Sodium transport in red blood cells from dialyzed uremic patients

Studies on red blood cell (RBC) sodium (Na) transport in chronic renal failure have described abnormalities in the ouabain-sensitive Na, K pump. We now report Na transport in RBC using cation flux methodology, measuring both the ouabain-sensitive Na, K pump and the ouabain-insensitive Na, K cotransport (CoT) and Na, lithium (Li) countertransport (CTT) in 28 subjects on hemodialysis, eight subjects on chronic ambulatory peritoneal dialysis (CAPD) and 29 control subjects. Intracellular cation content and passive permeability of Na were also examined. Mean Na efflux through the ouabain-sensitive Na, K pump was not reduced in dialysis patients when compared to normal subjects, whether measured in fresh cells (1.41 +/- 0.05 vs. 1.30 +/- 0.03 mmole/liter RBC/hr; P less than 0.05) or in Na-loaded cells (7.10 +/- 0.24 vs. 6.90 +/- 0.22; NS). There was, however, a marked and uniform suppression of the CoT pathway in Na-loaded cells from dialysis patients versus controls (0.14 +/- 0.02 vs. 0.41 +/- 0.05 mmole/liter RBC/hr; P less than 0.001). Mean CTT activity, as measured by Li efflux, was not different between dialysis and normal subjects. Uremic and normal RBC had similar intracellular Na or K content as well as passive permeability for either ion. This indicates that intracellular cationic homeostasis is maintained, perhaps secondary to balanced changes in cationic flux activity through these transport pathways.     

AQP1 in red blood cells of uremic patients during hemodialytic treatment

Hemodialysis influences the transport of water through the erythrocytic membrane, and induces morphologic and functional modifications. Recently water channels, called aquaporins (AQP), have been identified on the membrane of red blood cells. The aim of the present study was, therefore, to evaluate any relationships between volumetric changes in erythrocytes (MCV), plasma osmolarity and membrane expression of AQP1 in 22 uremic patients during a hemodialysis session, and compare value with those in a control group of 22 healthy volunteers. Membranal AQP1 expression was evaluated using three methods: indirect immunofluorescence under confocal microscopy, immunoenzymatic method after membrane extraction, and immunoblotting. In uremic subjects, at baseline membrane AQP1 expression was significantly lower, whereas plasma osmolality was higher than in controls. At 1 and 2 h of replacement therapy, a progressive increase was observed in erythrocytic AQP1, values similar to those in controls being attained after 3.5 h. During the session osmolality values reduced progressively, becoming significantly lower than basal values. The mean erythrocytic corpuscular volume in patients with ESRD was significantly lower than in cntrols at baseline. This value increased during hemodialysis, attaining statistical significance with respect to the basal value at 3.5 h of dialysis. Close correlations were found between plasma osmolality and AQP1 values (r = -0.930; p < 0.05), and also between MCV and plasma osmolality trend (r = -0.909; p < 0.05). There was a linear correlation (r = 0.63, p < 0.05) between plasma AVP concentrations and plasma osmolality. The variations found in plasma osmolarity during hemodialysis, may induce AQP1 expression on the membrane of intact red blood cells.     

Oral and parenteral essential amino acid therapy in malnourished hemodialysis patients

BACKGROUND/AIMS: Malnutrition has been encountered more frequently than expected and is associated with increased morbidity and mortality in hemodialysis (HD) patients. Until the last few years, only oral and enteral nutritional supplies have been used in the treatment of malnutrition in HD patients. However, intradialytic parenteral essential amino acid (EAA) nutrition has recently been introduced to treat these patients. The present study was conducted to compare both methods of EAA nutrition, oral and parenteral, in malnourished HD patients. METHODS: Half of the 20 malnourished HD patients in this study received 0.9 g/kg/week of oral EAA (oral group), while the other half of the patients were treated with the same dose of parenteral EAA (parenteral group) for 4 months. However, at the very beginning of the study, 4 patients from the oral group were transferred to the parenteral group because of complaints such as nausea and vomiting. Therefore, this study was completed with 6 patients in the oral group and 14 patients in the parenteral group. Some biochemical parameters, including blood lymphocyte counts and anthropometric measurements as indicators of the nutritional status, were obtained from both of the groups in the pre- and posttreatment periods. RESULTS: Following the treatment, there were no statistically significant differences between the groups with respect to anthropometric measurements. However, statistically significant increases were observed in serum albumin (p = 0.048) and creatinine (p = 0.006) levels and blood lymphocyte counts (p = 0.006) in the parenteral group, while there were statistically significant increases only in serum calcium (p = 0.028) levels and blood lymphocyte counts (p = 0.038) in the oral group following the treatment when compared to pretreatment values. CONCLUSION: These results show that parenteral EAA therapy is more comfortable and effective than oral EAA therapy in the treatment of malnourished HD patients.     

