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This experiment was carried out to evaluate the possibility of degradation of d-lactate into formate and acetaldehyde. In order to induce hyperproduction of d-lactate in rats. Donryu male albino rats were fed diets containing 0.064% 3'-methyl-4-dimethylaminoazobenzene (3'-MDAB), 4'-methyl-4-dimethylaminoazobenzene (4'-MDAB) or 2-methyl-4-dimethylaminoazobenzene (2-MDAB) for 10 weeks. During the experiment, body mass, food and water intake and volume of urine were documented. Methylglyoxal, d-lactate and formate in the urine samples were determined. On the first day of the eleventh week, methylglyoxal, d-lactate, glutathione and enzymatic activities of demethylation and glyoxalase I and II in liver were measured. Methylglyoxal, d-lactate and clinical chemistry parameters of blood plasma were also measured. The levels of methylglyoxal and d-lactate in livers of rats fed 3'-MDAB were very high, while those of 2-MDAB fed-rats and the control group were the same. The fact that glyoxalase I activity and the level of glutathione, a cofactor of glyoxalase I, were high in the livers of the 3'-MDAB-fed rats can explain the elevated levels of methylglyoxal and d-lactate in the liver. The most striking results were the elevated formate levels in the urine of rats fed 3'- and 4'-MDAB in a precancerous state. The degradation of d-lactate, an end product of the methylglyoxal bypass, into acetaldehyde and formate was suggested as a possible way to explain the results.

The Malaysian people consist of several ethnic groups including the Malay, the Chinese, the Indian and the Orang Asli (aboriginal Malaysians). We collected blood samples from outpatients of 2 hospitals in the State of Selangor and identified 27 glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects among these ethnic groups. In the Malay, G6PD Viangchan (871G＞A, 1311C＞T, IVS11 nt93T＞C) and G6PD Mahidol (487G＞A) types, which are common in Cambodia and Myanmar, respectively, were detected. The Malay also had both subtypes of G6PD Mediterranean:the Mediterranean subtype (563C＞T, 1311C＞T, IVS11 nt93T＞C) and the Indo-Pakistan subtype (563C＞T, 1311C, IVS11 nt93T). In Malaysians of Chinese background, G6PD Kaiping (1388G＞A), G6PD Canton (1376G＞T) and G6PD Gaohe (95A＞G), which are common in China, were detected. Indian Malaysians possessed G6PD Mediterranean (Indo-Pakistan subtype) and G6PD Namoru (208T＞C), a few cases of which had been reported in Vanuatu and many in India. Our findings indicate that G6PD Namoru occurs in India and flows to Malaysia up to Vanuatu. We also discovered 5 G6PD-deficient cases with 2 nucleotide substitutions of 1311C＞T and IVS11 nt93T＞C, but without amino-acid substitution in the G6PD molecule. These results indicate that the Malaysian people have incorporated many ancestors in terms of G6PD variants.

Our study aimed to investigate the potential radioprotective effects of N-acetylcysteine (NAC) by comparing its biochemical effects with those of WR-2721, as a representative of clinically used radioprotectors, in preventing oxidative damage caused by gamma irradiation (single dose, 6Gy) in normal rat tissue. The rats (n=40) were divided randomly and equally into 4 groups:Control (C), Radiation (R), R+NAC (received irradiation and 1,000mg/kg NAC) and R＋WR-2721 (received irradiation and 200mg/kg WR-2721) rats. Liver tissues and blood samples were harvested and utilized for reduced glutathione (GSH), malondialdehyde (MDA) and myeloperoxidase (MPO) detection. Serum and tissue GSH levels of R rats decreased compared to those of other groups (p<0.01). Tissue MDA levels of R+NAC and R+WR-2721 rats decreased compared to R rats (p<0.01;p<0.05, respectively). Tissue MPO activities of R+NAC and R+WR-2721 rats were higher than those of R rats (p<0.001). Serum MPO levels of R+WR-2721 rats were lower than those of C rats and R rats (p<0.01, p<0.001, respectively). In conclusion, the study suggests that the radioprotective effect against radiation-induced oxidative damage of NAC may be similar to that of WR-2721.

