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Background: Tandem mass spectrometry (MS/MS) is rapidly being adopted by newborn screening programs to screen dried blood spots for >20 markers of disease in a single assay. Limited information is available for setting the marker cutoffs and for the resulting positive predictive values., Methods: We screened >160 000 newborns by MS/MS. The markers were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Multiple reaction monitoring of each sample for the markers of interest was accomplished in approximately 1.9 min. Cutoffs for each marker were set at 6-13 SD above the population mean., Results: We identified 22 babies with amino acid disorders (7 phenylketonuria, 11 hyperphenylalaninemia, 1 maple syrup urine disease, 1 hypermethioninemia, 1 arginosuccinate lyase deficiency, and 1 argininemia) and 20 infants with fatty and organic acid disorders (10 medium-chain acyl-CoA dehydrogenase deficiencies, 5 presumptive short-chain acyl-CoA dehydrogenase deficiencies, 2 propionic acidemias, 1 carnitine palmitoyltransferase II deficiency, 1 methylcrotonyl-CoA carboxylase deficiency, and 1 presumptive very-long chain acyl-CoA dehydrogenase deficiency). Approximately 0.3% of all newborns screened were flagged for either amino acid or acylcarnitine markers; approximately one-half of all the flagged infants were from the 5% of newborns who required neonatal intensive care or had birth weights <1500 g., Conclusions: In screening for 23 metabolic disorders by MS/MS, an mean positive predictive value of 8% can be achieved when using cutoffs for individual markers determined empirically on newborns.

A carcinogen binding protein (CBP) that is implicated in controlling the expression of rat cytochrome P-450c which is closely associated with aryl hydrocarbon hydroxylase (AHH) was examined in hepatic and extrahepatic tissues of the neonatal and adult New Zealand White (NZW) rabbits, the hepatic tissue of the BALB/cJ and DBA/2J mice, Brown Norway rat, Golden Syrian hamster and Hartley guinea pig. These animals and tissues were examined in order to determine whether there was a correlation of CBP levels and the reported presence or absence of inducibility of AHH in these tissues. The CBP was found in hepatic and extrahepatic tissue of the NZW rabbit and the hepatic tissues of all animals except the Hartley guinea pig. The Hartley guinea pig may provide a useful animal with which to further examine the role of CBP in cytochrome induction. Since the CBP is not a tissue specific protein and because it is found in both neonatal and adult NZW rabbit tissue, the data suggests that the CBP is not the limiting factor in the tissue specific induction of cytochromes nor in developmentally controlled induction of cytochromes previously reported in the rabbit.

Nuclear proteins from AKR-2B mouse embryo cells incubated 24 hr with [3H]benzo(a)pyrene (1 microgram/ml; 10 microCi/ml) were purified to remove nucleic acids and characterized by isoelectric focusing and sodium dodecyl sulfate gel electrophoresis on polyacrylamide gels. Analysis of the nuclear proteins by isoelectric focusing revealed a distribution of protein in the pH gradient with two prominent bands of proteins, one focusing at pH 5 and the other at pH 11, both of which bound significant amounts of the carcinogen. The acid-soluble proteins from the nuclear preparation were analyzed by isoelectric focusing, and the results also revealed two peaks of protein and radioactivity at pH's of 5 and 11. Further analysis of the acid-soluble fraction on acid urea gels revealed that significant radioactivity was associated with proteins that migrated near but slower than histone H1 which may be the high-mobility group proteins 1 and 2. Radioactivity also comigrated with histones H3 and H2B. Low levels of radioactivity were associated with histone H1 and H4.

The distribution of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid was examined in proteins from a variety of sources. Proteins examined include purified rat and bovine coagulation proteins, barium citrate-adsorbing proteins from trout plasma, lamprey plasma, earthworm hemolymph, army worm hemolymph, lobster hemolymph, E. coli B/5, soybean leaf, the protein lysate from the hemolymph cell of the horseshoe crab and parathyroid extract. Other purified proteins examined included human alpha-1-antitrypsin, pepsinogen, S-100, fetuin, tropomyosin-troponin and complement protein C-3. Of these, only the blood-cotting proteins and the vertebrate plasma samples were shown to contain gamma-carboxyglutamic acid.

