Band 2, Heft 3

β,γ-Methano derivatives of methotrexate (MTX) and aminopterin (AMT) were synthesized with the aim of assessing the effect of this side-chain modification on dihydrofolate reductase (OHFR) inhibition, folylpolyglutamate synthetase (FPGS) inhibition, and tumor cell growth inhibition. Mixed carboxylic-carbonic anhydride (MCA) coupling of 4-amino-4-deoxy-N 10 -methylpteroic acid (mAPA) and dimethyl trans- α-(2-carboxycyclopropyl) glycinate, followed by alkaline hydrolysis, afforded N-( 4-amino-4-deoxy-N 10 -methylpteroyl)-α-( trans-2-carboxycyclopropyl)glycine (β,γ-methanoMTX) as a mixture of the four possible diastereomers with trans substitution on the cyclopropane ring. N-(4-Amino-4-deoxypteroyl)-trans-α.-(2-carboxycyclopropyl) glycine (β,γ-methanoAMT) , also as a diastereomer mixture, was obtained from 4-amino-4-deoxy-N 10 - formylpteroic acid (fAPA) and dimethyl trans-α-(2-carboxycyclopropyl)-glycinate by MCA coupling and alkaline hydrolysis of the ester and N 10 -formyl groups. β,γ-MethanoMTX and β,γ-methanoAMT may be viewed as MTX and AMT analogues with a conformationally restricted side chain. In vitro biological activity data for these novel compounds support the view that the active site of DHFR, already known for its ability to tolerate modification of the γ-carboxyl group of MTX and AMT, can likewise accommodate substitution on the β- and γ-carbons.

A novel and sensitive enzyme immunoassay for neopterin and biopterin using a microtitre plate has been developed. The antiserum against neopterin or biopterin was incubated with samples prepared from human urine or rat tissues, and was placed in a well of microtitre plate, where N 2 -(3-aminopropyl) derivative of neopterin or biopterin was kinked by poly-L-Iysine and glutaraldehyde. The antibody, which did not form the immunocomplex with the hapten in the sample, was bound to the fixed hapten on the plate, and then was measured with anti rabbit IgG and peroxidase anti-peroxidase complex. The detection limits of these pterins were both 0.1 pmol. The specificity of the assay was so high that this assay system with employing anti-neopterin antiserum completely distinguished neopterin from biopterin, as judged from the cross reactivity (0.002%) and vice versa. The amounts of neopterin and biopterin in human urine and rat tissues measured by the present assay showed a good agreement with those determined by high performance liquid chromatography.

To compare the involvement of cellular immunity in response to vaccination we have investigated urinary neopterin levels in daily follow-ups of children after vaccination with live measles/mumps vaccine and of adults after boosting with the soluble antigen tetanus toxoid. Neopterin levels distinctly peaked 8 - 11 days after vaccination with measles/mumps vaccine. In contrast, after boosting with soluble antigen tetanus toxoid neopterin levels remained unaffected. Large amounts of neopterin are produced by human monocytes/ macro phages on stimulation with gamma interferon. In patients neopterin concentrations reflect activation of cell mediated immunity. The data imply that distinct pathways of T cell activation are triggered in humans after immunization with live vaccine and with soluble antigen .

Enzymatic conversion of stereoisomers of biopterin (3a-d) to the 7-oxo-biopterin isomers (4a-d) was performed with a new pterin 7-oxidase extracted from carp skin. The chiral centers of the 6-side chains of biopterin isomers were preserved during the conversion, and the four possible stereoisomers of 7-oxo-biopterin (4a-d) were obtained from the corresponding biopterin isomers (3a-d). HPLC and CD spectra studies showed that natural ichthyopterin is 2-amino-6-(L-erythro -1',2' -dihydroxypropyl)-4, 7(3H,8H)-pteridinedione (4a) which has the same L- erythro structure at the side chain as natural biopterin (3a). This fact suggests that ichthyopterin is biologically derived from biopterin in these fishes.

We evaluated urinary neopterin in seven HIV positive children, aged 4 months to 4 years, 11 months, under Zidovudine (AZT) treatment, 300 mg/mq/12 hourly per os. The results were compared with HIV antigenaemia (HIV Ag) and CD4/CD8 ratios. Neopterin (μmol/mol creatinine) was determined by HPLC; HIV Ag assessed by enzyme-immunoassay and T-cell subsets by flow-citometry. Blood and urine samples were obtained before and at 3 and 6 months after starting therapy. There was no significant trend in CD4/CD8 ratios during AZT treatment. Neopterin and HIV Ag showed a statistically significant decrease (median values 3239, 1679, 2062 μmol/mol creatinine; 2.7, 1.3, 1.2 sample optic density/cut off optic density respectively; p < 0.05 by Friedman's test) and were also significantly correlated (p = 0.01 by Spearman's ranks correlation test). We conclude that urinary neopterin is useful in monitoring AZT therapy in HIV positive children.

