Fluorescence of the tri-cyclic adenine and isoguanine derivatives and their ribosides: possible analytical applications.

Fluorescent tri-cyclic purine analogs, derivatives of isoguanine and adenine, were examined as potential substrates of purine-nucleoside phosphorylase. It was found previously that etheno- derivatives of both compounds are ribosylated in phosphate-free media, but ribosylation places in some instances differ from purine N9. New ribosides are examined as potential substrates of human blood PNP and indicators of this enzyme. Of these, N6-riboside of 1,N6-etheno-adenine was found the most promising.

Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells.

OBJECTIVE: Systemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single-nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow-up of this polymorphism in human cells. METHODS: Type I IFN was measured in patient sera, using a reporter cell assay. Structural modeling of the PNP variant was performed using PyMOL software. PNP messenger RNA (mRNA) and protein levels and type I IFN-induced gene expression were measured in lymphoblastoid cell lines with known PNP rs1049564 genotypes. The cell cycle was assayed using flow cytometry. RESULTS: Structural modeling indicated no major disruption in folding related to rs1049564. We observed that homozygous rs1049564 TT lymphoblastoid cells had decreased PNP mRNA expression and protein levels, and that cells with the TT genotype had reduced PNP enzymatic activity even when the amount of PNP was controlled. Cells with the TT genotype had a 2-fold increase in S-phase block as compared with cells with the homozygous CC phenotype. The S-phase block could be pharmacologically reversed with hypoxanthine and adenosine, supporting the notion that relative PNP deficiency is the cause of the S-phase block. Type I IFN-induced transcripts were increased in a dose-response manner related to the rs1049564 T allele, at both baseline and after type I IFN stimulation. CONCLUSION: The PNP rs1049564 T allele is a loss-of-function variant that induces S-phase block and IFN pathway activation in lymphocytes. The S-phase block could be rescued in our in vitro experiments, suggesting the potential for personalized treatment.

Development of a new HPLC method using fluorescence detection without derivatization for determining purine nucleoside phosphorylase activity in human plasma.

Purine nucleoside phosphorylase (PNP) activity is involved in cell survival and function, since PNP is a key enzyme in the purine metabolic pathway where it catalyzes the phosphorolysis of the nucleosides to the corresponding nucleobases. Its dysfunction has been found in relevant pathological conditions (such as inflammation and cancer), so the detection of PNP activity in plasma could represent an attractive marker for early diagnosis or assessment of disease progression. Thus the aim of this study was to develop a simple, fast and sensitive HPLC method for the determination of PNP activity in plasma. The separation was achieved on a Phenomenex Kinetex PFP column using 0.1% formic acid in water and methanol as mobile phases in gradient elution mode at a flow rate of 1ml/min and purine compounds were detected using UV absorption and fluorescence. The analysis was fast since the run was achieved within 13min. This method improved the separation of the different purines, allowing the UV-based quantification of the natural PNP substrates (inosine and guanosine) or products (hypoxanthine and guanine) and its subsequent metabolic products (xanthine and uric acid) with a good precision and accuracy. The most interesting innovation is the simultaneous use of a fluorescence detector (excitation/emission wavelength of 260/375nm) that allowed the quantification of guanosine and guanine without derivatization. Compared with UV, the fluorescence detection improved the sensitivity for guanine detection by about 10-fold and abolished almost completely the baseline noise due to the presence of plasma in the enzymatic reaction mixture. Thus, the validated method allowed an excellent evaluation of PNP activity in plasma which could be useful as an indicator of several pathological conditions.

