Efficacy of atorvastatin therapy in ischaemic heart disease - effects on oxidized low-density lipoprotein and adiponectin.

The lipid-lowering and anti-atherosclerotic effects of atorvastatin (10 mg/day) were investigated by measuring changes in the levels of oxidized low-density lipoprotein (LDL), serum lipids (total cholesterol [TC], LDL-cholesterol [LDL-C] and triglycerides [TG]), and in the protein adiponectin. This was undertaken in 22 patients with ischaemic heart disease and serum LDL-C levels > 100 mg/dl. After 3 months of therapy, atorvastatin significantly decreased serum lipids, oxidized LDL was reduced from 457.0 +/- 148.6 to 286.9 +/- 88.5 nmol/l, and adiponectin increased from 9.7 +/- 7.4 to 13.9 +/- 9.98 microg/ml. No significant correlation was observed between adiponectin and LDL-C, TG and high-density lipoprotein cholesterol. Atorvastatin therapy was not associated with side-effects, such as myalgia and gastrointestinal disorders, and did not give abnormal laboratory test results. It is concluded that atorvastatin decreases serum lipid and oxidized LDL levels, and increases adiponectin levels in patients with ischaemic heart disease.

Interference of a methotrexate derivative with urinary oncopterin [N2-(3-aminopropyl)biopterin] measurement by high-performance liquid chromatography with fluorimetric detection.

We previously reported a HPLC assay method using fluorimetric detection for the simultaneous determination of urinary N2-(3-aminopropyl)biopterin (oncopterin, a natural pteridine newly found in urine from cancer patients), biopterin and neopterin. We now have observed that an unknown substance, which may be derived from methotrexate, in urine from a patient with stomach cancer interfered with the assay of oncopterin and demonstrated that oncopterin could be completely separated from the unidentified substance by HPLC using a Nucleosil 100-5SA strong cation-exchange column. Furthermore, oncopterin was not detectable by this HPLC-fluorimetric method in urine samples from patients with stomach cancer who were not treated with methotrexate. The content of urinary oncopterin from cancer patients is supposed to be very low, with less than 1 mumol/mol creatinine. The present results indicate that the peak found with elution from the C18 column was a methotrexate-derived compound and co-eluted with the analyte oncopterin.

Simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin), biopterin and neopterin by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method is described for the simultaneous determination of N2-(3-aminopropyl)biopterin (oncopterin, a newly found natural pteridine in urine from cancer patients), biopterin, and neopterin in urine. For the detection and quantification of the compounds, fluorometry was used. Using Develosil ODS K-5 and Develosil ODS HG-5 reversed-phase columns and a Nucleosil 100-SSA strong cation-exchange column, oncopterin, biopterin, and neopterin in urine were completely separated and assayed simultaneously by fluorescence detection. Similar values of oncopterin were obtained using each of the three columns, and the Develosil ODS K-5 reversed-phase column gave the most satisfactory separation. The sensitivity was high enough to measure 1 pmol of each pteridine. The HPLC method was highly reproducible. Our preliminary results indicate that oncopterin could be a most sensitive marker for cancer.

Elevated levels of oncopterin, N2-(3-aminopropyl)biopterin, a new pterin compound, in urine from patients with solid and blood cancers.

The concentrations of oncopterin, N2-(3-aminopropyl)biopterin, a new pterin compound, were determined in urine from various cancer patients by HPLC on a reverse-phase or ion-exchange column. The concentration of oncopterin increased after acid hydrolysis, indicating that it exists as an amide in urine. The oncopterin concentrations were very low in the urine of healthy controls. Among the urine samples examined, those from cases of solid cancers, e.g., hepatomas, prostatic cancer and bladder cancer, exhibited very high levels; and those from cases of blood cancers, e.g., myelomas, acute myelocytic leukemia, and lymphomas, showed moderate increases. Oncopterin may thus be a new biochemical marker of some types of cancer.

Diastereomers of neopterin and biopterin in human urine.

