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The genetic locus of human transcobalamin II (TC2) is not yet known. The mouse transcobalamin II gene has been assigned to mouse chromosome 11, linked to hemoglobin A. This fact suggested a similar linkage of transcobalamin II in man, assigning it thus to human chromosome 16. Our linkage investigation in a family material of more than 600 individuals demonstrated absence of linkage between transcobalamin II and phosphoglycolate phosphatase, which is very closely linked to hemoglobin A on chromosome 16. Additionally we confirmed absence of linkage with the chromosome 16 gene marker system haptoglobin. These two gene marker systems are located far from each other, and the total length of chromosome 16 is estimated only about 100 cM. Together with recent results of investigations in somatic mouse-man cell hybrids, we conclude that TC2 is not located on chromosome 16. Additionally we found absence of linkage between transcobalamin II and 6-phosphoglucona-te dehydrogenase, rhesus blood group (both on chromosome 1), GC (chromosome 4), Esterase D (chromosome 13) and AG; absence of close linkage with “debrisoquin polymorphism”.

Tetrahydrobiopterin (BH(4)) is widely used as a therapeutic agent in patients with BH(4) deficiencies and mild forms of phenylketonuria (PKU) and there is an increasing need for the measurement of its plasma concentrations in patients with cardiovascular disorders. We measured BH(4) and total biopterin in dithioerythritol (DTE) pretreated plasma from four adults after oral administration of BH(4) (2, 10, and 20mg/kg body weight) using the differential iodine oxidation method. About 80% (range 64.8-92.2% ) of total biopterin was found as BH(4) when analyzed immediately after blood sampling. Compared with ascorbic acid as an antioxidant, DTE was more protective against oxidation of BH(4), particularly in samples stored over a period of 8 months. Without antioxidant (DTE or ascorbic acid) almost no BH(4) was detected. Furthermore, BH(4) and total biopterin were measured at different time intervals (up to 33 h after oral administration) and pharmacokinetic parameters T(max) (1-4h), C(max) (258.7-259.0 nmol/L biopterin at a dosage of 10mg/kg), and area under the curve (AUC=1708-1958 nmol(*)h/L up to T=10h) were estimated. The elimination half-life time was calculated to be 3.3-5.1h. Doubling the BH(4) dosage to 20mg/kg resulted in 60% higher AUC while sublingual BH(4) application (2mg/kg) resulted in 58-76% higher BH(4) plasma concentrations when compared with oral administration. These preliminary data suggest that in patients with BH(4) cofactor defects and BH(4)-responsive phenylalanine hydroxylase deficiency, BH(4) should be given in at least two to three daily doses and that sublingual administration may lower the required BH(4) dosage and subsequently the cost of treatment. Due to inter individual differences in pharmacokinetic properties, in some patients with hyperphenylalaninemia and mild PKU plasma BH(4) levels may be not high enough to fully activate the liver phenylalanine hydroxylase and thus lower blood phenylalanine levels. Assessment of plasma BH(4) or total biopterin concentrations may be a good way to control the efficacy of the loading test.

Background: Dopa-responsive dystonia (DRD) and tetrahydrobiopterin (BH4) defects are inherited disorders characterized by monoamine neurotransmitter deficiency with decreased activity of one of the BH4-metabolizing enzymes. The aim of the study was to determine the utility of cultured skin fibroblasts for the diagnosis of these diseases. Methods: Neopterin and biopterin production and GTP cyclohydrolase I (GTPCH) activity were measured in cytokine-stimulated fibroblasts; 6-pyruvoyltetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) activities were measured in unstimulated fibroblasts. We examined 8 patients with DRD, 3 with autosomal recessive GTPCH deficiency, 7 with PTPS deficiency, 3 with DHPR deficiency, and 49 controls (35 fibroblast and 14 amniocyte samples). Results: Fibroblasts from patients with DRD and autosomal recessive GTPCH deficiency showed reduced GTPCH activity (15.4% and 30.7% of normal activity, respectively) compared with controls (P Conclusions: Cultured skin fibroblasts are a useful tool in the diagnosis of BH4 deficiencies. Intracellular neopterin and biopterin concentrations and GTPCH activity in cytokine-stimulated fibroblasts are particularly helpful in diagnosing patients with DRD.