Development and validation of a 2nd tier test for identification of purine nucleoside phosphorylase deficiency patients during expanded newborn screening by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Purine nucleoside phosphorylase (PNP) deficiency has been recently introduced in the newborn screening program in Tuscany. In order to improve the PNP screening efficiency, we developed a 2nd tier test to quantify PNP primary markers deoxyguanosine (dGuo) and deoxyinosine (dIno). METHODS: Dried blood spots (DBS) samples were extracted with 200 µL of methanol and 100 µL of water (by two steps). Internal standards were added at a final concentration of 10 µmol/L. After extraction, samples were analysed by LC-MS/MS. The chromatographic run was performed in gradient mode by using a Synergi Fusion column. RESULTS: The assay was linear over a concentration range of 0.05-50 µmol/L (R2>0.999) for dGuo and 0.5-50 µmol/L (R2>0.998) for dIno. Intra- and interassay imprecision (mean CVs) for dIno and dGuo ranged from 2.9% to 12%. Limit of quantitaion (LOQ) were found to be 0.05 µmol/L and 0.5 µmol/L for dGuo and dIno, respectively. The reference ranges, obtained by measuring dGuo and dIno concentrations on DBS, were close to zero for both biomarkers. Moreover, DBS samples from seven patients with confirmed PNP were retrospectively evaluated and correctly identified. CONCLUSIONS: The LC-MS/MS method can reliably measure dIno and dGuo in DBS for the diagnosis of PNP. Validation data confirm the present method is characterised by good reproducibility, accuracy and imprecision for the quantitation of dIno and dGuo. The assay also appears suitable for use in monitoring treatment of PNP patients.

Purine nucleoside phosphorylase and xanthine oxidase activities in erythrocytes and plasma from marine, semiaquatic and terrestrial mammals.

Purine nucleoside phosphorylase (PNP) and xanthine oxidase (XO) are key enzymes involved in the purine salvage pathway. PNP metabolizes purine bases to synthetize purine nucleotides whereas XO catalyzes the oxidation of purines to uric acid. In humans, PNP activity is reported to be high in erythrocytes and XO activity to be low in plasma; however, XO activity increases after ischemic events. XO activity in plasma of northern elephant seals has been reported during prolonged fasting and rest and voluntary associated apneas. The objective of this study was to analyze circulating PNP and XO activities in marine mammals adapted to tolerate repeated cycles of ischemia/reperfusion associated with diving (bottlenose dolphin, northern elephant seal) in comparison with semiaquatic (river otter) and terrestrial mammals (human, pig). PNP activities in plasma and erythrocytes, as well as XO activity in plasma, from all species were quantified by spectrophotometry. No clear relationship in circulating PNP or XO activity could be established between marine, semiaquatic and terrestrial mammals. Erythrocytes from bottlenose dolphins and humans are highly permeable to nucleosides and glucose, intraerythrocyte PNP activity may be related to a release of purine nucleotides from the liver. High-energy costs will probably mean a higher ATP degradation rate in river otters, as compared to northern elephant seals or dolphins. Lower erythrocyte PNP activity and elevated plasma XO activity in northern elephant seal could be associated with fasting and/or sleep- and dive-associated apneas.

[Effect of low-intensity 900 MHz frequency electromagnetic radiation on rat liver and blood serum enzyme activities].

The comparative analysis of the rat liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and purine nucleoside phosphorylase post-radiation activity levels after a total two-hour long single and fractional exposure of the animals to low-intensity 900 MHz frequency electromagnetic field showed that the most sensitive enzymes to the both schedules of radiation are the liver creatine kinase, as well as the blood serum creatine kinase and alkaline phosphatase. According to the comparative analysis of the dynamics of changes in the activity level of the liver and blood serum creatine kinase, alanine aminotransferase, aspartate aminotransferase and purine nucleoside phosphorylase, both single and fractional radiation schedules do not affect the permeability of a hepatocyte cell membrane, but rather cause changes in their energetic metabolism. The correlation analysis of the post-radiation activity level changes of the investigated enzymes did not reveal a clear relationship between them. The dynamics of post-radiation changes in the activity of investigated enzyme levels following a single and short-term fractional schedules of radiation did not differ essentially.

Assessment of emerging biomarkers of liver injury in human subjects.

