Kazuharu Ienaga

Production of Methylguanidine from Creatinine via Creatol by Active Oxygen Species: Analyses of the Catabolism in vitro

The chemical oxidative conversion of creatinine (Cr) into methylguanidine (MG) has been followed by 1H-NMR and HPLC. By using active oxygen species generated by Fentons reagent (Fe2+ and H2O2) or the similar reagent (Fe3+ and H2O2), creatol (CTL), a metabolite newly isolated from the urine of uremic patients, and creatones A and B were experimentally detected in the reaction mixture and implicated as successive intermediates in the pathway from CR to MG. An alternative oxidation of Cr to demethylcreatinine (glycocyamidine) was also observed. The importance of CTL in this in vitro oxidation mechanism is discussed.

Crosslines A and B as candidates for the fluorophores in age- and diabetes-related cross-linked proteins, and their diacetates produced by Maillard reaction of α-N-acetyl-L-lysine with D-glucose

A pair of novel fluorophores (N-diacetates of crosslines A and B), (3R,4S)-3,4-dihydroxy-5-[(1S or 1R,2S,3R)-1,2,3,4-tetrahydroxybutyl]-1,7-bis[6-(N-acetyl-L-norleucyl)]-1,2,3,4-tetrahydro-1,7-naphthyridinium chloride and its epimer, were isolated from the Maillard reaction mixture of α-N-acetyl-L-lysine and D-glucose, and their properties are similar to those of fluorophores in age- and diabetes-related cross-linked proteins, and a pair of homologues derived from n-pentylamine.

Neurotropin induces antinociceptive effect by enhancing descending pain inhibitory systems involving 5-HT3 and noradrenergic α2 receptors in spinal dorsal horn

Neurotropin, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug in Japan. Its analgesic effect has been demonstrated by reduced mechano-nociception in hyperalgesic rats exposed to SART-stress (a repeated cold stress) for 5 days. In order to clarify the mechanism of the analgesic effect of neurotropin at the spinal cord level, we examined the effects of several neurotransmitter receptor antagonists given by intrathecal (i.t.) injection on the antinociceptive effect of intraperitoneally (i.p.) injected neurotropin [100 and 200 Neurotropin Unit (NU)/kg]. The analgesic effect of neurotropin was significantly inhibited not only by methysergide (100 nmol/rat, i.t.), a non-selective antagonist against serotonin (5-HT), but also MDL 72222 (30 nmol/rat, i.t.), a selective 5-HT3 antagonist, but not influenced by ketanserin (100 nmol/rat, i.t.), a 5-HT2A antagonist. The antinociceptive effect of neurotropin (200 NU/kg, i. p.) was significantly inhibited also by yohimbine (30 nmol/rat, i.t.), a noradrenergic alpha2 antagonist. However, the analgesic effect of neurotropin (100 and 200 NU/kg, i.p.) was not influenced by naloxone (30 nmol/rat, i.t.), an opioid antagonist. These results suggest that the mechanism of the antinociceptive effect of neurotropin is via enhancement of endogenous descending pain inhibitory pathways of the serotonergic and noradrenergic systems, especially involving 5-HT3 and noradrenergic alpha2 receptors in spinal dorsal horn in which these neurons terminate. No influence of opioid receptors at the spinal cord level is indicated.

Crosslines, Fluorophores in the AGE-Related Cross-Linked Proteins

We can summarize our results as follows: (1) A pair of fluorescent crosslines were isolated from the Maillard reaction mixture; (2) AGE-proteins contained crossline-like structures, and (3) crossline-like immunoreactivities were accumulated in renal tissues of diabetic rats. From these results we concluded that fluorophores in AGE proteins have crossline-like structures and we had the first indication that XLs could be markers for renal disorders.

Bioactive compounds produced in animal tissues (I); two diketopiperadine plant growth regulators containing hydroxyproline isolated from rabbit skin tissue extract

Abstract A tricyclic diketopiperadine, L-hydroxyprolyl-L-proline anhydride and a bicyclic diketopiperadine, L-hydroxyprolyl-L-leucine anhydride, were isolated as plant growth regulators from skin tissue extract of rabbits.

Creatol, a creatinine metabolite, as a useful determinant of renal function.