Tetrahydrobiopterin (BH 4 ) is an obligatory cofactor and regulator of nitric oxide synthases (NOS). We evaluated the biosynthesis of BH 4 in human umbilical vein smooth muscle cells (HUVSMC). Trace amounts of BH 4 were found intra- and extracellularly in untreated cells. When HUVSMC were activated by individual inflammatory stimuli (IL-1 β , TNF α , IFN γ or LPS), both intra- and extracellular levels of BH 4 increased significantly, with TNF α being the most potent single stimulus. Combined inflammatory cytokines synergized in the induction of an up to 600-fold increase of BH 4 synthesis. Addition of LPS to the cytokine mixture led to a further increase of BH 4 synthesis. Neopterin, a product of the first intermediate in BH 4 biosynthesis, was also raised, but to a much lesser extent. The increase of BH 4 synthesis was paralleled by an enhanced expression of isoform-1 (the only isoform coding for the active enzyme) of GTP cyclohydrolase I in cytokine treated cells. Our results show for the first time that BH 4 biosynthesis is strongly induced by combinations of inflammatory stimuli in HUVSMC. The importance of BH 4 -dependent NO synthesis in HUVSMC needs, however, additional detailed studies.

The functional relationship of nitric oxide (NO) production and synthesis of tetrahydrobiopterin (BH 4 ), the requisite cofactor for NO synthase, was investigated in rat aortic smooth muscle cells (SMC). Inflammatory cytokines induced BH 4 and NO synthesis in different ratios, IL-1β induced mainly NO synthesis with concomitant but limiting amounts of BH 4 for maximal NO production. TNFα did not induce NO synthesis but induced BH 4 synthesis. IFNγ was ineffective on both the induction of NO and BH 4 synthesis. TGFβ downregulated NO production but did not affect BH 4 biosynthesis. IL-4 and IL-10 had no effect on both BH 4 and NO synthesis. Activating cytokines strongly synergized in induction of NO production, whereas endogenous BH 4 production became insufficient for maximal NO synthesis. Exogenous cofactor in the form of sepiapterin or authentic BH 4 , but not the natural isomer 7-BH 4 , enhanced NO production twofold. Inhibition of BH 4 synthesis with dicumarol abolished NO production that could be restored in the presence of BH 4 .

Tetrahydrobiopterin (BH4) is an obligatory cofactor of nitric oxide synthase and an essential regulator of its activity. The murine vascular endothelial cell line send1 constitutively secretes large amounts of BH4 as well as scant amounts of neopterin, an oxidized intermediate in the de novo biosynthesis of BH4. Further enhancement of BH4 and neopterin secretion is achieved by activation with endotoxin (LPS) and interferon-gamma. This finding is in accordance with previous described BH4 secretion by human endothelial cells. It supports the view that endothelial cells are the source of BH4 serving vascular needs in vivo. In septic conditions, BH4 released by endothelial cells in large amounts could serve induced nitric oxide synthase in smooth muscle cells, thereby acting as another endothelium-derived relaxing factor mediating vasodilatation.

We describe a new fully reliable method for the differential diagnosis of tetrahydrobiopterin-dependent hyperphenylalaninaemia (HPA). The method comprises the combined phenylalanine (Phe) plus tetrahydrobiopterin (BH4) oral loading test and enables the selective screening of BH4 deficiency when pterin analysis is not available or when a clear diagnosis has not been previously made. It should be performed together with the measurement of dihydropteridine reductase (DHPR) activity in blood. The new combined loading test was performed in nine patients with primary HPA, three with classical phenylketonuria (PKU), three with DHPR deficiency, and three with 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency. Three hours after oral Phe loading (100 mg/kg body weight), synthetic BH4 was administered orally at doses of either 7.5 or 20 mg/kg body weight. Amino acid (Phe and tyrosine) and pterin (neopterin and biopterin) metabolism and kinetics were analysed. By exploiting the decrease in serum Phe 4 and 8 h after administration, a clear response was obtained with the higher BH4 dose (20 mg/kg body weight), allowing detection of all cases of BH4 deficiency, as well as differentiation of BH4 synthesis from regeneration defects. Since DHPR deficient patients who were previously shown to be non-responsive to the simple BH4 loading test gave a positive response, the combined Phe plus BH4 loading test can be used as a more reliable tool for the differential diagnosis of HPA in these patients. Moreover, it takes advantage of being performed while patients are on a Phe-restricted diet.