Hepatotoxicity remains a major challenge in drug development. Although alanine aminotransferase (ALT) remains the gold standard biomarker of liver injury, alternative biomarker strategies to better predict the potential for severe drug-induced liver injury (DILI) are essential. In this study, we evaluated the utility of glutamate dehydrogenase (GLDH), purine nucleoside phosphorylase (PNP), malate dehydrogenase (MDH), and paraxonase 1 (PON1) as indicators of liver injury in cohorts of human subjects, including healthy subjects across age and gender, subjects with a variety of liver impairments, and several cases of acetaminophen poisoning. In the healthy subjects, levels of GLDH and MDH were not affected by age or gender. Reference ranges for GLDH and MDH in healthy subjects were 1-10 and 79-176U/L, respectively. In contrast, the levels of PON1 and PNP were not consistent across cohorts of healthy subjects. Furthermore, GLDH and MDH had a strong correlation with elevated ALT levels and possessed a high predictive power for liver injury, as determined by ROC analysis. In contrast, PON1 and PNP did not detect liver injury in our study. Finally, evaluation of patients with acetaminophen-induced liver injury provided evidence that both GLDH and MDH might have utility as biomarkers of DILI in humans. This study is the first to evaluate GLDH, MDH, PON1, and PNP in a large number of human subjects and, and it provides an impetus for prospective clinical studies to fully evaluate the diagnostic value of GLDH and MDH for detection of liver injury.

Tissue distribution and metabolism of guanosine in rats following intraperitoneal injection.

Guanosine has long been known as an endogenous purine nucleoside deeply involved in the modulation of several intracellular processes, especially G-protein activity. More recently, it has been reported to act as an extracellular signaling molecule released from neurons and, more markedly, from astrocytes either in basal conditions or after different kinds of stimulation including hypoxia. Moreover, in vivo studies have shown that guanosine plays an important role as both a neuroprotective and neurotrophic agent in the central nervous system. Specific high-affinity binding sites for this nucleoside have been found on membrane preparations from rat brain. The present study was undertaken to investigate the distribution and metabolic profiles of guanosine after administering the nucleoside to gain a better understanding of the biological effects of this potential drug candidate. Rats were given an intraperitonal (i.p.) injection of 2, 4, 8 or 16 mg/kg of guanosine combined with 0.05% of [3H]guanosine. Plasma samples were collected 7.5, 15, 30, 60 and 90 min after the guanosine-mixture administration and analyzed by either a liquid scintillation counter or by HPLC connected to a UV and to an on-line radiochemical detector to measure the levels of guanosine and its metabolic products guanine, xanthine and uric acid. The levels of guanosine, guanine and xanthine were also measured in brain, lung, heart, kidney and liver tissue homogenates at the defined time points after the injection of 8 mg/kg of the guanosine-mixture. We found that the levels of radioactivity in plasma increased linearly in a dose- and time-dependent manner. Guanosine was widely distributed in all tissues examined in the present study, at almost twice its usual levels. In addition, guanine levels dramatically increased in all the organs. Interestingly, enzymatic analysis of the plasma samples showed the presence of a soluble purine nucleoside phosphorylase, a key enzyme in the purine salvage pathway and nucleoside catabolism. Since guanosine has been shown to be neuroprotective and astrocytes have been reported to play critical roles in mediating neuronal survival and functions in different neurodegenerative disorders, we also performed uptake and release.

Purine nucleoside phosphorylase deficiency: a mutation update.

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.

Metabolites of purine nucleoside phosphorylase (NP) in serum have the potential to delineate pancreatic adenocarcinoma.

