Roman Safranek

Effects of proteasome inhibitors MG132, ZL3VS and AdaAhx3L3VS on protein metabolism in septic rats

Proteasome inhibitors are novel therapeutic agents for the treatment of cancer and other severe disorders. One of the possible side effects is influencing the metabolism of proteins. The aim of our study was to evaluate the influence of three proteasome inhibitors MG132, ZL3VS and AdaAhx3L3VS on protein metabolism and leucine oxidation in incubated skeletal muscle of control and septic rats. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of protein synthesis and leucine oxidation were measured in a medium containing L‐[1‐14C]leucine. Protein synthesis was determined as the amount of L‐[1‐14C]leucine incorporated into proteins, and leucine oxidation was evaluated according to the release of 14CO2 during incubation. Sepsis was induced in rats by means of caecal ligation and puncture. MG132 reduced proteolysis by more than 50% and protein synthesis by 10–20% in the muscles of healthy rats. In septic rats, proteasome inhibitors, except ZL3VS, decreased proteolysis in both soleus and extensor digitorum longus (EDL) muscles, although none of the inhibitors had any effect on protein synthesis. Leucine oxidation was increased by AdaAhx3L3VS in the septic EDL muscle and decreased by MG132 in intact EDL muscle. We conclude that MG132 and AdaAhx3L3VS reversed protein catabolism in septic rat muscles.

Effect of acute acidosis on protein and amino acid metabolism in rats

BACKGROUND & AIMS Metabolic acidosis is a common finding in critical illness. The aim of the present study was to evaluate acute acidosis as a signal that induces changes in protein metabolism. METHODS In the first study, Wistar rats were infused for 6h with HCl or saline resulting in blood pH7.30+/-0.03 and 7.46+/-0.02, respectively. The whole body protein metabolism was evaluated using L-[1-(14)C]leucine. In the second study, soleus and extensor digitorum longus muscles from normal rats were incubated in medium, pH7.4, 7.3 or 7.0. Protein metabolism was evaluated using L-[1-(14)C]leucine and tyrosine release. RESULTS In the in vivo study we observed increased protein turnover-protein synthesis, proteolysis and leucine oxidation and more negative protein balance in rats with acidosis. There was no change in protein synthesis in gastrocnemius muscle. We observed an increase in plasma levels of most amino acids including branched-chain amino acids and a decrease in intracellular amino acid pool in skeletal muscle. In vitro decrease in pH of 0.1 had no effect on protein metabolism, decrease of 0.4 decreased protein turnover and leucine oxidation. CONCLUSION Acute metabolic acidosis is a protein wasting condition. Direct effect of acidosis on skeletal muscle is under condition in vivo modified by neurohumoral regulations.

Vitamin D Binding Protein Is Not Involved in Vitamin D Deficiency in Patients with Chronic Kidney Disease

Objective. This study was designed to evaluate vitamin D status with separate determination of 25-OH D2 and 25-OH D3 and its relationship to vitamin D binding protein (VDBP) in patients with chronic kidney disease (CKD) and long-term haemodialysis patients (HD). Methods. 45 CKD patients, 103 HD patients, and 25 controls (C) were included. Plasma vitamin D concentrations were determined using chromatography and VDBP in serum and urine in CKD using enzyme immunoassay. Results. Plasma vitamin D levels were lower in CKD (30.16 ± 16.74 ng/mL) and HD (18.85 ± 15.85 ng/mL) versus C (48.72 ± 18.35 ng/mL), P < 0.0001. 25-OH D3 was the dominant form of vitamin D. Serum VDBP was higher in CKD (273.2 ± 93.8 ug/mL) versus C (222 ± 87.6 ug/mL) and HD (213.8 ± 70.9 ug/mL), P = 0.0003. Vitamin D/VDBP ratio was the highest in C and the lowest in HD; however, there was no correlation between vitamin D and VDBP. Urinary concentration of VDBP in CKD (0.25 ± 0.13 ug/mL) correlated with proteinuria (r = 0.43, P = 0.003). Conclusions. Plasma levels of vitamin D are decreased in CKD patients and especially in HD patients. 25-OH D3 was the major form of vitamin D. Despite urinary losses of VDBP, CKD patients had higher serum VDBP concentrations, indicating compensatory enhanced production. Vitamin D binding protein is not involved in vitamin D deficiency.