The relationship between blood phenylalanine concentrations and serum and erythrocyte biopterin and neopterin concentrations was investigated in 20 phenylketonuric patients with different dietary compliance. At serum phenylalanine concentrations ranging from 43 to 1004 μmol/l, a good correlation was found with serum biopterin (r = 0.76, P < 0.001) and with red blood cell biopterin (r = 0.62, P < 0.001). A similar correlation was found between serum neopterin and phenylalanine (r = 0.60, P < 0.001). The correlation between red blood cell neopterin and serum phenylalanine was less evident, however (r = 0.47, P < 0.005). After oral loading with phenylalanine (100 mg/kg body weight), serum and red blood cell biopterin concentrations increased in patients with classical phenylketonuria as well as in one patient with dihydropteridine reductase deficiency in response to the induced acute hyperphenylalaninemia. One patient suffering from 6-pyruvoyl tetrahydropterin synthase deficiency was loaded orally with tetrahydrobiopterin (20 mg/kg body weight). The kinetics of administered cofactor confirmed its rapid absorption, with early increase of serum concentrations followed by its transport into the red blood cells. The half-life of biopterin was approximately 7 h in serum and 15 h in red blood cells. Because both values are less than the half-life of phenylalanine (20–30 h) in serum, biopterin measurement offers no advantage in monitoring dietary control in hyperphenylalaninemic patients.

Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper.

Tetrahydrobiopterin (BH4) is the cofactor for phenylalanine hydroxylase (PAH) and is essential to treat different forms of hyperphenylalaninaemia, i.e. patients with defects in the biosynthesis of BH4, but also in the PAH gene [1, 4,5]. Responsiveness of mutant PAH to BH4 administration relies on a chemical chaperon effect of BH4 preventing PAH from protein misfolding and inactivation [6], but several other mechanisms may also be involved [2]. Recently, we reported that sublingual administration of BH4 tablets in a single control person resulted in 58%–67% higher plasma BH4 concentrations than the usual oral route [3]. Drawbacks included rapid decomposition of tablets, acidic taste, and increased salivation. Since sublingual application could reduce therapy costs, orange flavoured artificially sweetened BH4 pastilles were tested on four healthy volunteers and a child with mild phenylketonuria (PKU). BH4 (Schircks, Switzerland) was administered to four healthy male volunteers (age 28–57 years) on 2 subsequent days either as tablets (50 mg) or pastilles (100 mg, containing 5 mg aspartame and orange aroma) with a 1 week wash-out between the two trials. Blood was collected before, and 1, 2, 3, and 4 h after administration. An 8-year-old patient with mild PKU (genotype P281L/R261Q) who was already on regular BH4 therapy was treated with BH4 pastilles (sublingually, 12 mg/kg/day; bid) in addition to P-AM 2 formula for 2 weeks. Written consensus was obtained from the family. In the four healthy male volunteers, total biopterin values before and up to 4 h after oral and sublingual administration were compared (Fig. 1A). No statistically significant difference between oral and sublingual administration was observed. Pterin concentrations increased after both oral and sublingual administration, reflecting instability of BH4 in blood. The ratio of pterin to cumulated biopterin and pterin ranged between 22.8% and 33.5%. Summing biopterin and pterin instead of total biopterin did not alter the ratio between oral and sublingual administration (data not shown). Changing from standard therapy to sublingual administration in a child with mild PKU resulted in almost unchanged plasma phenylalanine concentrations (Fig. 1B). After 2 weeks of twice daily sublingual BH4 administration, the patient complained of a painful prickling sensation at the tip of the tongue. He nevertheless continued the treatment for a further 10 days, moving the tablets in his mouth from one side to the other, which resulted in severe mucitis. His condition normalised rapidly upon discontinuation of the sublingual form. No mucitis appeared in healthy volunteers. A clinical trial with sublingual BH4 in a patient with mild PKU failed despite pharmacological effectiveness on blood phenylalanine levels because of an adverse pastille-related side-effect. Formulation of buffered tablets approaching neutral pH and further investigations on BH4 metabolism and pharmacokinetics are needed.