Pancreatic Adenocarcinoma (PDAC), the fourth highest cause of cancer related deaths in the United States, has the most aggressive presentation resulting in a very short median survival time for the affected patients. Early detection of PDAC is confounded by lack of specific markers that has motivated the use of high throughput molecular approaches to delineate potential biomarkers. To pursue identification of a distinct marker, this study profiled the secretory proteome in 16 PDAC, 2 carcinoma in situ (CIS) and 7 benign patients using label-free mass spectrometry coupled to 1D-SDS-PAGE and Strong Cation-Exchange Chromatography (SCX). A total of 431 proteins were detected of which 56 were found to be significantly elevated in PDAC. Included in this differential set were Parkinson disease autosomal recessive, early onset 7 (PARK 7) and Alpha Synuclein (aSyn), both of which are known to be pathognomonic to Parkinson's disease as well as metabolic enzymes like Purine Nucleoside Phosphorylase (NP) which has been exploited as therapeutic target in cancers. Tissue Microarray analysis confirmed higher expression of aSyn and NP in ductal epithelia of pancreatic tumors compared to benign ducts. Furthermore, extent of both aSyn and NP staining positively correlated with tumor stage and perineural invasion while their intensity of staining correlated with the existence of metastatic lesions in the PDAC tissues. From the biomarker perspective, NP protein levels were higher in PDAC sera and furthermore serum levels of its downstream metabolites guanosine and adenosine were able to distinguish PDAC from benign in an unsupervised hierarchical classification model. Overall, this study for the first time describes elevated levels of aSyn in PDAC as well as highlights the potential of evaluating NP protein expression and levels of its downstream metabolites to develop a multiplex panel for non-invasive detection of PDAC.

Identification of purine nucleoside phosphorylase deficiency in dried blood spots by a non-radiochemical assay using reversed-phase high-performance liquid chromatography.

Purine nucleoside phosphorylase (PNP) deficiency results in severe T cell dysfunction and hypouricemia. An assay to measure PNP activity in dried blood spots was developed using reversed-phase HPLC. The assay was linear with reaction times between 5 and 12.5 minutes, and protein concentrations ranging from 0.4 to 1.8 mg/ml. The intra-assay CV and the inter-assay CV for the complete assay was <3.6%. The PNP activity in a control blood spot, stored at 4 degrees C, remained stable for at least one year. In a patient suffering from a PNP deficiency, the residual PNP activity was only 0.3% compared to that observed in controls (1431 +/- 238 nmol/mg/h, n = 114). The PNP activity (483 +/- 35 nmol/mg/h, n = 3) in heterozygotes for the c.614A > C mutation (p.E205A) in the PNP gene was 34% compared to controls. Thus, the analysis of the PNP activity in blood spots can readily detect patients with a PNP deficiency.

[The clinical diagnostic value of a study of the activity of enzymes of the guanyl branch of purine metabolism in patients with ankylosing spondyloarthritis].

AIM: To assess the diagnosis of the activity of a pathological process in patients with ankylosing spondyloarthritis (AS) and to reveal purine metabolic (PM) changes in relation to the clinical features of AS. MATERIALS AND METHODS: The serum activities of the PM enzymes: xanthine oxidase (XO), guanine deaminase (GDA), guanosine deaminase (GSDA), purine nucleoside phosphorylase (PNP), guanosine phosphorylase (GP), and adenosine deaminase (ADA) were determined in 55 patients (51 males and 4 females) aged 36.0 +/- 1.4 years). A control group comprised 30 apparently age- and gender-matched healthy individuals, as in the study group. RESULTS: On admission, the patients were found to have increased XO, GDA, PNP, and GD activities and decreased GSDA activity. The higher activity of the process was observed, the greater activities of GDA, XO, PNP, GP and the lower activity of GSDA and ADA were. There were the enzymatic activity differences that depended on the degree of pathological process activity, clinical form, the magnitude of X-ray changes, and the degree of joint functional insufficiency. CONCLUSION: The findings suggest that there may be PM disturbances in the immunocompetent cells.

[Purine nucleoside phosphorylase inhibitors and their clinical significance].