The content of beta-hydroxyaspartic acid was measured in the urine of man and several species of animals. The configuration of urinary beta-hydroxyaspartic acid was deduced to be L-erythro in form by chromatographic comparisons with authentic samples. An increased excretion of urinary beta-hydroxyaspartic acid was observed in cats when serine or thiamine was administered with glycine. Glycine-1-14C administered to rats was incorporated into the urinary beta-hydroxyaspartic acid. The formation of beta-hydroxyaspartic acid in pig-liver homogenate increased in the presence of glutamate and thiamine pyrophosphate. These results were discussed in relation to the author's working hypothesis on the biosynthesis of beta-hydroxyaspartic acid.

Sprague-Dawley rats, 6 with aminonucleoside nephrosis and 6 controls, were intravenously injected with human liver ferritin isolated from post mortem liver, and their 24-h urine samples were examined for human ferritin by immunoradiometric assay. In rats with aminonucleoside nephrosis, the amount of excreted ferritin in urine was forty times greater than in control rats. Much more monomeric ferritin was excreted than that of polymeric ferritin. We are the first to have utilized human liver ferritin as a tracer to measure a minor amount of ferritin by a commercially available kit. Our present study seems to indicate a critical role for glomerular basement membrane as a size barrier.

Even after successful operations, ugly postoperative skin scars are often distressing to patients and their parents. To judge the success of surgical methods and postoperative treatment, postoperative scars should be evaluated using a quantitative system. Height and width are easily measured, but scar redness is not. We have developed a simple and effective method for evaluating scar redness. According to the color definitions employed in computer graphics, each color can be expressed as RGB (red, green or blue) coordinates (r, g, b): 0 ≦ r, g, b ≦ 10. The degree of scar redness is defined by the following formula: redness score (RS) = (r₁ - r₀)² + (g₁ - g₀)² + (b₁ - b₀)². Here, (R₁, g₁, b₁) = coordinates of the scar color and (r₀, g₀, b₀) = coordinates of the surrounding skin color. RS was evaluated in 59 children (35 males, 24 females; ages 1 month to 12 years old) who had scar redness after congenital cardiac surgery. For each patient, scar color and surrounding skin color was identified on the color sample table. Scar redness was also evaluated by the conventional grading method: 1 = mild, 2 = moderate and 3 = severe. The RS of the colored scars ranged from 4 to 100 (38 ± 27). By the conventional grading method, 44 scars were grade 1, 15 grade 2 and none grade 3. RS was significantly higher among grade 2 than grade 1 patients, 52 ± 25 and 33 ± 27, respectively (P < 0.05). Given its subjectivity, the conventional grading method yields variable data; surrounding skin color, moreover, is not considered. Our new evaluation method using RS effectively and accurately defines scar and skin colors, and allows quantitative studies of these factors.