Objective: To describe three unrelated children with a distinctive variant of Aicardi-Goutières syndrome (AGS) characterized by microcephaly, severe mental and motor retardation, dyskinesia or spasticity, and occasional seizures.Results: Neuroimaging showed bilateral calcification of basal ganglia and white matter. CSF glucose, protein, cell count, and interferon alpha were normal. Abnormal CSF findings included extremely high neopterin (293 to 814 nmol/L; normal 12 to 30 nmol/L) and biopterin (226 to 416 nmol/L; normal 15 to 40 nmol/L) combined with lowered 5-methyltetrahydrofolate (23 to 48 nmol/L; normal 64 to 182 nmol/L) concentrations in two patients. The absence of pleocytosis and normal CSF interferon alpha was a characteristic finding compared to the classic AGS syndrome. Genetic and enzymatic tests excluded disorders of tetrahydrobiopterin metabolism, including mutation analysis of GTP cyclohydrolase feed-back regulatory protein. CSF investigations in three patients with classic AGS also showed increased pterins and partially lowered folate levels.Conclusions: Intrathecal overproduction of pterins is the first biochemical abnormality identified in patients with AGS variants. Long-term substitution with folinic acid (2-4 mg/kg/day) resulted in substantial clinical recovery with normalization of CSF folates and pterins in one patient and clinical improvement in another. The underlying defect remains unknown.

Tetrahydrobiopterin deficiency among newborns with hyperphenylalaninaemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU), because patients in the two groups require different treatment to prevent irreversible neurological damage (Blau 1988; Dhondt 1991). The characteristic pattern of pterins excreted in urine makes it possible to identify all four variants of tetrahydrobiopterin deficiency

Four patients with primapterinuria, postulated to be due to pterin-4alpha-carbinolamine dehydratase (PCD) deficiency, were diagnosed by biochemical and DNA analysis. All four patients presented in the neonatal period with hyperphenylalaninemia, and elevated neopterin and decreased biopterin levels in the urine. These symptoms are common to 6-pyruvoyltetrahydropterin synthase deficiency and thus there is a danger of misdiagnosis. In addition, all four patients had elevated urinary excretion of primapterin (7-biopterin), the only persistent biochemical abnormality. Analysis of fibroblast DNA from the patients identified the following mutations in the PCBD gene: one patient homozygous for the missense mutation E96K and one homozygous for the nonsense mutation Q97X, both in exon 4; one compound heterozygote with the mutations E96K and Q97X; and one patient with two different homozygous mutations: E26X in exon 2 and R87Q in exon 4. In two families, the parents were investigated and found to be obligate heterozygotes for particular mutations. One sibling was found to be unaffected. These results further substantiate the idea that primapterinuria is associated with mutations in the PCBD gene.

The objectives of this study were to find additional diagnostic information for the evaluation of xanthine dehydrogenase deficiency and molybdenum cofactor deficiency. Patients were given an oral loading test with 10 mg/kg 5,6,7,8-tetrahydrobiopterin. Urine excretion of pterin and isoxanthopterin was measured by HPLC. Control subjects had a fairly constant ratio of urinary pterin/isoxanthopterin before (0.57-5.32) and after (0.55-4.55) 5,6,7,8-tetrahydrobiopterin loading. These ratios were increased to 33 and 22 in a patient with hereditary xanthinuria and to 570 and 8030 in a patient with molybdenum cofactor deficiency. Obligate heterozygotes had an entirely normal test result. Evidence was obtained for the in vivo involvement of xanthine dehydrogenase in the conversion of pterin to isoxanthopterin. This test could be a sensitive marker for the establishment of residual enzyme activity.