PNP catalyzes a reversible phosphorolysis of purine deoxy- and ribonucleosides with formation of (d)Rib-1-P and appropriate bases. PNP plays a leading role in the cell metabolism of nucleosides and nucleotides, as well as in maintaining the immune status of an organism. The major purpose of the majority of studies on the PNP is the detection of high-performance enzyme inhibitors, derivatives of the purine nucleosides, which are used in medicine as immunosuppressors. It is well known that the latter are necessary for creating a selective T-cell immunodeficiency in a human body under organs and tissue transplantation. The review discusses the issues related to deliberate synthesis of effective, metabolically inert, and low-toxic PNP inhibitors. It also analyzes the available studies on substrate and inhibitory properties of the analogues of purine nucleosides, as well as research on the structural factors which reinforce the inhibitor activity of those analogues. The inhibitors which are either used in medical practice or are currently at a stage of preclinical testing are described. The inhibitors which are more efficient in their influence on the PNF from tumorous tissues are of special interest. Using PNP inhibitors in case of a number of pathologies denotes the importance and promise of research on both the enzyme and the compounds affecting its activity.

Synthesis and biological evaluation of 9-deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid as multi-substrate analogue inhibitors of PNP.

9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) was designed as a multi-substrate analogue inhibitor against purine nucleoside phosphorylase (PNP) on the basis of X-ray crystallographic data obtained for a binary complex of 9-(5',5'-difluoro-5'-phosphonopentyl)guanine (DFPP-G) with calf spleen PNP. DFPP-DG and its analogous compounds were adjusted by length of the linker achieved by the Sonogashira-coupling reaction between a 9-deaza-9-iodoguanine derivative and omega-alkynyldifluoromethylene phosphonates as a key reaction. DFPP-DG is a very potent PNP inhibitor with apparent inhibition constants (in the presence of 1 mM phosphate) of 4.4 and 8.1 nM versus calf spleen and human erythrocyte PNPs, respectively. One of its analogues, homo-DFPP-DG, with longer chain linking phosphonate and 9-deazaguanine is even more potent versus human enzyme, with an apparent inhibition constant of 5.3 nM (in the presence of 1mM phosphate).

An HPLC method for determination of inosine and hypoxanthine in human plasma from healthy volunteers and patients presenting with potential acute cardiac ischemia.

A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing ultraviolet (UV) detection was developed for the determination of inosine and hypoxanthine in human plasma. For component separation, a monolithic C(18) column at a flow rate of 1.0 mL/min with an aqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and methanol gradient was used. The method employed a one-step sample preparation utilizing centrifugal filtration with high component recoveries (approximately 98%) from plasma, which eliminated the need of an internal standard. The method demonstrated excellent linearity (0.25-5 microg/mL, R>0.9990) for both inosine and hypoxanthine with detection limits of 100 ng/mL. This simple and cost effective method was utilized to evaluate potential endogenous plasma biomarker(s), which may aid hospital emergency personnel in the early detection of acute cardiac ischemia in patients presenting with non-traumatic chest pain.

Plasma purine nucleoside phosphorylase in cancer patients.

BACKGROUND: Purine nucleoside phosphorylase (PNP) is the purine salvage enzyme that converts guanosine to guanine and inosine to hypoxanthine. METHODS: 279 samples from patients with differing cancers were collected during treatment at both pre- and post-dose stages for plasma PNP activity and compared with a normal population. RESULTS: Normal plasma PNP activity was found to be 3.2+/-1.4 U/l (n=55) as compared with the cancer patients (pre-dose 12.3+/-7.4 U/l [n=215] and post-dose 11.2+/-5.9 U/l [n=64]). Levels of plasma PNP did not differ greatly between the different cancer types but were on average four times greater than that found in the reference population.

Capillary electrophoresis in diagnosis and monitoring of adenosine deaminase deficiency.