Analysis of creatol (CTL, 5-hydroxycreatinine), an oxidative creatinine (Cr) metabolite, in serum and urine of human subjects has indicated that CTL is a useful determinant of renal function. The existence itself of serum CTL (s-CTL) could be a diagnostic sign for chronic renal failure (CRF): in all normal subjects, s-CTL was undetectable, but s-CTL was detectable in sera of all patients with CRF (s-Cr: > 2.0 mg/dl). And the s-CTL values increased in proportion to the severity of CRF in such patients. Furthermore, the molar ratio (CTL/Cr) in both urine and serum increased significantly in proportion to the severity of CRF. Our results indicated not only hyperproduction of CTL but also higher oxygen stress in patients according to the progression of CRF. The diagnostic importance of the CTL value and the CTL/Cr ratio are discussed.

The use of 13C-labelling to prove that creatinine is oxidized by mammals into creatol and 5-hydroxy-1-methylhydantoin

Creatinine 1 is not an end-metabolite but a precursor for three heterocycles, 2-amino-5-hydroxy-1-methyl-4(5H)-imidazolone (creatol)2,1-methylimidazolidine-2,4-dione 4 and its 5-hydroxy derivative 5; this is now proven directly by administration of [Me-13C]creatinine 1, to a uraemic rat and subsequent isolation of the corresponding labelled metabolites 2, 4 and 5 from its urine.

Bioactive compounds produced in animal tissues (II); Two hydantoin plant growth regulators isolated from inflamed rabbit skin tissue☆

Abstract 1-Methylhydantoin and 5-hydroxy-1-methylhydantoin were isolated as plant growth regulators from rabbit skin tissue which had been inflamed by inoculation with vaccinia virus.

Urinary Excretion of Creatol, an In Vivo Biomarker of Hydroxyl Radical, in Patients with Chronic Renal Failure

Creatol (CTL) is a hydroxyl radical adduct of creatinine (Cr). The serum methylguanidine (MG) level and the MG/Cr molar ratio are reported to be biomarkers for oxidative stress. The aim of this study was to examine whether urinary excretion of CTL, another oxidative stress-related marker, is increased in patients with chronic renal failure (CRF). One hundred twenty-four non-dialyzed patients with chronic renal failure (serum Cr level, 1.3–10.0 mg/dL) were recruited from our hospitals. Urine and serum levels of CTL and MG were determined by high-performance liquid chromatography with the use of 9, 10- phenanthrenequinone as a fluorogenic reagent. The CTL/Cr and (CTL+MG)/Cr molar ratios in spot urine samples were also compared with those in 24-h urine samples. The urinary CTL/Cr and (CTL+MG)/Cr molar ratios increased with decreases in Cr clearance in patients with CRF. Correlations between serum and spot urine (CTL+MG)/Cr and between serum and spot urine CTL/Cr were quite similar to those in 24-h urine samples. CTL/Cr and (CTL+MG)/Cr molar ratios in both 24-h urine and spot urine samples appear to be useful indices of the severity of CRF.

Crossline levels in serum and erythrocyte membrane proteins from patients with diabetic nephropathy

Crossline is one of the structurally defined adducts of advanced glycation endproducts (AGEs) which has both a crosslink and fluorescence similar to AGE-protein in vivo. Crossline was measured in serum and erythrocyte membrane proteins (EMP) from 52 type 2 diabetic patients using a specific enzyme-linked immunosorbent assay system. Serum and EMP crossline levels in the diabetic patients were significantly higher than those in normal control. The patients with advanced diabetic nephropathy (serum creatinine levels of more than 1.2 mg/dl) had markedly elevated serum crossline levels compared to those with moderate diabetic nephropathy (clinical proteinuria) (180. 7+/-51.7 vs. 71.8+/-18.4 pmol/ml; P<0.01). On the other hand, there were no significant differences in EMP crossline levels between the two. EMP crossline levels in the patients with moderate diabetic nephropathy (8.8+/-2.9 pmol/mg protein) and those with advanced diabetic nephropathy (9.7+/-3.0 pmol/mg protein) were significantly higher than those without clinical proteinuria (6.4+/-1.9 pmol/mg protein; P<0.01). The present study demonstrated that EMP crossline levels were associated with the presence of nephropathy in patients with type 2 diabetes mellitus. Serum crossline levels were significantly influenced by remaining renal function. The measurement of crossline from a blood sample could provide us with important information for the study of clinical evaluation and pathogenesis of diabetic complications.