Prenatal diagnosis of tetrahydrobiopterin (BH4) deficiency was undertaken by evaluating the pterin patterns in amniotic fluid and the specific enzyme activities in fetal or extrafetal tissues. This allowed the prenatal diagnosis in 19 pregnancies at risk. In 8 families with a child already affected by dihydropteridine reductase deficiency 4 fetuses were diagnosed as homozygotes and 4 as heterozygotes for the defect. In 11 families with a child affected by 6-pyruvoyl tetrahydropterin synthase deficiency 4 fetuses were homozygous, 4 heterozygous and 3 normal. This study also advanced our knowledge of tetrahydrobiopterin metabolism during fetal development. The key enzymes involved in the biosynthesis of BH4 are expressed early and allow the fetus to be autotrophous for its cofactor requirement. In a twin pregnancy, both fetuses were diagnosed to be heterozygotes for dihydropteridine reductase deficiency and primapterin (7-biopterin) in amniotic fluid was increased. This indicates that pterin-4 alpha-carbinolamine dehydratase activity seems to be differently expressed during fetal life. As a consequence, pterins detected in amniotic fluid are of fetal origin and 6- and 7-substituted pterins can be present in amniotic fluid in higher proportions when compared with other body fluids.

Primapterinuria, a recently discovered variant of hyperphenylalaninaemia, is characterized by the excretion of 7-substituted pterins in the patient's urine (Curtius et al 1988). Although 9 patients have already been diagnosed worldwide (Dhondt et al 1987, 1988; Blaskovics and Giudici 1988; Blau et al 1988), the exact metabolic defect is still hypothetical. Patients with primapterinuria show an increased ratio of neopterin to biopterin excretion, excretion of subnormal levels of biopterins, and normal levels of biogenic amines in CSF (Blau et al 1989; Curtius et al 1990a)

Accessible online at: www.karger.com/journals/crd Dear Sir, The vascular endothelium has important secretory, metabolic, and immunologic functions. The impairment of such functions is an early step in the pathogenesis of several diseases including atherosclerosis [1], and endothelium-dependent vasodilation induced by nitric oxide (NO) is certainly a key function. Tetrahydrobiopterin (BH4) is intimately involved in NO-mediated vasomotion. The exact role of BH4 in NO catalysis remains debated, but may include stabilization of the active conformation of the enzyme, action as an allosteric effector enhancing substrate binding, coupling of substrate oxidation to NADPH consumption thereby reducing the ratio of superoxide to NO production, and posttranscriptional stabilization of NO synthase mRNA [2, 3]. In addition, BH4 is a potent antioxidant and scavenger of oxygen-derived free radicals [2, 4]. Impaired NO production and increased free radical formation have been observed in several diseases predisposing to atherosclerosis [4]. In states of BH4 depletion, endotheliumlocated NO synthase switches from NO to superoxide production. Thus, a local BH4 deficiency in endothelial cells may be of major importance in the pathogenesis of a dysfunctional endothelium as a mechanism of oxidative vascular injury [3]. This consideration is corroborated by several in vitro and Table 1. BH4 levels in 17 patients with normal coronary vessels and in 17 patients with 3-vessel disease

Transcobalamin 2 is the only vitamin B12-binding protein found in mouse serum. Two allelic forms of mouse transcobalamin 2 are described. The two forms differ in their mobilities on polyacrylamide gel electrophoresis. The slowly migrating form has been found in serum from 25 inbred mouse strains. The more rapidly migrating form was detected in 3 inbred mouse strains (NZB, ST/bJ, and CPB-WV). Both parental variants were expressed in F1 progeny of appropriate interstrain crosses, showing codominant expression of the transcobalamin 2 alleles. In backcrosses between F1 and parental individuals, the two electrophoretic variants were inherited as single Mendelian traits. The strain distribution pattern of the two variants in recombinant inbred lines likewise suggested a single-gene mode of inheritance and indicated a lack of close linkage with a number of genetic loci on chromosomes 1, 2, 4, 5, 6, 7, 9, 12, 14, 15, and 17. We propose the symbol Tcn-2 for the polymorphic gene locus coding for transcobalamin 2 in the mouse and Tcn-2s and Tcn-2f for the two alleles.