This study included 45 patients with intentional insecticide intoxication and 21 with accidental intoxication who were treated at the First-Aid and Emergency Department of Balcali Hospital at the Faculty of Medicine in the Cukurova University, Adana, Turkey, while the control group consisted of 25 people selected from university personnel known to be healthy. Patients with a history of X-ray exposure in the last 6 months or of any virus disease as well as continuous drug users and smokers were excluded, leaving a total of 49 patients. Acetylcholine esterase (Pseudocholinesterase) enzyme (AchE), sister-chromatid exchanges (SCE), the mitotic index (MI), and the replication index (RI) were evaluated. Blood samples were cultured for SCE evaluation and sera separated for AchE levels. Insecticide exposure was generally intentional for suicide in adolescents and at older ages, but accidental for children. AchE levels were found to be significantly lower in organophosphorus (OP) and carbamated (CB) insecticide poisoning groups in comparison with the control group (p<0.001), while the pyrethroid (PY) group was not statistically different for the AchE effect (p>0.05). SCE was found to be significantly higher in OP and CB groups (p<0.001), while the PY and control groups were statistically similar for SCE levels (p>0.05). This study showed an increase in SCE in response to orally ingested insecticides. These findings indicate that insecticide exposure results in cell abnormalities, with resulting impediments to the division and replication of cells, as suggested by MI decreases and RI increases, while the speed of the division cycles of stimulated cells increases.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) causes acute hemolytic anemia triggered by oxidative drugs such as primaquine. It is therefore essential in malaria-endemic areas for malaria patients to be confirmed for their G6PD activity before taking primaquine. The WST-8 method, a newly established screening method for G6PD deficiency, has been demonstrated to be suitable for field conditions, particularly for on-site malaria surveys. Here we report a laboratory evaluation by this method of the reactivity of blood-spotted filters. A time-course experiment was conducted to evaluate the reactivity of blood samples spotted onto 4 types of filter paper, Whatman 31ET Chr (ET), 3MM Chr (3MM), P81, and Advantec No. 2 (AD2). The rank of the relative reaction intensity was ET > 3MM = AD2 > P81. Blood-spotted filters stored at 4 degrees centigrade gradually decreased G6PD reactivity with the passage of storage time, whereas those stored at room temperature rapidly reduced their reactivity. Unexpectedly, saponin supplementation reduced the reactivity of blood-spotted filters. In conclusion, 1) ET is the most suitable filter for the WST-8 method ; 2) blood-spotted filters stored in cold condition can be assayed within 14 days, or those stored at room temperature should be tested within 3 days ; and 3) reaction mixtures should not contain saponin.

This study investigated the optimal conditions for detection of nucleotides in blood using an IP-1B capillary isotachophoretic apparatus. The system used 10 mM HCl-beta-alanine (pH 4.2) as the leading electrolyte and n-caproic acid as the terminal electrolyte. Direct application of lysed red blood cells was shown to be inaccurate, and a method of deproteinization based on heat in a microwave oven was developed. The zones for 2,3-diphosphoglycerate, ATP, inorganic phosphate, and lactate were identified enzymatically by withdrawal of pure samples of each zone via a special withdrawal cell. The quantitative values obtained by isotachophoresis were also confirmed enzymatically. The technique is now available for convenient and accurate identification of these metabolites simultaneously.

The microvasculature of peripheral nerves in dog and human samples was studied by an injection replica scanning electron microscope (SEM) method. The three-dimensional relationship of epineural and intraneural vessels was well demonstrated by this method. Epineural vessels were made of both longitudinal arterioles and venules and also a close meshwork of capillaries. In the intraneural microvasculature, longitudinal arterioles and venules made up the perineural vessels, and grouped capillaries corresponded to the fine vasculature of intrafascicular vessels. Between epineural and intraneural vessels, there was little anastomosis but nutrient arteries ran through the epineural vasculature into the intraneural vessels. There was little interconnection of vessels among the intraneural vasculature. It was postulated that the close meshwork of the epineural layer can resist pressure from outside the peripheral nerves but that longitudinal venules seemed to be affected by pressure and tension at the localized area.

Circulating hepatitis C virus (HCV) particles can be fractionated by means of differential flotation centrifugation. It is reported that in the bottom fraction HCV is in the form immune complexes, whereas in the top, it is free of antibodies. We evaluated the significance of circulating complex and free HCV in chronic hepatitis C, and assessed the relationship in terms of the response to interferon (IFN) therapy. We examined sera before, just after, and 1 year after administering IFN to 18 patients with chronic hepatitis C, 10 of whom responded (group CR), and 8 did not (group NR). The amounts of virus were similar between both groups before therapy. After differential flotation centrifugation with 1.063 g/ml of NaCl, the top and bottom fractions were assayed for HCV RNA. Before therapy, HCV RNA was detected in the top fraction in 1 of 10 in group CR, and in 6 of 8 in group NR (P < 0.05, chi-square test). HCV RNA was positive in the bottom fraction of all samples. In a follow-up study of group NR, HCV RNA was detected in the top fraction in 3 of 8 just after IFN therapy, and in 7 of 8 after 1 year. This study suggests that the presence of HCV in the top fraction can predict a poor response to IFN therapy.