Aspects of Protein and Amino Acid Metabolism in a Model of Severe Glutamine Deficiency in Sepsis

Background/Aims: Growth hormone (GH) could have the potential to improve protein metabolism in sepsis but glutamine deficiency has been reported after GH treatment. The aim was to investigate the effects of glutamine deficiency in sepsis with and without GH treatment on protein and amino acid metabolism. Methods: Cecal ligation and puncture (CLP) was used as a model of sepsis. Serious glutamine deficiency was induced by administration of glutamine synthetase inhibitor, methionine sulfoximine (MSO). Young Wistar rats were divided into 5 groups: control; CLP; CLP+MSO; CLP+GH, and CLP+MSO+GH. Parameters of protein metabolism were measured on incubated soleus and extensor digitorum longus muscles: [1-14C]leucine was used to estimate protein synthesis and leucine oxidation, tyrosine release was used to evaluate protein breakdown. Amino acid concentrations in plasma, skeletal muscle and incubation media were measured by HPLC. Results/Conclusions: A reduced muscle glutamine concentration after MSO treatment is not associated with changes in the rates of protein synthesis or breakdown. MSO treatment decreased glutamine release from skeletal muscle and plasma glutamine concentration. Severe glutamine deficiency in GH-treated septic rats resulted in increased release of branched-chain amino acids from skeletal muscle.

DENOSUMAB ASSOCIATED WITH BONE DENSITY INCREASE AND CLINICAL IMPROVEMENT IN A LONG-TERM HEMODIALYSIS PATIENT. CASE REPORT AND REVIEW OF THE LITERATURE

Denosumab is a human monoclonal antibody representing a novel therapy of osteoporosis. Contrary to always other antiosteoporotic drugs, it is not contraindicated in advanced chronic kidney disease, as its pharmacokinetic does not differ from patients with normal kidney function. However, published case reports in chronic kidney disease (CKD) patients stopped the therapy after single dose because of hypocalcemia. We present a case of successful treatment of osteoporosis in a young hemodialysis patient with repeated denosumab doses.

Acute effects of decreased glutamine supply on protein and amino acid metabolism in hepatic tissue: a study using isolated perfused rat liver

Glutamine deficiency, a common finding in severe illness, has a negative influence on immune status, protein metabolism, and disease outcome. In several studies, a close relationship between glutamine, branched-chain amino acid (BCAA), and protein metabolism was demonstrated. The aim of the present study was to investigate the effect of glutamine deficiency on amino acid and protein metabolism in hepatic tissue using a model of isolated perfused rat liver (IPRL). Parameters of protein metabolism and amino acid metabolism were measured using both recirculation and single pass technique with L-[1-(14)C]leucine and [1-(14)C]ketoisocaproate (KIC) as a tracer. Glutamine concentration in perfusion solution was 0.5 mmol/L in control and 0 mmol/L in the glutamine-deficient group. The net release of glutamine (about 11 micromol/g/h) and higher net uptake of most of the amino acids was observed in the glutamine-deficient group. There was an insignificant effect of lack of glutamine on hepatic protein synthesis, proteolysis, and the release of urea. However, significantly lower release of proteins by the liver perfused with glutamine-deficient solution was observed. The lack of glutamine in perfusion solution caused a significant decrease in leucine oxidation (6.66 +/- 1.04 v 13.67 +/- 2.38, micromol/g dry liver/h, P <.05) and an increase in KIC oxidation (163.7 +/- 16.5 v 92.0 +/- 12.9 micromL/g dry liver/h, P <.05). We conclude that decreased delivery of glutamine to hepatic tissue activates glutamine synthesis, decreases resynthesis of essential BCAA from branched-chain keto acids (BCKA), increases catabolism of BCKA, and has an insignificant effect on protein turnover in hepatic tissue.

Acute effects of acidosis on protein and amino acid metabolism in perfused rat liver.

Summary.  Acidosis is frequently associated with protein wasting and derangements in amino acid metabolism. As its effect on protein metabolism is significantly modulated by other abnormal metabolic conditions caused by specific illnesses, it is difficult to separate out the effects on protein metabolism solely due to acidosis. The aim of the present study was to evaluate, using a model of isolated perfused rat liver, the direct response of hepatic tissue to acidosis. We have compared hepatic response to perfusion with a solution of pH 7.2 and 7.4 (controls). Parameters of protein and amino acid metabolism were measured using both recirculation and single‐pass technique with 4,5‐[3H]leucine, [1–14C]leucine and [1–14C]ketoisocaproate (ketoleucine) as tracers and on the basis of difference of amino acid levels in perfusion solution at the beginning and end of perfusion. In liver perfused with a solution of pH 7.2, we observed higher rates of proteolysis, protein synthesis, amino acid utilization and urea production. Furthermore, the liver perfused with a solution of pH 7.2 released a higher amount of proteins to perfusate than the liver perfused with a solution of pH 7.4. Enhanced decarboxylation of ketoisocaproate in liver perfused by a solution of a lower pH indicates increased catabolism of branched‐chain amino acids (leucine, valine and isoleucine), decreased reamination of branched‐chain keto acids to corresponding essential amino acids and increased ketogenesis from leucine.