A solid-phase radio immunoassay was developed for total immunoreactive transcobalamin II (TC II). Rabbit antihuman TC II antiserum (which recognizes both apo- and holo-TC II), was immobilized by covalent binding to acrylamide-acrylic acid copolymer beads. A normal mean and SD for immunoreactive TC II in serum was determined in 130 healthy adult individuals and found to be 1150 ± 250 ng/liter cobalamin equivalent. Mean holo-TC II (N = 30), estimated by substraction of apo-TC II from total TC II, was 137 ng/liter bound cobalamin (or 12% of total TC II). Three patients with lack of functional TC II had immunoreactive TC II levels between 22 and 39% of normal mean, which demonstrated that the solid-phase bound antiserum recognized deficient TC II molecules, whereas the same antiserum in its soluble form did not. Eight out of nine individuals, recognized as heterozygous for TC II deficiency, had TC II levels below the normal range, on the order of 50% of the normal mean. The stability of immunoreactive TC II was strongly enhanced by the presence of an unknown serum factor not corresponding to serum albumin.

We have found normal concentrations of neopterin, monapterin, isoxanthopterin, biopterin and pterin in the urine of 10 patients with Rett syndrome, and normal values for total biopterin and neopterin in the blood of 4 subjects. Thus, there is no biochemical evidence of a generalized tetrahydrobiopterin deficiency in this syndrome. Since we had no opportunity so far to study cerebrospinal fluid, a defect in the metabolism of pterins in the central nervous system is not yet fully excluded.

Atypical phenylketonuria (PKU) is a group of very rare and severe diseases caused by tetrahydrobiopterin (BH4) deficiency (Niederwieser and Curtius, 1987). So far three inborn errors of metabolism are known to cause BH4 deficiency. defects in: GTP cyclohydrolase I (GTPCH); 6-pyruvoyl tetrahydropterin synthase (PPH4S); and dihydropteridine reductase (DHPR) (Blau, 1988). Recently a new form of atypical PKU with unusual 7-iso-biopterin excretion in the urine of patients was described (Curtius et al., 1988). Prenatal diagnosis of BH4 deficiency can be achieved mainly by measurement of pterin metabolites in amniotic fluid and of enzyme activities in cultured fluid cells and fetal blood (Blau et al., 1987).

Phenotypes of transcobalamin II (TC2) were determined in 95 maternal- cord serum pairs in order to identify the origin of TC2 in human cord blood. Unsaturated (apo) TC2 in serum was labeled with radioactive (57Co) cobalamin (CbI) and separated into isoproteins by polyacrylamide gel electrophoresis and autoradiography. Discordancy between the maternal and the cord serum type was observed in 45% of the pairs. The results demonstrated that, at the end of pregnancy, the fetus is capable of TC2 synthesis and that there is no detectable transplacental passage of maternal apo-TC2. Presence of maternal saturated (holo) TC2 in cord serum could be excluded in 9 informative discordant pairs by exchanging endogenously bound CbI with 57Co-CbI. Our finding that TC2 in human cord serum is of fetal rather than maternal origin suggests an essential role for fetal TC2 in CbI utilization and appears to contradict the hypothesis that transplacental passage of maternal TC2 may explain the normal fetal development in cases of congenital TC2 deficiency. The total immunoreactive TC2 content in 23 maternal serum samples collected at the end of pregnancy (812 +/- 175 pM CbI equivalent) was significantly higher than in the corresponding cord sera (605 +/- 148 pM; p less than 0.001) and did not significantly differ from the value in a control group of healthy male and female adults (841 +/- 192 pM). At the end of pregnancy, the apo-TC2 content in 12 maternal serum samples (760 +/- 347 pM) was significantly higher than in the corresponding cord sera (501 +/- 254 pM; p less than 0.05) and did not significantly differ from the value in the control group (747 +/- 137 pM).