BACKGROUND: The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attributable to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphorylase (PNP), and S-adenosyl-L-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evaluation of enzyme activities. METHODS: Deoxyadenosine metabolites were separated in 30 mmol/L sodium borate-10 mmol/L sodium dodecyl sulfate (pH 9.80) at 25 degrees C on a 60-cm uncoated capillary. For determination of enzyme activities, substrate-product separation and measurements were carried out in 20 mmol/L sodium borate (pH 10.00) at 25 degrees C on a 42-cm uncoated capillary. RESULTS: Deoxynucleotides and deoxyadenosine were readily detectable in erythrocytes and urine, respectively. Both methods were linear in the range 2-500 micro mol/L (r >0.99). Intra- and interassay CV were <4%. Enzyme activities were linear with respect to sample amounts in the incubation mixture and to incubation time (r >0.99 for both). In erythrocytes from healthy individuals, mean (SD) ADA activity was 5619 (2584) nmol/s per liter of packed cells. In erythrocytes of SCID patients at diagnosis, ADA activity was 56.9 (48.3) nmol/s per liter of packed cells; SAHH activity was also much reduced. PNP activity was similar in patients and controls. CONCLUSIONS: CE can be used to test ADA deficiency and enables rapid assessment of ADA expression in hematopoietic cells of SCID patients during therapy.

Purine metabolism in leukocytes and erythrocytes in Graves' or Hashimoto's disease.

INTRODUCTION: Adenosine deaminase (ADA), purine nucleoside phosphorylase (PNPase), S-adenosylhomocysteine hydrolase (SAHH), 5'-nucleotidase (5N), and deoxycytidine kinase (dCK) are involved in purine salvage metabolism. Changes of the activities of the above enzymes have been observed in blood cells in patients with immunological disorders. MATERIALS AND METHODS: The activities of ADA, PNPase, SAHH, 5'N, and dCK in lysates of leukocytes and erythrocytes, obtained from patients with Graves' or Hashimoto's disease, were measured, using chromatographic analysis. Serum concentrations of antithyroglobulin (Tg Ab) and antithyroperoxidase (TPO Ab) antibodies were measured by an immunoenzymatic method. RESULTS: (1) ADA activity in leukocytes, obtained from patients with Hashimoto's disease, was significantly higher than in control leukocytes, as well as in leukocytes from patients with Graves' disease; (2) dCK activities in leukocytes from patients with both Graves' and Hashimoto's diseases were approximately four and five times higher, respectively, than in leukocytes of control subjects; (3) a positive correlation was observed between dCK activity in leukocytes and serum Tg Ab concentration in patients with Graves' disease. In conclusion, the increased ADA and dCK activities in leukocytes from patients with Graves' and Hashimoto's diseases may be regarded as indicators of autoimmunological thyroid diseases.

Blood transfusion causes deterioration in liver regeneration after partial hepatectomy in rats.

BACKGROUND: This study investigated the effects of blood transfusion on liver regeneration and function after hepatectomy in rats. METHODS: Inbred male Sprague-Dawley rats underwent a sham operation or a 70% hepatectomy (PHx) and were randomly divided into seven groups according to transfusion type: groups I and II underwent a sham operation and received saline (I) or whole blood (II). Groups III to VII underwent PHx with saline (III), whole blood (IV), irradiated/leukocyte-depleted whole blood (V), plasma (VI), or autologous blood (VII). The liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling index, serum aspartate aminotransferase, alanine aminotransferase, purine nucleoside phosphorylase (PNP) activity, hepatocyte growth factor (HGF), and activated transforming growth factor beta1 (TGF-beta(1)) were measured 6 and 24 h and 5 days after PHx. RESULTS: The liver regeneration rate and PCNA labeling index were lower in groups IV and V than in the other groups. Serum liver enzymes 6 h after PHx were worst in groups IV and V. PNP activity increased most in group IV, 6 and 24 h after PHx. The HGF values 6 h after PHx in all the transfused groups were lower than in group III. The activated TGF-beta(1) level 6 h after surgery was highest in group IV. CONCLUSION: Whole blood or irradiated/leukocyte-depleted whole blood impaired liver regeneration after PHx, probably through the production of activated TGF-beta(1) and HGF outside the liver, and plasma or autologous blood reduced the deleterious effects.