additional vitamin K is needed, the starting dose should not exceed 1 mg daily.

Detection of vitamin K deficiency by use of an enzyme-linked immunosorbent assay for circulating abnormal prothrombin.

A monoclonal antibody was raised against an abnormal decarboxylated prothrombin by a cell fusion technique. A cell line which produces an IgG1 murine antibody to the abnormal prothrombin, but not to prothrombin, was selected. Using this antibody we developed an enzyme-linked sandwich immunoassay for the abnormal prothrombin. The detection range was 0.5 X 10(-1) approximately 0.5 X 10(-3) micrograms protein of decarboxylated prothrombin and 0.5 approximately 0.5 X 10(-2) micrograms protein of abnormal prothrombin in vitamin K-deficient subjects. This discrepancy is attributable to a heterogeneity of decarboxylated prothrombin, depending on the number of gamma-carboxyglutamic acid residues. The antibody obtained had a higher affinity to a protein possessing less gamma-carboxyglutamic acid residues. The assay system developed may be useful for the detection of vitamin K deficiency, since a severe deficiency may result in less gamma-carboxyglutamic acid residues in the protein.

Oral supplementation of vitamin K for pregnant women and effects on levels of plasma vitamin K and PIVKA-II in the neonate.

Levels of plasma vitamin K1 (VK1) and vitamin K2 (VK2) and protein-induced vitamin K absence-II (PIVKA-II) were measured in Japanese mothers and their newborn (N = 33). Twenty milligrams of VK1 (N = 11) or VK2 (N = 12) were given orally to randomly selected mothers 7 to 10 days prior to delivery. Means of plasma VK1 and VK2 concentrations were significantly higher in VK1 (p less than 0.01) and VK2 (p less than 0.01) treated mothers than in the controls at delivery, respectively. Similarly, these levels were significantly elevated in cord plasma in VK1 (p less than 0.05) and VK2 (p less than 0.05) treated groups, compared with findings in the control group, although there was a large concentration gradient between maternal and cord plasma (mostly less than one-tenth). A significant positive correlation was found in VK1 concentration between maternal and cord plasma (N = 33, p less than 0.01), and the proportion of PIVKA-II-positive infants was significantly lower in the VK treated groups than in the control group at birth (p less than 0.05). On the fifth postnatal day, mean levels of VK1 (p less than 0.01) and VK2 (p less than 0.01) in breast milk were significantly higher in the VK1 and VK2 treated mothers than in the control mothers, respectively. In the control group, 9 of 10 infants had a positive PIVKA-II, but no one in the treated groups was positive, thereby indicating significant differences between control and treated groups (p less than 0.01 and p less than 0.01, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the type of feeding on the presence of PIVKA-II in infants.

PIVKA-II levels were studied by a highly sensitive immunological method in two groups of infants, breast-fed and bottle-fed, at the age of 4 days, 1 month, 2 months, and 3 months. PIVKA-II could be demonstrated in 9 infants after the age of 1 month when they were breast-fed. In none of the bottle-fed infants PIVKA-II was present during the same period. This significant difference can probably be explained by the lower vitamin K1 content of human milk compared to commercial formulas. The frequently occurring biochemical deficiency of vitamin K indicates the need of prophylactic administration of vitamin K to all newborns.