AKR-2B mouse embryo cells were incubated for 24 hr with [3H]benzo(a)pyrene, and the histones were isolated and analyzed using one- and two-dimensional gel electrophoresis and autoradiography. The results revealed that (a) histones H1, H2A, and H3 incorporated significant amounts of label whereas little or no label was associated with histones H2B and H4 and (b) electrophoresis of the histones in the Triton:acid:urea gel system caused labeled histones to have a slower migration than did the corresponding unlabeled histones. Additional studies such as incubation of (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene with nuclei resulted in radioactive labeling of histones H1, H2A, H2B, and H3 and of high-mobility-group proteins HMG1 and HMG2. The low levels of label associated with histone H4 in the whole-cell and nuclear studies were further investigated by incubating isolated histones with (+/-)-7 beta,8 alpha-[3H]dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene. Under these conditions, negligible amounts of radioactivity were associated with H4, while significant labeling of H1, H2A, H2B, and H3 and other nuclear proteins was observed. The results suggest that factors other than the presence of suitable nucleophilic acceptor sites on the histones may be necessary for carcinogen binding.

A protein that binds the chemical carcinogen, benzo(a)pyrene (BP), in a high-affinity and saturable manner has been identified in cell extracts from the AKR-2B mouse embryo cell line. The BP bound to the carcinogen-binding protein (CBP) was exchangeable in a time- and temperature-dependent fashion and has a Kd of 1.8 X 10(-9) M. Competitive binding studies indicate that chemical carcinogens [3-methylcholanthrene, benz(a)anthracene, dibenz(a,c)anthracene] and other inducers of aryl hydrocarbon hydroxylase (5,6- and 7,8-benzoflavone) compete to varying degrees with BP for binding. Steroids, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and phenobarbital did not compete with BP for binding to the CBP, BP prevented the heat inactivation of CBP. Additional properties of CBP reveal it to have a Stokes' radius of 31 A, molecular weight of 61,000, frictional ratio of 1.2, an apparent isoelectric point of 5.2, and an S value of 4.8 in linear sucrose gradients. It was estimated that there are about 20,000 molecules of CBP per AKR-2B mouse embryo cell. The CBP was found in two nontransformed and one chemically transformed cell line; a fourth cell line A-431 (human vaginal carcinoma) had significantly reduced amounts of CBP.

Twelve tissues from the BDA/2J mouse were examined in order to assess the tissue distribution of a soluble 4-5S carcinogen binding protein (CBP). The CBP binds numerous polycyclic aromatic hydrocarbons, regulates the expression of cytochrome P-450c and is a carrier protein of carcinogen metabolites. The highest specific binding was found in hepatic tissue (600 fmol per mg protein). The amount of CBP in the kidney and testis was about 60% of the value observed for the liver. Lower levels of CBP were observed in the reproductive tract (uterus and vagina), spleen, stomach, heart, urinary bladder, skin, large and small intestine and lung. It was shown that the CBP bound the environmental carcinogen 1-nitropyrene, its reduced metabolite 1-aminopyrene and the ultimate carcinogenic metabolite of benzo[a]pyrene or (+)-r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene but not the smaller coplanar PCB molecule, 3,3',4,4'-tetrachlorobiphenyl. The results suggest that (a) the CBP is not a tissue-specific protein in the mouse, (b) the ligands for the CBP are not limited to the unmetabolized procarcinogens but include both positively and negatively charged ligands, and (c) in vitro the CBP covalently binds the ultimate carcinogenic metabolite of benzo[a]pyrene.

