Kunihiko Motohara

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.

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 ∼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 ∼1 month of age.

Late neonatal vitamin K deficiency associated with subclinical liver dysfunction in human milk-fed infants

because of the small number of epidermal appendages, or to other causes is not known. Scaling of the skin did not seem to be as consistent a feature of any form of ectodermal dysplasia other than X-L HED, although the numbers are too small to be conclusive. Early diagnosis of X-L HED has several advantages. Life-threatening complications can be prevented, genetic counseling can be given early, and the parents have an opportunity to plan appropriately for the future (e.g., changing the environment to control heat exposure, obtaining and maintaining adequate dental and medical insurance). Scaling or peeling of the skin of a newborn infant, especially a male infant, should alert the examiner to the possibility of X-L HED. Careful, directed examination is warranted and a detailed family history should be obtained. Examination of the mother may be helpful. If suspicion is high, dental radiographs will reveal diagnostic tooth abnormalities. Skin biopsy is usually not necessary for confirmation.

Immunochemical Studies of Human Prolidase with Monoclonal and Polyclonal Antibodies:Absence of the Subunit of Prolidase in Erythrocytes from a Patient with Prolidase Deficiency

ABSTRACT. 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.

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 < 0.01) and VK2 (p < 0.01) treated mothers than in the controls at delivery, respectively. Similarly, these levels were significantly elevated in cord plasma in VK1 (p < 0.05) and VK2 (p < 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 < 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 < 0.05). On the fifth postnatal day, mean levels of VK1 (p < 0.01) and VK2 (p < 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 < 0.01 and p < 0.01, respectively). The administration of both VK,1 and VK2 to mothers prior to delivery may prevent hypoprothrombinemia in infants, at birth, and during neonatal stages.

Immunochemical analysis of prolidase deficiency and molecular cloning of cDNA for prolidase of human liver

Prolidase (imidodipeptidase, EC 3.4.13.9) splits imidodipeptides with C-terminal proline or hydroxyproline residues. In humans, prolidase activity that is absent or markedly decreased results in prolidase deficiency (McKusick 26413), a genetic disease transmitted through autosomal recessive inheritance (Scriver et al., 1983). This biochemical defect has been associated with mental retardation, imidodipeptiduria, and, in some instances, deep skin ulcers (Ogata et al., 1981). The properties of prolidase from mammalian, including human, tissues have been reported. Most of the evidence obtained indicates that prolidase is a ubiquitous enzyme whose substrate specificity is restricted to imidodipeptides (Endo et al., 1981, Yoshimoto et al., 1983). However, no immunological analysis of human prolidase with a specific antibody has yet been described and the nature of the mutation has not been analysed at the molecular level. In this study, we isolated human prolidase and prepared monoclonal and polyclonal antibody. We were able to demonstrate the absence of the subunit of prolidase in a patient with prolidase deficiency. Further, a cDNA expression bank of human liver Was screened immunologically by using antibody against prolidase and a cDNA clone for the enzyme was isolated and identified.

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.

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.

Absence of the subunit of prolidase in a patient with prolidase deficiency

Prolidase deficiency (McKusick 26413) is a rare inherited disorder characterized by ulceration of the skin, mental retardation and massive urinary excretion of imidodipeptides (Scriver, 1978). Some of the properties of human prolidase derived from erythrocytes have been studied by Adams and Smith (1952). The purification of human prolidase has previously been described by Endo and colleagues (1982). However, no immunological analysis of human prolidase with a specific antibody has yet been described and the nature of prolidase mutation has not been analysed in man at the protein level. Recently we have prepared monoclonal and polyclonal antibodies directed against human prolidase. We have used the antibodies to demonstrate the subunit of prolidase in crude extracts of tissues and cells.