Association of plasma pentosidine concentrations with renal function in kidney graft recipients.

BACKGROUND: Advanced glycation end-products accumulate in the plasma of uremic patients. We aimed to assess the changes of free (P(free)) and total (P(tot)) plasma pentosidine concentrations in kidney graft recipients, create a model describing their profile and analyze associations with clinical parameters. MATERIAL AND METHODS: We measured P(free) and P(tot) in the plasma of 12 non-diabetic patients before and after kidney transplantation by HPLC. RESULTS: P (tot) concentrations were significantly decreasing after transplantation. The changes were well described by the exponential model assuming an asymptotic fall until the steady-state concentration is attained. P(tot) before and after transplantation displayed a strong negative correlation with mean daily diuresis (Rs = -0.64, p < 0.05 before; Rs = -0.94, p < 0.01 after 20 d). The rate of fall of the P(tot) was positively correlated with the mean daily volume of urine passed in the second week after operation (Rs = +0.58, p < 0.05). CONCLUSIONS: The rate of fall of the P(tot) after transplantation displays a strong correlation with diuresis, and the P(tot) before transplantation is a potential prognostic factor for diuresis after the operation. Further prospective studies are necessary to demonstrate whether an effort to reduce carbonyl stress and pentosidine concentration before transplantation improves renal outcome.

The effect of dialysis membrane type on plasma concentrations of pentosidine, a marker of carbonyl stress, in hemodialyzed patients.

INTRODUCTION: Advanced glycation end products (AGEs), which accumulate in plasma of hemodialyzed patients, participate in the development of complications associated with hemodialysis (HD). Carbonyl stress plays an essential role in the formation of AGEs, including pentosidine. OBJECTIVES: The aim of the study was to assess the effect of various low-flux dialysis membranes on plasma concentrations of total (P(tot)) and free (P(free)) pentosidine. PATIENTS AND METHODS: We examined 56 adult patients (aged 50 +/-13 years) on chronic HD. Plasma pentosidine concentrations were measured with high-performance liquid chromatography with fluorescence detection. Plasma proteins were subjected to acid hydrolysis or precipitation with trichloroacetic acid before measurement of P(tot) and P(free), respectively. RESULTS: Significantly lower concentrations of P(tot) were observed in patients dialyzed with polysulfone than non-polysulfone membranes before the HD session (22.0 +/-11.9 vs 34.4 +/-12.5 pmol/mg protein, respectively, p = 0.0008) and after the HD session (22.5 +/-12.9 vs 32.9 +/-12.0 pmol/mg protein, p = 0.004). Moreover, there was a strong inverse correlation between the percentage of HD sessions performed with polysulfone membranes during the last 3 months and P(tot) concentration before HD (Rs = -0.44, p = 0.0011) and after HD (Rs = -0.45, p = 0.00073). CONCLUSIONS: The results suggest that polysulfone membranes reduce carbonyl stress more effectively than modified or unmodified cellulose membranes in patients on chronic HD. Determination of plasma pentosidine in hemodialyzed patients may help in comparing the physicochemical and biological properties of dialysis membranes. It may also contribute to the development of optimal strategies for renal replacement therapy.

[Physiological and pathological consequences of a presence of germ line stem cells in adult tissues]. / Konsekwencje fizjologiczne i patologiczne wystepowania komórek linii zarodkowych w doroslych tkankach.

Various therapheutic strategies employing stem cells have been proposed as the alternative, effective methods for therapy of multitude diseases, difficult to treat using standard, well-established methods. Advancing regenerative medicine, which is becoming a novel branch of clinical medicine, has high hopes of stem cells which could be used in treatment of injuried organs such as myocardium after heart infarction, brain after stroke, spinal cord after mechanical injury as well as in treatment of diabetes and Parkinson disease. Application of embryonic stem cells, harvested from developing embryos, is highly controversial. Hence, the stem/primitive cells isolated from adult tissuses are considered to be an optimal source of cells for therapy. Recently our research team has isolated a population of very primitive stem cells from adult tissues (very small embryonic-like stem cells - VSELs) that show several embryonic-like features. These cells can become an alternative and more ethical source of the stem cells for therapy when compared to those isolated from the developing embryos.

Characterisation of rat and human tissue alkaline phosphatase isoforms by high-performance liquid chromatography and agarose gel electrophoresis.

Alkaline phosphatase (ALP) exists as several isoenzymes and many isoforms present in tissues and serum. The objective of this study was to separate tissue ALP forms in rats and humans and characterise their properties. The materials for the investigation were intestinal, bone, and liver tissue of rats and commercially available human preparations of tissue ALP. Two methods of separation were used: high-performance liquid chromatography (HPLC) and agarose gel electrophoresis. Using HPLC in the rat tissues, two ALP isoforms in the intestine, one in the bone, and three in the liver were identified. In humans three intestinal, two bone, and one liver isoform were resolved. Electrophoresis showed two ALP activity bands in rat intestine, one wide band in the bone, and three bands in the liver. ALP of human tissues was visualised as a single wide band, with a different mobility observed for each organ. In both species the presence of a form with properties characteristic of the bone isoform of the tissue-nonspecific isoenzyme was observed in the intestine. HPLC offers a higher resolution than electrophoresis with respect to tissue ALP fractions in rats and in humans, but electrophoresis visualises high-molecular-mass insoluble enzyme forms.