The marked localization of a carcinogenic polycyclic aromatic hydrocarbon, benzo(alpha)pyrene, and its metabolites and a carcinogenic alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, to a specific subnuclear fraction (fraction I) from AKR-2B mouse embryo cells in culture is described. Fraction I is isolated by sucrose gradient centrifugation of sheared nuclei from cells exposed to the carcinogens. The association of tritiated benzo(alpha)-pyrene to fraction I consisted of loosely associated radioactivity which is extractable by organic solvents, and of tightly bound (termed "covalently" bound) radioactivity which is not extractable by organic solvents. Increases in the extent of metabolism of benzo(alpha)pyrene and in the amount of "covalently" bound radioactivity occur with increasing periods of incubation of the cells with the labelled carcinogen. This observation, together with the fact that these increases are dramatically reduced by inhibiting polycyclic aromatic hydrocarbon metabolism (using the inhibitor 7,8-benzo-flavone), suggests that a time-dependent metabolism of benzo(alpha)pyrene is required for "covalent" binding to muclear material. Data are presented suggesting that a two-step reaction may be involved in the binding of benzo(alpha)pyrene to subnuclear macromolecules. The fraction I localization of such structurally diverse chemical carcinogens as benzo(alpha)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine suggests that this fraction may localize all species of chemical carcinogens and that this localization may be involved in the chemically induced malignant transformation of cells.

A high-affinity localization of [2H]methylcholanthrene and/or its metabolites to a specific nuclear fraction (Fraction l) between 4 and 72 hr of exposure is described. Other carcinogenic hydrocarbons, such as benzo(a)pyrene and dibenz(a,h)anthracene, demonstrate a similar markded localization in Fraction l after 24 hr of incubation. The weak carcinogen dibenz(a,c)anthracene, as well as steroid hormones, sho little localization in this fraction. Two types of binding are measured:and organic solvent: extractable (non-covalent) binding and a nonextractable (covalent) binding. Maximal levels of the combined extractable and nonextractable binding per mass DNA are found at 24 hr of exposure, while at 48 and 72 hr of exposure the binding is reduced. The highest level of the nonextractable binding per mass DNA is also observed at the 24-hr exposure period. However, as the period of exposure increases, the proportion of the total nuclear-bound radioactivity representing the non-extractable type increases. Analysis by high-pressure liquid chromatography of the extractable radioactivity from the fractions indicates that the longer the period of exposure, the greater the extent of metabolism of 3-methylchol-anthrene. When 7,8-benzoflavone (a flavanoid hydroxylase inhibitor) is included in the incubations, practically all metabolic alterations of the parent compound are prevented. In addition, the time-dependent increase in nonextractable radioactivity from all nuclear subfractions is prevented. A metabolic-dependent "covalent" binding of carcinogenic polycyclic aromatic hydrocarbons to the barious nuclear subfractions of chromatin is suggested. This covalent binding is markedly localized in a specific fraction of the chromatin containing rapidly labeled nascent RNA.

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.

gamma-Carboxyglutamic acid has recently been identified as a component of the vitamin K-dependent region of bovine prothrombin (Nelsestuen, G. L., Zytkovicz, T. H., and Howard J. B. (1974) J. Biol Chem. 249, 6347-6350). The presence of this amino acid has been substantiated here by the reduction of vitamin K-dependent proteins with [3H]-DIBORANE. The reduction product of gamma-carboxyglutamic acid, 5,5'-[3H]dihydroxyleucine, was shown to be present in hydrolysates of reduced rat prothrombin, bovine prothrombin, and bovine factor X. The results are consistent with a minimum of 10 gamma-carboxyglutamic acid residues in the nonthrombin-generating region of bovine prothrombin but no such residues in the thrombin precursor portion of prothrombin. It is concluded that amino acid analyses of [3H]diborane-reduced proteins provides a sensitive, qualitative method for the identification of proteins which contain gamma-carboxyglutamic acid and are vitamin K-dependent.