A new pteridine compound, named umanopterin, was isolated from human urine both of cancer patients and non-cancer controls. The structure was confirmed to be 2-amino-4(3H)-oxo-6-[(1'R,2'R)-1',2',3'-trihydroxypropyl]pteridine , a diastereomer of neopterin. The amount of umanopterin relative to neopterin was about 10%, which was practically the same among the non-cancer controls and the patients of various cancers. A small amount of a threo diastereomer of biopterin, named orinapterin, was isolated from human urine for the first time. Its structure was shown to be 2-amino-4(3H)-oxo-6-[(1'S,2'S)-1',2'-dihydroxypropyl]pteridine. A non-enzymatic transformation of 7,8-dihydroneopterin and 7,8-dihydrobiopterin by a mechanism analogous to keto-enol tautomerism is postulated for the formation of umanopterin and orinapterin in human body.

Highly sensitive, specific enzyme-linked immunosorbent assay of neopterin and biopterin in biological samples.

An enzyme immunosorbent assay of neopterin and biopterin on a polystyrene microtiter plate has been developed. A conjugate of neopterin or biopterin to bovine serum albumin was used to raise a specific antiserum against neopterin or biopterin in rabbits. An incubation mixture of the antiserum and samples prepared from human serum underwent another antigen-antibody reaction with the hapten fixed on the microtiter plate. The amount of antibody bound to the fixed hapten, which is inverse to the amount of hapten in the sample, was determined by using anti-rabbit IgG-horseradish peroxidase conjugate in a usual manner by measuring absorbance at 490 nm after reaction with o-phenylenediamine and hydrogen peroxide. The minimal detectable amounts of neopterin and biopterin were approximately 0.1 pmol. The specificity of the assay was so high that the assay system for neopterin completely distinguished it from biopterin, as judged from the cross-reaction of 0.002%, and vice versa. The amounts of neopterin and biopterin in human serum determined by the present method agreed well with those determined by high-performance liquid chromatography. We used the present method to determine the concentrations of neopterin in serum from healthy control subjects and patients with cancers and systemic lupus erythematosus; the results were consistent with literature data.

Simple, sensitive assay of polyamines by high-performance liquid chromatography with electrochemical detection after post-column reaction with immobilized polyamine oxidase.

This simple, rapid liquid-chromatographic assay of urinary polyamines (putrescine, spermidine, spermine, and cadaverine) involves electrochemical detection with a post-column immobilized enzyme, polyamine oxidase (EC 1.4.3.6) from soybean seedlings. Polyamines are separated by isocratic ion-pairing reversed-phase chromatography, then enzymatically converted, with release of hydrogen peroxide, via the post-column reactor with immobilized polyamine oxidase; the hydrogen peroxide is detected by electrochemical oxidation on a platinum electrode. The detection limits for injected putrescine, spermidine, and spermine were 0.3, 0.5, 0.6, and 4 pmol, respectively, with linear ranges of two to three orders of magnitude. Reproducibility was also good, with CV values less than 7%. The efficiency of the immobilized enzyme column was not decreased after analysis of 300 urine samples. Putrescine and spermidine excretion in urine from patients with blood cancers and solid cancers was significantly increased.

Sandwich enzyme immunoassay of dopamine-?-hydroxylase in cerebrospinal fluid from control and parkinsonian patients.

A sandwich enzyme immunoassay (EIA) was established by using purified human serum dopamine-?-hydroxylase (DBH) as a standard protein and a monospecific polyclonal antibody raised against human DBH purified from human pheochromocytoma. The EIA was applied to measuring DBH levels in human CSF from Parkinsonian patients and control patients devoid of neurological diseases. The control group had DBH content of 21.1 +/- 3.1 ng/ml CSF and DBH activity of 24.0 +/- 3.7 ? U/ml CSF, and Parkinsonian group 3.3 ? 0.7 ng/ml CSF (16% of control) and 4.6 +/- 0.7 ?U/ml CSF (19% of control) (mean +/- SEM). Thus, both DBH content and DBH activity in CSF were reduced in Parkinsonian patients to less than 20% of the control values (P $ ? 0.005 ). However, the specific activity (units of enzyme activity/mg of DBH protein) in CSF of Parkinsonian patients was similar to that of control patients. These results suggest that the reduced DBH activity in CSF from Parkinsonian patients is caused by a reduction in DBH protein content, and is not due to production of an inactive form of DBH, for example, by combining with endogenous inhibitor(s). These data support our previous findings that DBH activities in the Parkinsonian brain and CSF are decreased.