Earlier investigations have shown that the activity of autoimmune diseases appears to correlate with increased levels of the vitamin B12 (cobalamin)-binding serum protein, transcobalamin II (M. Fráter-Schröder et al., Schweìz. Med. Wochensch 110, 1441, 1980; M. Fráter-Schröde et al., Lancet 2, 238, 1978). These preliminary findings were confirmed and extended with regard to SLE in the present prospective study. The correlation of serum levels with the degree of disease activity (determined by clinical scoring of 44 patients with systemic lupus erythematosus (SLE), fulfilling four or more of the American Rheumatoid Association criteria) was shown to be most reliable for transcobalamin II (P less than 0.001), when compared to other serological parameters. An answer to the question "what induces increased levels of transcobalamin II in active SLE?" was sought by injecting 15 SLE patients with cyanocobalamin which influences plasma dynamics of transcobalamin II. Results indicate that transcobalamin II-cobalamin "clearance" is probably unchanged in SLE, and that increased production or stimulation of transcobalamin II secretion may be the cause of elevated plasma levels in active SLE.

Summary Pterin-4a-carbinolamine dehydratase (PCD) is required for efficient tetrahydrobiopterin regeneration after phenylalanine hydroxylase activity. This catalytic function was proposed to be specifically defective in newborns with a mild form of hyperphenylalaninemia (HPA) and persistent high urinary levels of primapterin (7-biopterin). A second regulatory task of the same protein is DCoH, a coactivation of transcription by hepatocyte nuclear factor 1α (HNF-1α), a function that is apparently not impaired in these HPA individuals. It has been shown elsewhere that the human PCD/DCoH bifunctional protein is encoded by a single 4-exon–containing gene, PCBD , located on chromosome 10q22. We have now examined the PCBD gene for mutations at the genomic level in six such HPA patients from four different families. By the use of new intron-specific primers, we detected, in all six patients, single, homozygous nucleotide alterations, in exon 4, that were inherited from their parents. These homozygous alterations predicted mutant PCD/DCoH with a single amino acid exchange, in two cases (alleles T78I), or premature stop codons, in the other four patients (alleles E86X and Q97X). Recombinant expression in Escherichia coli revealed that the mutant proteins—T78I, E86X, and Q97X—are almost entirely in the insoluble fraction, in contrast to wild type, which is expressed as a soluble protein. These data support the proposal that HPA in combination with urinary primapterin may be due to autosomal recessive inheritance of mutations in the PCBD gene specifically affecting the dehydratase activity.

Phenotypes of transcobalamin II (TC2) were determined in 95 maternal- cord serum pairs in order to identify the origin of TC2 in human cord blood. Unsaturated (apo) TC2 in serum was labeled with radioactive (57Co) cobalamin (CbI) and separated into isoproteins by polyacrylamide gel electrophoresis and autoradiography. Discordancy between the maternal and the cord serum type was observed in 45% of the pairs. The results demonstrated that, at the end of pregnancy, the fetus is capable of TC2 synthesis and that there is no detectable transplacental passage of maternal apo-TC2. Presence of maternal saturated (holo) TC2 in cord serum could be excluded in 9 informative discordant pairs by exchanging endogenously bound CbI with 57Co-CbI. Our finding that TC2 in human cord serum is of fetal rather than maternal origin suggests an essential role for fetal TC2 in CbI utilization and appears to contradict the hypothesis that transplacental passage of maternal TC2 may explain the normal fetal development in cases of congenital TC2 deficiency. The total immunoreactive TC2 content in 23 maternal serum samples collected at the end of pregnancy (812 +/- 175 pM CbI equivalent) was significantly higher than in the corresponding cord sera (605 +/- 148 pM; p less than 0.001) and did not significantly differ from the value in a control group of healthy male and female adults (841 +/- 192 pM). At the end of pregnancy, the apo-TC2 content in 12 maternal serum samples (760 +/- 347 pM) was significantly higher than in the corresponding cord sera (501 +/- 254 pM; p less than 0.05) and did not significantly differ from the value in the control group (747 +/- 137 pM).