The sensitivity of five kinds of cytotoxicity assays using ethanol on human hepatoblastoma cells (HUH-6 line), which were cultured as monolayers or spheroids, was compared. Ethanol was chosen as a test because it acts on cell membranes directly without being metabolized and exerts its cytotoxicity. The assay methods used were as follows: 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH), colony formation, cell growth and DNA assays. The sensitivity of the assays was: LDH < DNA < cell growth < MTT < colony formation. LDH assay had the advantage that the same culture could be used for multiple assays, but when a small number of cells were assayed, no significant increase in the release of LDH was detected in the assay cultures compared with the control cultures. Although the DNA and cell growth assays were more sensitive than the LDH assay, the extent of cell damage may be underestimated because the damaged cells and DNA present in the cultures are included in the assay samples. On the other hand, both MTT and colony formation assays showed a high sensitivity. The MTT assay was done within 24 h after ethanol was added to the cultures and was applicable to both monolayer and spheroid cultures, while the colony formation assay required 1-2 weeks and it was applicable only to monolayer cultures. Taken together, the MTT assay was the most suitable method to evaluate the cytotoxic effects of ethanol on HUH-6 cells cultured as either monolayers or spheroids.

The blood vascular bed, perivascular space and intercellular space of the rat parathyroid gland were studied using scanning electron microscopy of vascular casts, freeze-cracked tissue samples, and NaOH-digested tissue blocks. The findings were supplemented by transmission light and electron microscopy of iron colloid-treated or enzyme-digested tissue sections. The rat parathyroid gland contained a rich network of capillaries. These capillaries were surrounded by marked pericapillary spaces which were demarcated by basal lamina of both capillaries and parenchymal cells. The pericapillary spaces contained numerous collagen fibrils, and issued many crista-like projections which ran deep into the sheets of parenchymal cells. The intercellular spaces of parenchymal cells contained neither basal lamina nor collagen fibrils. The surfaces of the parenchymal cells showed strong negative charging, and maintained the intercellular spaces. The luminal surfaces of the capillary endothelium also showed strong negative charging, and maintained the capillary lumen.

To clarify the relation between systemic and cutaneous vascular endothelial injury in progressive systemic sclerosis (PSS), we examined thrombomodulin (TM) expression in PSS skin lesions immuno-histopathologically and compared it with plasma soluble TM levels measured by specific enzyme-linked immunosorbent assay. The plasma soluble TM level in PSS patients was significantly higher than that of normal controls and was as high as the levels of SLE patients. In relation to disease activities, the plasma TM levels of sclerotic phase PSS patients were significantly higher than that of atrophic phase PSS patients. The plasma samples with anti-Scl-70 antibody showed a high TM level than samples with anti-centromere antibody or anti-RNP antibody. Barnett's types or systemic corticosteroid treatment did not affect the TM level. Histopathologically, the dermal endothelial TM expression significantly increased in the sclerotic skin and moderately increased in the non-sclerotic skin of PSS compared with that of normal control skin. In addition, immunoreactive TM expression in the epidermis also increased in PSS. Disease activity-dependent elevation of plasma TM levels and immuno-histopathological expression of TM suggested generalized endothelial and epidermal cell involvement in PSS, and compensation in part by overproduction of TM by endothelial cells.