Changes of Serum Calcium, Magnesium and Parathyroid Hormone Induced by Hemodialysis with Citrate-Enriched Dialysis Solution

Background/Aims: In recent years, one of technical attempts to improve biocompatibility and tolerability of the hemodialysis procedure is the substitution of acetate in dialysis solution with citrate. The aim of our study was to compare two dialysis solutions: traditional bicarbonate dialysis solution containing acetate (3 mmol/L) (solution A); and (solution C) commercially produced citrate-enriched bicarbonate dialysis solution (0.8 mmol/L citrate). Methods: Patients from a single hemodialysis center (N=126) were included in the study. Both conventional low-flux hemodialysis and on-line hemodiafiltration procedures were studied. Both dialysis solutions contained identical calcium (1.5 mmol/L) and magnesium (0.5 mmol/L) concentrations. Results: Parathyroid hormone (iPTH) concentration decreased during procedures with solution A by 64%. On the contrary, when solution C was used, iPTH concentration increased insignificantly by 4%. For solution A, serum calcium and magnesium increased during procedures in patients with predialysis concentrations lower than 2.33 and 0.76 mmol/L, respectively. In procedures with dialysis solution C these concentrations were significantly lower: 2.19 mmol/L for Ca and 0.68 mmol/L for Mg. Conclusion: Our study clearly shows that the substitution of part of acetate with citrate in dialysis solution significantly influences changes of serum calcium, magnesium and parathyroid hormone concentrations during hemodialysis and hemodiafiltration procedures.

Indoxyl Sulfate Elimination in Renal Replacement Therapy: Influence of Citrate- versus Acetate-Buffering Component during Bicarbonate Dialysis

Indoxyl sulfate has been identified as a major factor in the dysregulation of several genes. It is classified as a poorly dialyzable uremic toxin and thus a leading cause in the poor survival rate of dialysis patients. A monocentric, prospective, open cohort study was performed in 43 male patients undergoing chronic renal replacement therapy in a single hemodialysis center. The aim of the study was to determine the influence of acetate- versus citrate-buffered dialysis fluids in hemodialysis (HD) and postdilution hemodiafiltration (HDF) settings on the elimination of indoxyl sulfate. Also, additional factors potentially influencing the serum concentration of indoxyl sulfate were evaluated. For this purpose, the predialysis and postdialysis concentration ratio of indoxyl sulfate and total protein was determined. The difference was of 1.15 (0.61; 2.10), 0.89 (0.53; 1.66), 0.32 (0.07; 0.63), and 0.44 (0.27; 0.77) μmol/g in acetate HD and HDF and citrate HD and HDF, respectively. Acetate HD and HDF were superior when concerning IS elimination when compared to citrate HD and HDF. Moreover, residual diuresis was determined as the only predictor of lower indoxyl sulfate concentration, suggesting that it should be preserved as long as possible. This trial is registered with EU PAS Register of Studies EUPAS23714.

Parathyroidectomy in Hyperparathyroidism-Associated Calciphylaxis in End-Stage Renal Disease Should be Prompt and Radical – a Case Report with Two Original Therapeutic Modifications and Successful Outcome

We present a case of severe calciphylaxis in both thighs and calves in a patient with end-stage renal disease and advanced secondary hyperparathyroidism with successful outcome after modified therapeutic approach. The cause of calciphylaxis is multifactorial. In our case, not only severe hyperparathyroidism and mediocalcinosis, but also medication (warfarin, calcium and active vitamin D) was involved. Because the initial conservative therapy was not successful, we indicated parathyroidectomy. However, we were not able to localize parathyroid glands and we contraindicated bilateral neck exploration due to the patients critical status. Therefore, we decided for total thyroidectomy with total parathyroidectomy. Surgery was uncomplicated and histology confirmed that all four parathyroid glands were removed. The expected post-operative hypocalcaemia was asymptomatic and we did not use any calcium supplementation or vitamin D. Thyroid hormone replacement was easy. After surgery, the large and multiple subcutaneous defects started to heal. We achieved complete healing within several months of continuing dedicated care. There is no recurrence after three years. Prompt and radical surgical parathyroidectomy was extremely useful in our patient.