Eleven mothers of 12 infants with human immunodeficiency virus type 1 (HIV-1) infection (all intravenous drug users) were followed throughout their pregnancies for evidence of clinical and immunological abnormalities. Intrauterine death occurred in one, growth retardation in 7/12 pregnancies. Moderate progression of HIV-l infection according to the Walter Reed Staging Classification was observed in 5/ 11 women during pregnancy. Two of them developed AIDS during follow up after delivery. Mean neopterin levels in the first, second and third trimester were significantly higher as compared to HIV-l seronegative pregnant women. Furthermore, concentrations of urinary neopterin increased significantly with the time of pregnancy in all HIV-1 seropositive women. Since higher urinary neopterin concentrations are predictive for more rapid disease progression in HIV infected patients the data may indicate that pregnancy increases the risk for developing AIDS. However, a possible influence of continuous drug use has to be considered.

Fifteen patients with clinical diagnosis of dilated cardiomyopathy (DCMP) were followed for 3 to 34 months. Serum neopterin and β 2 -microglobulin concentrations were measured by radioimmunoassay. According to the changes of neopterin and β 2 microglobulin levels in the follow-up study patients were divided into three groups. The course of disease in group 1 (5 patients with constant normal neopterin level <9 nmol/L) during the study) was stable without deterioration and increase of New York Heart Association (NYHA) Class. In group 2 (2 patients with raised neopterin in levels at the beginning of the study) some clinical improvement was associated with normalization of neopterin concentration. In group 3 (8 patients) the relation between the disease progression and increasing neopterin level was observed. The data of this study clearly indicated that neopterin measurements had a predictive value in patients with clinical diagnosis of DCMP.

Isotope-labeled dihydroneopterin 3'-triphosphate with 3 H at positions C-1' and C-2', respectively, has been prepared from isotope-labeled glucose as starting material. Glucose was first converted enzymatically to ribose 5-phosphate. GMP was subsequently obtained by the action of phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase. It was subsequently phosphorylated to GTP in two steps using adenylate kinase and guanylate kinase. Dihydroneopterin triphosphate was prepared from GTP by the action of recombinant GTP-cyclohydrolase I from Escherichia coli. The method allows the incorporation of 3 H and 14 C isotope labels into any desired position of dihydroneopterin triphosphate. Rapid purfication procedures for phosphoribosylpyrophosphate synthetase and guanosine phosphoribosyl transferase as well as HPLC assays for their determinations are described.

A new method for assay of sepiapterin deaminase (3.5.4.24) activity by use of high performance liquid chromatography (HPLC) was developed. By this sensitive method the enzyme activity in the cell organelle was assayed. After cell fractionation, the enzyme was extracted with deoxycholate from pteridine granules of epidermal cells of the lemon mutant silkworm. On stepwise sucrose gradient centrifugation, most of the enzyme activity localized in the aggregated pteridine granules fraction, while the soluble fraction contained only one fourth of the total enzyme activity. The enzyme in the granule fraction had the same properties as the previously reported sepiapterin deaminase. These data show that the enzyme is localized in pteridine granules in the living cells. The attachment of the enzyme to the granule membrane is rather loose and previous papers studied the enzyme released from the granules. Cell fractionation and morphological observation showed that sepiapterin, sepialumazine and other pteridines were also localized in the granules together with uric acid.

The effects of the hormones thyroxine, glucagon and hydrocortisone, and of protein level on hepatic histidase, urocanase, and certain folate enzymes were studied in rats. Hypothyroidism, induced either by feeding thiouracil or by thyroidectomy, increased histidase, urocanase, and the folate dependent enzyme formiminotransferase, and increased histidine metabolism as measured by oxidation of the 2-ring carbon of histidine to carbon dioxide. Hyperthyroidism, produced by feeding thyroid powder, decreased histidine oxidation and liver levels of this enzyme. Glucagon and hydrocortisone increased histidine oxidation and increased levels of histidase, urocanase and formiminotransferase. Elevated levels of casein and soy protein produced four to eight fold increases in histidase and urocanase, and 50% increases in formiminotransferase. Thus, increases in histidase and urocanase involved in the formation of formiminoglutamic acid (FIGlu) were accompanied by increases in the FIGlu metabolizing enzyme formiminotransferase.