Giancarlo Severini

Reliability of oxidative stress biomarkers in hemodialysis patients: a comparative study

Abstract Background: Oxidative stress (OS) is considered to play a major role in the development of end-stage renal disease (ESRD) complications. However, conflicting and inconsistent data have been reported on OS in ESRD patients. Our aim was to investigate the reliability of the most popular non-enzymatic plasma OS biomarkers in ESRD. Methods: Vitamins A (VitA), E and C (VitC), uric acid, plasma antioxidant and ferric-reducing potential (PAP and PRP), thiols (SH), malondialdehyde (MDA) and lipid hydroperoxides (HPO) were determined before and after dialysis in plasma from 33 ESRD patients on hemodialysis, hemodiafiltration or peritoneal dialysis and 20 control subjects. Results: In ESRD patients, high PRP and normal PAP values were positively correlated with VitC levels. After dialysis, PRP levels decreased, while unchanged PAP levels correlated positively with high VitA and transiently recovered SH values. All patients showed high levels of both MDA and cholesterol-normalized HPO. However, while the former significantly decreased after dialysis, the latter were unaffected by treatment. Paradoxical correlations of MDA with both VitA and HPO were found. Conclusions: Plasma PRP and MDA levels may be dramatically affected by both uremia and dialysis; their use in ESRD patients may therefore lead to OS misevaluation and should be avoided. More reliable results can be obtained using physiologically relevant OS functional tests, such as PAP, and early biomarkers of OS damage, such as SH and HPO. Clin Chem Lab Med 2007;45:1211–8.

Glutathione S-transferase activity in patients with cancer of the digestive tract

GlutathioneS-transferase (GST) and carcinoembryonic antigen were measured in the plasma of 95 patients with neoplasm of digestive tract, in 40 patients suffering from non-neoplastic diseases and in 40 healthy subjects. The mean value of the GST activity was significantly (P<0.001) elevated in patients with gastric, liver and colorectal cancer (10.4 U/l, 14.1 U/l and 12.3 U/l respectively) as compared with the reference population (3.2 U/l). GST elevations above normal were observed in 26 (90%) patients with gastric cancer, in 18 (100%) with liver cancer and in 25 (89%) with colorectal cancer. Carcinoembryonic antigen appeared less sensitive. In 15 patients the postoperative levels of serum GST were increased after surgery then gradually declined, and after 1 month showed a normalization in 10 patients. Our data suggest that GST measurement may be useful as a tumour marker in gastric, liver and colorectal cancer. Moreover the combined determination of GST and other markers increase the sensitivity for cancer detection.

Influence of uremic middle molecules on in vitro stimulated lymphocytes and interleukin-2 production

The influence of uremic middle molecules on in vitro lymphocyte blast transformation and interleukin-2 production was studied in 12 patients on long-term hemodialysis and 12 healthy control subjects with normal kidney function. Middle molecules inhibit phytohemoagglutinin induced lymphocyte proliferation in a concentration dependent fashion, achieving more than 50% inhibition of tritiated thymidine incorporation after 72 hrs in culture. In addition, there was less interleukin-2 production in the long-term hemodialysis patients studied compared with healthy control subjects.

A study of serum glycosidases in cancer

N-Acetyl-β-glucosaminidase (NAG) and β-glucuronidase were measured in the serum of 70 patients with breast and digestive-tract neoplasms and in 70 healthy subjects. The mean value of the NAG activity was significantly (P<0.001) elevated in patients with gastric, liver and pancreas cancer as compared with the reference population. In patients with liver and pancreas cancer the very high sensitivity contrasted with a low specificity. NAG elevations above normal were observed in 14 (78%) patients with breast cancer, in 11 (100%) with gastric cancer, in 17 (70%) with colorectal cancer, in 8 (100%) with liver cancer and in 9 (100%) with pancreas cancer. In patients with breast and gastric cancer the enzyme shows a good specificity and sufficient sensitivity as a tumor marker. β-Glucuronidase appeared less sensitive and was significantly elevated (100%) only in patients with pancreas cancer.

Diagnostic significance of urinary enzymes: Development of a high performance liquid chromatographic method for the measurement of urinary lysozyme

We describe a new high pressure liquid chromatography (HPLC) method for quantitative determination of urinary lysozyme. The method is simple, reproducible and with detection limit of 0.2 microgram. Before HPLC analysis the urine was purified using a Sep-Pak C18 cartridge. Lysozyme concentration was significantly higher in patients with chronic renal failure than in a control group (p less than 0.001). The concentration of lysozyme in urine is shown to be a sensitive indicator of renal damage.