We observed differences in the capillary architecture of the skeletal muscles that have different fiber metabolism. The soleus, the vastus intermedius and the tibialis anterior muscles of adult Wistar rats were prepared using two different techniques. Samples for adenosine triphosphatase (ATPase) staining were prepared following Dubovitz's method, and the distributions of fiber type, Types 1, 2A and 2B, were analyzed. Then, corrosion casts of capillary architecture of these muscles prepared following Murakami's method were observed with a scanning electron microscope (SEM) and compared with the fiber distribution. The fiber type composition of the soleus muscle showed Type 1 (slow-twitch) dominance and that of the vastus intermedius and the tibialis anterior muscle showed Type 2 (fast-twitch) dominance. The capillaries of the soleus muscle were tortuous, and this was thought to be advantageous for blood supply. In contrast, the capillaries of the vastus intermedius and tibialis anterior muscles had a relatively parallel pattern. Additionally, two different patterns of capillary architecture that appeared to correspond to certain metabolic characteristic of different muscle fiber types were preserved with corrosion casting. In conclusion, comparative studies on capillary architecture of the skeletal muscles are useful for analyses of its function.

The effects of intraperitoneal administration of 2-(4-carboxy-D-gluco-tetrahydroxybutyl)thiazolidine-4-carboxylic acid (CGUA), a cysteine derivative conjugated with glucuronic acid, on total glutathione and total cysteine contents in rat tissues were investigated. Total glutathione (GSH and GSSG) and total cysteine (cysteine and cystine) were determined by a new method consisting of preparation of S-carboxymethylglutathione (CMSG) and S-carboxymethylcysteine (CMC), respectively, and subsequent analyses with an amino acid analyzer. CGUA was determined by a coloration method employing an acidic ninhydrin reagent. Total cysteine contents in liver, kidney and plasma rapidly increased to 2.3, 2.7 and 6.5 times the levels of the controls, respectively, after CGUA administration at a dose of 5 mmol/kg of body weight. Total glutathione content did not change significantly in the liver or blood except for the kidney with a significant increase during the first 1-h period after administration. CGUA content increased markedly in these tissues, especially in the kidney, and 30% of administered CGUA was excreted in urine within 2h. These results indicate that CGUA is converted into cysteine in vivo, suggesting the usefulness of this compound for protection of the kidney and the liver.

"Free" iron, a potentially radical-generating low mass iron, and not found in normal human blood, was increased in the serum of blood-transfused thalassemia major patients seen in the Yangon General Hospital, Yangon, Myanmar (Burma). The low mass iron was detected by the bleomycin assay. Fifty-one blood samples were analyzed (from 28 males and 23 females). High "free" iron was detected in 47 sera samples from thalassemia patients. Serum ferritin, which reflects the body store iron, was higher than the normal range (10-200 ng/ml) in 49 patients. On the other hand, serum iron of 39 sera samples fell within the normal range (50-150 micrograms/dl). Four were less than 50 micrograms/dl and eight were more than 150 micrograms/dl. Almost all the patients' sera of normal or higher serum iron level contained "free" iron. Thus, almost all the sera from thalassemic patients from Myanmar contain bleomycin-detectable iron, even when serum iron is within the normal range. In developing countries where undernutrition is prevalent (serum albumin in these patients was 3.6 +/- 0.4 g/dl, P < 0.0001 vs. control value of 4.0 - 4.8 g/dl), normal serum iron does not preclude the presence of free iron in the serum.

Examination was made of the in vitro response of human peripheral blood mononuclear cells (PBNMCs) to phytohemagglutinin (PHA) following treatment with varicella zoster virus (VZV) or herpes simplex virus type 1 (HSV 1). Cell proliferation was determined by colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. The response to PHA was depressed in all cases by virus infection of PBMNCs prior to PHA treatment. When the infection with the viruses was after PHA treatment, PHA response differed. For VZV infection, the response increased in four out of six samples, but was reduced in the other two. The response to PHA was depressed in all six samples by HSV 1 infection.