Clinica Chimica Acta

The gut is considered an important target organ of injury after severe insult such as sepsis, trauma and shock. Hydroxyethyl starch (HES) 130/0.4 has been developed to improve the pharmacokinetics of current medium molecular weight HES solutions. We investigated the protective effects of HES 130/0.4 on intestinal inflammatory response and survival in a rat polymicrobial sepsis model induced by cecal ligation and puncture. Animals were treated with HES 130/0.4 or saline at 4, 10, 16 or 22 h after the induction of sepsis or sham-operation and were sacrificed 2 h after resuscitation. Intestines were harvested for measurement of tumour necrosis factor alpha (TNF-alpha), interleukin (IL)-10 and macrophage inflammatory protein-2 (MIP-2) production by EELISA; intercellular adhesion molecule-1 (ICAM-1) mRNA expression by reverse-transcription PCR; nuclear factor-kappa B (NF-kappaB) by electrophoretic mobility shift assay; neutrophil sequestration by myeloperoxidase (MPO) assay; intestinal permeability by fluorescein isothiocyanate-labeled dextran assay. In addition, the role of HES 130/0.4 in rat survival was observed. Intestinal permeability was significantly decreased after HES 130/0.4 administration in septic rats, which was associated with a reduction in inflammatory mediators and NF-kappaB activation. Furthermore, early administration of HES 130/0.4 after septic insult resulted in greater decrease in inflammatory mediators. In addition, HES 130/0.4 co-administrated with antibiotics not HES 130/0.4 alone greatly improved the survival of septic rats. HES 130/0.4 reduced intestinal permeability by modulating inflammatory response and had a promising effect on survival together with antibiotics under septic conditions.

The amount of small intestinal calcium-binding protein was studied in biopsies of human jejunum sectioned perpendicular to the long axis of the villi. Brush-border aminopeptidase N activity was measured to distinguish undifferentiated crypt cells from mature villus cells. The amount of calcium-binding protein as measured by enzyme-linked immunoadsorbent assay was highest at the villus tip and gradually decreased to near zero values in the crypt. The distribution of calcium-binding protein observed is parallel to that found in vitamin D repleted rats. The present study indicates that the mature enterocyte in man is fully committed to express calcium-binding protein in accordance with the view that these cells are considered the most active in the calcium absorptive mechanism.

Between-laboratory agreement for 6 specific protein assays was better in two surveys (total 8 specimens) in which a calibration material was provided than in the preceding five surveys (20 specimens) in a national external quality assessment scheme. CVs for immunoglobulins G, A and M (200 participants) and alpha 1-antitrypsin and complement components C3 and C4 (70 participants) were reduced from 18.3% (range 11.6-23.8%) to 10.7% (range 7.5-13.6%). This improvement was not due to changes in within-laboratory precision or in the concentrations of protein surveyed. Improvement was maintained in the following four surveys (3 normal and 5 pathological sera) without calibration material for immunoglobulins G and A and for alpha 1-antitrypsin. Agreement for immunoglobulin M, C3 and C4 returned to values close to the original CVs, but subsequently improved. The role of a common calibration material in improving between-laboratory agreement is discussed.

A reference method for tonometry of blood is described. The document covers the theory of tonometry, the materials and equipment needed, and essential aspects of the tonometry procedure for blood. The partial pressures of oxygen and carbon dioxide in tonometered blood are accurately known and therefore this blood is recommended for assessing the accuracy of blood gas analyzers. Tonometry of blood samples from patients may also be used in the determination of acid-base quantities and hemoglobin-oxygen affinity, e.g. p50.

A technique has been developed to measure the diphosphonate ethane-1-hydroxy-1, 1-diphosphonate (EHDP) quantitatively in 5 ml of urine or 2 ml of plasma. The procedure is based on a coprecipitation of EHDP with calcium phosphate, elimination of inorganic phosphate as an insoluble triethylamine-phosphomolybdate complex, decomposition of the P-C-P bond with ultraviolet light and spectrophotometric determination of the inorganic phosphate released. A trace amount of [14C] EHDP is used to correct for losses. The method appears specific for the diphosphonate, exhibits quantitative recoveries, and has a mean coefficient of variation of 3.7% for urine and 7.3% for plasma. The limit of detection is in the order of 2.5 mumol/1 in 5 ml urine and 0.5 mumol/1 in 2 ml of plasma.