Variation of urinary enzymes N-acetyl-beta-glucosaminidase, alanine-aminopeptidase, and lysozyme in patients receiving radio-contrast agents

A urinary enzyme pattern consisting of two lysosomal enzymes, N-acetyl-beta-glucosaminidase and lysozyme, and one enzyme originating from kidney tubular brush border membrane, alanine-aminopeptidase, were studied in 30 patients undergoing intravenous urography and arteriography. N-acetyl-beta-glucosaminidase and lysozyme showed the greatest diagnostic sensitivity and were still abnormal on the fifth day after the administration of radio contrast agent. The results, which are statistically significant (Students t test), suggest that radio-contrast agents are potentially nephotoxic.

Homocysteinemia Correlates with Plasma Thiol Redox Status in Patients with End-Stage Renal Disease

Background/Aims: In end-stage renal disease (ESRD), hyperhomocysteinemia is a common finding associated with increased cardiovascular risk. However, the pathogenic role of homocysteine is still unclear. In vitro studies show that thiol redox status affects endothelial cell functions. We therefore investigated the possible association between homocysteinemia and plasma thiol redox status in ESRD patients. Methods: Total plasma homocysteine (Hcy), cysteine (Cys) and free thiols (SH) were measured both before and after a dialytic session in 54 ESRD patients receiving (n = 15) or not receiving (n = 39) folate supplementation, and 17 control subjects. Results: High predialysis levels of both Hcy and Cys were found to be negatively correlated with low SH levels both in supplemented (r = –0.680, p < 0.01 and r = –0.624, p < 0.02, respectively) and unsupplemented (r = –0.698, p < 0.001 and r = –0.445, p < 0.01, respectively) patients. Following dialysis, SH values returned to normal and the above correlations were no longer appreciable. Conclusion: A strong, folate therapy-insensitive association between homocysteinemia and plasma free thiol levels was found in ESRD patients. These results support a role for oxidative stress in ESRD-related hyperhomocysteinemia and suggest the plasma thiol redox status alteration as a possible pathogenic mechanism underlying the cardiovascular toxicity of hyperhomocysteinemia in these patients.

Clinical evaluation of serum N-acetyl-β-d-glucosaminidase as a liver function test

Abstract Serum N-acetyl-β- d -glucosaminidase has been shown to be a sensitive indicator of liver function. The enzyme activity in serum from patients with different forms of hepatic disease was found to be elevated. A comparison is also made with routine liver parameters. The frequencies of pathological serum levels of the routine measurements made are in relatively good agreement with earlier reports.

Determination of ethyl-p-hydroxybenzoate in sow pancreatic juice by reversed-phase high-performance liquid chromatography

We have developed a high-performance liquid chromatographic-UV-Vis-diode-array detection (HPLC-DAD) method for the determination of ethyl-p-hydroxybenzoate, a hydrolytic degradation product of the synthetic protease inhibitor, gabexate-mesilate ethyl-p-(6-guanidinohexanoyloxy) benzoate methanesulfonate (GM) (FOY) in sow pancreatic juice. Methyl-p-hydroxybenzoate (I) was used as the internal standard. The pancreatic juice was deproteinised by acetonitrile and the analytes were chromatographed on a reversed-phase C18 LC column using the gradient elution method. The mobile phase consisted of a solution of 0.017 M orthophosphoric acid and another solution of acetonitrile-water (80:20, v/v). The wavelength of detection was 237 nm. The limit of quantification of the method was 0.20 microM at a 9:1 signal-to-noise ratio. The overall intra- and inter-day accuracy (relative error, RE) ranged from 14.2 to 8.3% and from 13.3 to 9.8, respectively. The overall intra- and inter-day precision (relative standard deviation, RSD) ranged from 7.6 to 2.62% and from 6.7 to 3.1%, respectively. The method proved to be sensitive, specific, accurate and precise and was successfully used to determine the ethyl-p-hydroxybenzoate (II) in sow pancreatic juice.

Uremic toxins and adenosine deaminase activity

Predialysis erythrocyte adenosine deaminase activity was depressed in 12 of 20 patients receiving hemodialysis. In contrast, in 17 patients a marked rise in enzyme activity was observed subsequent to dialysis. Six patients had evidence of neuropathy. Predialysis plasma inhibited normal enzyme activity by an average of 36%, but postdialysis plasma had minimal inhibitory effect. A low molecular weight substance or substances, isolated from the dialysate of uremics, inhibited adenosine deaminase activity by 38%. Three patients having the longest courses of dialysis had no neuropathy and showed no depression of activity, with their plasma failing to inhibit normal activity. It is suggested that accumulation of low molecular weight toxins that depress adenosine deaminase activity may lead to myelin sheath degeneration with subsequent neuropathy. By removing the inhibitor, dialysis may permit nerve repair and eventual recovery.