Comparison of rat and human alkaline phosphatase isoenzymes and isoforms using HPLC and electrophoresis.

Total activity of alkaline phosphatase (ALP) in serum represents the sum of activities of some isoenzymes and their numerous isoforms derived from various tissues of the organism. The aim of this work was to separate ALP isoenzymes and their isoforms in rat and human serum, compare the properties of serum ALP isoforms in rats and humans, and determine the usefulness of some analytical methods for specific measurements of ALP isoenzyme and isoform activities. Two methods of separation, i.e. high-performance liquid chromatography (HPLC) and agarose gel electrophoresis were chosen. The combination of HPLC with electrophoresis of the eluted fractions and with ALP inhibition methods (urea, l-phenylalanine), inactivation (heat) and precipitation (wheat-germ lectins) enabled the identification of isoenzymes and isoforms of ALP in serum. Using chromatography and a post-column reactor, three isoforms of the intestinal isoenzyme and one bone isoform of a tissue non-specific isoenzyme were detected. Rat serum differs significantly from human as regards activities of intestinal and hepatic isoforms, whereas the properties of the bone isoform are similar in both species. Our HPLC method offers a higher resolution than agarose gel electrophoresis with respect to ALP subfractions in rat serum.

A sensitive and specific HPLC method for the determination of total pentosidine concentration in plasma.

Pentosidine is an advanced glycation end-product (AGE) appearing when arginine and lysine residues in proteins are cross-linked with carbonyl derivatives. This paper presents an improved method for the synthesis of pentosidine and reversed-phase chromatography of this substance with fluorometric detection that enables sensitive (0.01 pmol/mg protein) and specific determination of pentosidine in plasma. Separation is done twice on the same C(18) Vydac 218TP54 column, first with trifluoroacetic acid and next with heptafluorobutyric acid as ion pair. The inter-day coefficient of variation is 6.4% at pentosidine concentration in plasma of 25 pmol/mg protein and 8% at 1.7 pmol/mg protein. Spectral properties of pentosidine exploited during identification of the substance with UV absorption and fluorescence detectors are described. Maximum of absorbance was observed at 325 nm, maximum fluorescence at lambda(ex)/lambda(em)=330/373 nm. The method may prove useful for the study of processes associated with generation and accumulation of pentosidine in the body as a marker of AGE production in healthy subjects and patients with chronic renal failure.

[Reduction of carbonyl stress with renal replacement therapy in chronic renal failure]. / Skutecznosc leczenia nerkozastepczego w redukcji stresu karbonylowego u chorych z przewlekla niewydolnoscia nerek.

Advanced glycation end products (AGEs) play a significant role in the pathogenesis of chronic renal failure and diabetic complications. Their accumulation in these disorders leads to the formation of intermolecular crosslinks and lowers susceptibility to protein denaturation and proteolysis. By binding with specific receptors, AGEs initiate processes that result in the generation and release of cytokines, growth factors, and reactive oxygen species (ROS). This work concentrated on pentosidine as a representative AGE. High tissue and plasma concentrations of pentosidine observed in chronic renal failure result from accumulation of pentosidine precursors containing highly reactive carbonyl groups and responsible for carbonyl stress in uremia. The aim of this work was to evaluate clinical and biochemical parameters that affect pentosidine concentration in plasma of chronic renal failure patients and renal graft recipients. A pentosidine standard was synthesized and purified and its chromatographic separation method was optimized. RP-HPLC was applied using an octadecylsilane (ODS) column and HFBA or TFA as solvents. 53 chronically haemodialyzed patients and 14 renal graft recipients were studied. The chromatographic method proved to be sensitive and specific enough for precise measurements of plasma concentrations of both total and free pentosidine. The results served to evaluate the efficiency of renal replacement therapy for the reduction of carbonyl stress in patients with chronic renal failure. Numerous factors influencing the concentration of pentosidine in plasma were identified. Some are not amenable (gender, age), while others, like type of dialysis membrane, efficiency of dialysotherapy, surface of dialyser, or body mass index (BMI) can be controlled. Patients undergoing more dialysis sessions with polysulphone membranes three months prior to the study demonstrated lower plasma concentrations of total pentosidine. Lower concentrations of total and free pentosidine were observed in patients with higher BMI values. On the other hand, there was a positive correlation between pentosidine concentration, efficiency of dialysotherapy and ultrafiltration. Modification of these parameters may decrease carbonyl stress. The study demonstrated that renal transplantation is the only effective therapy for decreasing pentosidine concentrations and the intensity of carbonyl stress. A transplanted kidney efficiently and quickly eliminates carbonyl derivatives, immediately reduces the rate of pentosidine synthesis, and in consequence leads to a slow decrease in its concentra- tion resulting from whitch elimination half-time of plasma proteins. The study showed that the concentration of total pentosidine prior to transplantation has a significant influence on diuresis after transplantation. It is possible that haemodialysotherapy whitch effectively decreases carbonyl stress in patients awaiting renal grafting and contributes to improved function of the transplanted organ.