Colon cancer is one of the most common forms of malignant tumors in humans, and its incidence is increasing. Since the intestinal microflora is directly in contact with the colonic cells, the enzymes of the bacterial microflora may also play a role in colon carcinogenesis. We studied the activity of bacterial enzymes in experimental colon cancer. Twenty milligrams per kilogram body weight of 1,2-dimethyl hydrazine (DMH) was administered subcutaneously once a week for first 15 weeks and then discontinued. Coconut cake (25%) was mixed in the diet and given to 30 rats to study the diet effect throughout the experimental period. After 30 weeks, the macroscopic findings in the colon as well as the incidence of tumors in 30 rats was recorded in each group and the activity of beta-glucuronidase and mucinase was estimated in the tissues, colon and fecal contents of 10 rats per group. Average number of tumors in the colon as well as the incidence of cancer was significantly increased in DMH-treated rats which was markedly reduced on supplementing coconut cake. DMH injections significantly elevated both the activities of beta-glucuronidase (distal colon, distal intestine, liver and colon contents) and mucinase (colon and fecal contents) as compared to the control rats. The increase in beta-glucuronidase activity may augment the hydrolysis of glucuronide conjugates, liberating the toxins, while the increase in mucinase activity may enhance the hydrolysis of the protective mucins in the colon. Coconut cake supplementation to DMH-treated rats significantly decreased the incidence and number of tumors as well as the activity of beta-glucuronidase and mucinase. Coconut cake has a protective effect against DMH induced colon cancer by virtue of its ability to lower the activities of the microbial enzymes beta-glucuronidase and mucinase.

Background: The primary mode of action for cis-diamminedichloroplatinum (II), referred to as cisplatin, toward the treatment of solid malignancies is through formation of cross-links with DNA at purine sites, especially guanines. Methods: We prepared oligodeoxyribonucleotides (ODNs) containing a 1,2-GpG, 1,2-ApG, or 1,3-GpXpG cisplatin intrastrand cross-link and the corresponding ODNs modified with (15)N2-labeled cisplatin, and characterized these ODNs with electrospray ionization mass spectrometry (ESI-MS) and tandem MS (MS/MS). We also employed LC-MS/MS to characterize the digestion products of these ODNs after treatment with a cocktail of 4 enzymes (nuclease P1, phosphodiesterases I and II, and alkaline phosphatase). Results: 1,2-GpG was released from the ODNs as a dinucleoside monophosphate or a dinucleotide. Analyses of the digestion products of ODNs containing a 1,2-GpG cross-link on the 5' or 3' terminus revealed that the dinucleotide carries a terminal 5' phosphate. On the other hand, digestion of the 1,3-GpXpG intrastrand cross-link yielded 3 dinucleoside products with 0, 1, or 2 phosphate groups. Conclusion: The availability of the ODNs carrying the stable isotope-labeled lesions, MS/MS analyses of the cisplatin-modified ODNs, and the characterization of the enzymatic digestion products of these ODNs set the stage for the future LC-MS/MS quantification of the 1,2-GpG, 1,2-ApG, and 1,3-GpXpG lesions in cellular DNA.

Ginger (Zingiber officinale Rosc) is a natural dietary component, which has antioxidant and anticarcinogenic properties. We investigated the effect of ginger on the initiation and post-initiation stages of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in male Wistar rats. Rats were given a weekly subcutaneous injection of DMH (20 mg/kg body weight) in the groin for 15 weeks. Ginger (50 mg/kg body weight/everyday p.o.) was given to the rats at the initiation, post-initiation stages of carcinogenesis. The activity of lipid peroxidation was studied by measuring the formation of thiobarbituric acid reactive substances (TBARS), lipid hydroperoxides (LOOH) and conjugated dienes (CD), and the antioxidant status by measuring superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST), glutathione reductase (GR), reduced glutathione (GSH), vitamins C, E, and A concentrations in the circulation of 1,2-dimethylhydrazine-induced experimental colon cancer. In the presence of a known colon carcinogen, DMH, plasma lipid peroxidation (TBARS, lipid hydroperoxides and conjugated dienes) and cancer incidence were significantly increased whereas enzymic (GPx, GST, GR, SOD and CAT) and non-enzymic antioxidant concentrations (GSH, vitamins C, E, and A) were decreased as compared to control rats. The number of tumors as well as the incidence of cancer was significantly decreased on treatment with ginger. In addition, ginger supplementation at the initiation stage and also at the post-initiation stages of carcinogenesis significantly reduced circulating lipid peroxidation and significantly enhanced the enzymic and non-enzymic antioxidants as compared to unsupplemented DMH-treated rats. Ginger supplementation suppresses colon carcinogenesis in the presence of the procarcinogen DMH.