Galactose-particle receptor on liver macrophages. Quantitation of particle uptake.

Liver macrophages have been shown previously to bind and ingest gold particles coated with asialoglycoproteins via a N-acetyl-D-galactosamine / D-galactose-specific lectin (Kolb-Bachofen, V., Schlepper-Schäfer, J., Vogell, W. and Kolb, H. (1982) Cell 29, 859-866). We present here a quantitative analysis of lectin-dependent particle endocytosis. We used a conjugate of asialofetuin with colloidal gold as ligand, the cellular uptake of which could be followed by spectrophotometry. Freshly isolated Kupffer cells from the rat liver ingest asialofetuin at a rate of approx. 4200 particles/cell per min. Uptake is inhibited by saccharides related in structure to D-galactose and depends on the presence of Ca2+. The rate of endocytosis is zero below 10 degrees C, shows a modest increase until 20 degrees C and a steep increase between 20 and 37 degrees C. Uptake is energy-dependent and strongly inhibited by cytochalasin B but only slightly by colchicine.

Comparative measurements of urinary polyamines in early morning and 24-hour urine specimens.

Detailed comparative measurements of urinary polyamines were made between an early morning spot urine and 24-hour urine from normal controls and from patients with cancers. Urinary putrescine and spermidine concentrations in samples of morning spot urine were similar to those in 24-hour urine samples, and they were significantly increased in patients with various types of cancer. A morning spot urine can replace 24-hour urine in polyamine assay.

[Absorption, distribution and excretion of 14C-putrescine in mice (author's transl)].

In order to know in detail the distribution, absorption and excretion of putrescine (1,4-diaminobutane), after a single subcutaneous injection of 14C-putrescine (carrier free) in mice, localization of radioactivity in various tissues was followed by whole-body autoradiography and changes in radioactivity levels in blood, various tissues, expiration, urine and feces were examined. During 6 hours after injection, 22.5 +/- 3.2% of radioactivity was excreted in the urine, and after that the percentage excretion increased only slightly. The percentage of fecal excretion was small, being 2.7 +/- 0.2% during 5 days after injection. A considerable amount of putrescine was rapidly degraded to 14CO2; during 2 hours 34.4 +/- 4.9% of radioactivity was expired as 14CO2. In the studies on distribution in the tissues, at 10 minutes after injection, the highest radioactivity per g wet tissue was found in the kidney, but the radioactivity decreased rapidly. At 10 minutes after injection, the next highest distribution was found in the intestine. The high radioactivity in the intestine was detected also at 1 hour and 24 hours. The radioactivity in the pancreas was highest at 1 hour and 24 hours. A high radioactivity was also detected in the bone marrow by autoradiography. Therefore, the present study suggest that the accumulation of putrescine in mice take place mainly in the pancreas, bone marrow, intestine and liver.

Improved analysis for urinary polyamines by use of high-voltage electrophoresis on paper.

This method for assay of polyamines (putrescine, spermidine, and spermine) in large numbers of urine samples is based on preliminary analyte isolation by use of a Dowex 50W column, separation by high-voltage paper electrophoresis, reaction with ninhydrin to detect the separated amines, and subsequent direct assay with a dual-wave-length thin-layer chromatography scanner by the zigzag scanning method. By optimizing the conditions for the Dowex 50W column and high-voltage paper electrophoresis, putrescine, cadaverine, spermidine, and spermine were completely separated. The major technical improvements are room-temperature collection of urine with toluene, shorter hydrolysis time, complete separation of putrescine from cadaverine and histamine, and elimination of interference with spermine measurement. Polyamine concentrations as measured by this method agreed with those obtained by use of an amino acid analyzer. Reference intervals by this method were similar to those reported by Russell (Clin. Chem. 23: 22, 1977) with an automated amino acid analyzer. Putrescine, spermidine, and spermine excretion in urine from patients with blood cancers and solid cancers was significantly increased; information on concentrations of putrescine and spermidine were especially useful for following the clinical efficacy of cancer treatments.