Increased serum levels of the essential vitamin B12 binding protein, transcobalamin II (TC2) were previously observed in autoimmune disease. The periods of raised serum level correlated with clinical disease activity in patients with SLE and dermatomyositis. The correlation of serum levels with disease activity in a large group of 44 Swiss SLE patients was shown to be most reliable for TC2, when compared to certain established serological markers such as complement factors C3 and C4, antinuclear antibody titer or antinative DNA antibodies. Several questions were raised: Why is the TC2 level elevated in active SLE? Is the accumulation in serum due to lack of TC2 uptake by the cell or is it due to stimulation of synthesis? Answers were sought by applying a plasma turnover test for TC2 to the SLE patients. After 400 ng/kg cyanocobalamin (i.m.) the TC2 level decreased, due to preferential uptake of holo TC2 by the cells. Total TC2 levels were determined by radioimmunoassay. Normalisation of the plasma level and corresponding reappearance of apo TC2 was interpreted as newly synthesized TC2. Twelve SLE patients and six healthy controls were investigated. One SLE patient was treated with a higher cobalamin dose (200 micrograms) to ensure complete saturation of TC2. The TC2 level decrease after cobalamin injection was comparable in controls and patients, independently of the state of the disease. Normalisation of plasma levels was significantly faster, elevation above starting levels was observed, in 3 of 5 SLE patients exhibiting active disease. In the remaining 9 patients normalisation of the plasma level was comparable to the control group. Our conclusions are that TC2 uptake, in other words TC2 consumption by the cell, is unchanged in SLE, and that an increased rate of TC2 synthesis may be the cause of elevated plasma levels in active SLE.

Human skin fibroblasts and bone marrow cells were tested for their ability to synthesize the cobalamin-binding protein transcobalamin II. Cobalamin binders secreted in the media of cultured fibroblasts and of dextran-sedimented bone marrow cells in liquid culture could be identified as transcobalamin II on the basis of immunological, electrophoretical and chromatographical identity with serum transcobalamin II. The net secretion of transcobalamin II increased linearly with time of culture, up to 30 days after confluence. The reversible inhibition of transcobalamin II secretion by cycloheximide demonstrated that human fibroblasts are capable of de novo transcobalamin II synthesis. Addition of cyanocobalamin to the fibroblast culture medium induced a reduction of transcobalamin II net secretion, most likely due to preferred uptake of transcobalamin II saturated with cobalamin, as opposed to unsaturated protein. Addition of lysozymal enzyme inhibitors, ammonium chloride and chloroquine, resulted in a markedly increased secretion of transcobalamin II. In the culture medium of fibroblasts, obtained from two transcobalamin II-deficient patients, functionally deficient transcobalamin II was demonstrated on the basis of strongly reduced secretion of immunoreactive transcobalamin II, and the absence of apotranscobalamin II. Individual phenotypes in the culture media of the fibroblasts and bone marrow cells were identical to the corresponding serum transcobalamin II types.

Studies concerning the site of synthesis of the vitamin B12 binding serum protein, transcobalamin II, have been done in various mammalian animals. The actual site of biosynthesis in man has not yet been defined. The finding that bone marrow derived cells release apo-transcobalamin II in the mouse led us to examine the genetic patterns of transcobalamin II in man, both before and after marrow transplantation. A gradual but incomplete transformation of the recipient's transcobalamin II type into donor's type, corresponding to 75% or less of the total activity, was registered in 4 cases. Surprisingly, persistent host-type TC II, in spite of different donor type, was observed in 4 further marrow recipients. We conclude that hematopoietic cells transferred with the transplanted marrow participate in the biosynthesis of human transcobalamin II.