We present a novel method for the analysis of ethylene glycol (ethane-1,2-diol) in serum. This method employs the ability of glycerol dehydrogenase (EC 1.1.1.6) to oxidize ethylene glycol. Two measurements, five and 30 min after addition of enzyme, are used to circumvent interferences from endogenous glycerol and from propylene glycol (propane-1,2-diol). Ethanol does not interfere in the assay. The method requires commonly available equipment only, making it suitable for all emergency laboratories.

A method for the analysis of 1,2-diacylglycerols in biological samples is presented. After tissue extraction and derivatisation with 3,5-dinitrobenzoyl chloride, samples are analysed by normal phase HPLC, using a 3.9 x 300 mm microPorasil column, and ultraviolet detection at 254 nm. The method gives quantitative recovery of 1,2-diacylglycerol, and is of sufficient sensitivity to allow quantitation of 1,2-diacylglycerol in human muscle needle biopsy specimens, from as little as 10 mg muscle. Human skeletal muscle from fasted control subjects was found to have a 1,2-diacylglycerol content of 455 +/- 78 nmol/g wet weight. The method is robust, giving intra- and inter-assay coefficients of variation of 2.9% and 5.9%, respectively, and should prove useful for the analysis of 1,2-diacylglycerol levels in human disease states, such as diabetes, in which no measurements of 1,2-diacylglycerol have yet been undertaken.

The relative influence of some endogenous corticosteroids and of synthetic prednisolone on competitive protein-binding radioassay was compared with that of cortisol, using as a source of transcortin pooled plasmas from various species and at different dilutions. Human (different clinical situations), cow, dog, sheep, hog, rabbit and chicken plasma were examined. The ability of corticosterone, cortisone, 11-deoxycortisol and prednisolone to displace [1,2-3-H] cortisol from corticosteroid-binding globulin (CBG) was measured: (1) by assessing the amounts (ng/tube) which produce a displacement equal to 10 ng/tube of cortisol, and (2) by calculating the integrated areas of displacement defined by the binding curves and by expressing them as a percentage of the cortisol curve area. While corticosterone, 11-deoxycortisol and prednisolone have a binding potency not very different from that of cortisol in almost the entire set of competitive protein-binding assays tested, the binding ability of cortisone was found to be particularly dependent upon the species of diluted plasma. Differences in the relative specificity of binding are apparent also within the human species, depending on the source of diluted plasma, whereas the concentration of endogenous steroids does not seem to significantly affect binding curves under the conditions examined. It is suggested that binding proteins in various conditions differ from those of healthy adults not only quantitatively but also qualitatively.

Antihypertensive and tissue-protective properties of vitamin D metabolites are increasingly attributed to the inhibition of renin synthesis by 1,25-dihydroxyvitamin D [1,25(OH)2D] in the kidney. We aimed to document a potential association between 25-hydroxyvitamin D [25(OH)D], 1,25(OH)2D and the circulating renin-angiotensin system (RAS) in a large cohort of patients referred (n=3316) to coronary angiography. Of the 3316 subjects, 3296 (median age: 63.5 (56.3-70.6)years; 30.2% women) had a baseline measurement of 25(OH)D [median: 15.6(10.1-23.0)microg/L)], 1,25(OH)2D [median: 33.2(25.2-42.9)pg/mL], plasma renin concentration [PRC; median: 11.4(6.0-24.6)pg/mL] and angiotensin 2 [median: 20.0(12.0-35.0)ng/L]. Multivariate adjusted ANCOVA showed a steady increase of PRC values across declining deciles of 25(OH)D and 1,25(OH)2D values (P=0.013 and P=0.045), respectively. Additionally, mean angiotensin 2 values increased significantly across decreasing 25(OH)D and 1,25(OH)2D values (P=0.020 and P=0.024, respectively). In contrast, multivariate adjusted ANCOVA revealed no significant associations between aldosterone, aldosterone-to-renin ratio and 25(OH)D/1,25(OH)2D values. In multivariate stepwise regression analyses both, 25(OH)D and 1,25(OH)2D emerged as independent predictors of plasma renin and angiotensin 2 concentrations. Our data showed for the first time in humans that both, lower 25(OH)D and 1,25(OH)2D values are independently related to an upregulated circulating RAS.