y+ cationic amino acid transport of arginine in packed red blood cells

BackgroundTransfusion of packed red blood cells (PRBCs) is associated with morbidity and mortality. The mechanisms are not fully understood. Packed red blood cells deplete extracellular arginine and possess transporters for arginine, an amino acid essential for normal immunity. We hypothesize that the membrane y+ amino acid transporter contributes to arginine depletion in PRBCs.Materials and methodsWe titrated PRBCs to a 10% hematocrit with phosphate-buffered saline, blocked PRBC y+ transporters using n-ethylmaleimide (0.2 mM), and measured arginine and ornithine levels using liquid chromatography–mass spectroscopy. We added radiolabeled L-arginine [4,5-³H] (10 μmol/L) added to similar culture conditions and measured arginine uptake in counts per minute (CPM). We examined storage periods of 6–9 d, 1–4 wk, and 6 wk, and correlated donor demographics with arginine uptake.Resultsn-Ethylmaleimide blockade of y+ transporters impaired PRBC arginine depletion from culture media (117.6 ± 8.6 μM versus 76.9 ± 5.8 μM; P < 0.001) and reduced intracellular L-arginine (7,574 ± 955 CPM versus 18,192 ± 1,376 CPM; P < 0.01). Arginine depletion increased with storage duration (1 wk versus 6 wk; P < 0.002). With n-ethylmaleimide treatment, 6-wk-old PRBCs preserved more culture arginine (P < 0.008) than at shorter durations. Nine-day storage duration increased L-arginine uptake compared with 6- to 8-day storage (n = 77, R = 0.225, P < 0.05). Extracellular arginine depletion and extracellular ornithine synthesis varied among donors and correlated inversely (R = ?0.5, P = 0.01).ConclusionsMembrane y+ transporters are responsible for arginine depletion by PRBCs. Membrane y+ activity increases with storage duration. Arginine uptake varies among donors. Membrane biology of RBCs may have a role in the negative clinical effects associated with PRBC transfusion.     

Plasma amino acids in patients with hepatic encephalopathy. Effects of amino acid infusions

Amino acid patterns in a group of patients with severe liver disease and hepatic encephalopathy were studied when patients were taking proteinrestricted diets per os and being infused with two solutions used for total parenteral nutrition. A somewhat constant amino acid pattern was found, with elevated levels of phenylalanine and methionine and decreased levels of the branched chain amino acids isoleucine, leucine, and valine.Patients with severe liver disease are apparently unable to utilize some essential amino acids, creating an amino acid imbalance. The significance of these findings and their possible relation to hepatic encephalopathy are discussed.     

Inhibited muscle amino acid uptake in sepsis.

Amino acid uptake in vivo was determined in soleus (SOL) muscle, diaphragm, heart, and liver following intravenous injection of [3H]-alpha-amino-isobutyric acid ([3H]-AIB) in rats made septic by cecal ligation and puncture (CLP) and in sham-operated controls. Muscle amino acid transport was also measured in vitro by determining uptake of [3H]-AIB in incubated extensor digitorum longus (EDL) and SOL muscles. Results were expressed as distribution ratio between [3H]-AIB in intracellular and extracellular fluid. AIB uptake in vivo was reduced by 90% in SOL and cardiac muscle and by 45% in diaphragm 16 hours after CLP. In contrast, AIB uptake by liver was almost four times higher in septic than in control animals. AIB uptake in vitro was reduced by 18% in EDL 8 hours after CLP but was not significantly altered in SOL at the same time point. Sixteen hours after CLP, AIB uptake was significantly reduced in both muscles, i.e., by 17% in EDL and by 65% in SOL. When muscles from untreated rats were incubated in the presence of plasma from septic animals (16 hours CLP) or from animals injected with endotoxin (2 mg/kg body weight), AIB uptake was reduced. Addition of endotoxin in vitro (2-200 micrograms/ml) to incubated muscles did not affect AIB uptake. The results suggest that sepsis leads to marked impairment of amino acid transport system A in muscle and that this impairment is mediated by a circulating factor that is not endotoxin. Reduced uptake of amino acids by skeletal muscle during sepsis may divert amino acids to the liver for increased gluconeogenesis and protein synthesis.     

增加精氨酸摄入量对烧伤患者血浆氨基酸谱的影响

目的观察摄入大剂量精氨酸对烧伤患者血浆氨基酸谱的影响。方法10例烧伤患者随机分为烧伤试验组和烧伤对照组(各5例),前者给予占总热量2%的盐酸精氨酸静脉注射液,后者给予占总热量2%的14复合氨基酸静脉注射液。两组患者其他营养成分的摄入量均相同。营养方案自伤后第3天起执行,给予总量的1/4;伤后4、5d给予总量的1/2;伤后6~21d给予全量。在两组患者伤后3d(方案实施前)及7、14、21、28d清晨取其静脉血标本,检测血浆氨基酸谱水平。以10名健康志愿献血者的血浆氨基酸谱水平作为正常参考值。结果伤后3d,两组患者血浆瓜氨酸水平显著低于正常参考值(P<0.05),而鸟氨酸和精氨酸水平与正常参考值比较差异无统计学意义(P>0.05)。烧伤对照组伤后3d后精氨酸、瓜氨酸和鸟氨酸水平呈现下降趋势。而烧伤试验组伤后14、21、28d血浆精氨酸水平分别为(280±121)、(223±106)、(110±44)μmol/L,与同期烧伤对照组(124±21)、(59±15)、(50±26)μmol/L相比明显偏高;伤后21d瓜氨酸和鸟氨酸水平分别为(30±5)、(162±44)μmol/L,明显高于同期烧伤对照组(8±7)、(66±4)μmol/L(P<0.05或0.01)。两组患者伤后血浆其他氨基酸水平差异均无统计学意义(P>0.05)。结论机体烧伤后血浆瓜氨酸转化为精氨酸的过程加速。增加外源性精氨酸摄入量可显著提高血浆精氨酸、鸟氨酸、瓜氨酸水平,但对其他氨基酸水平无明显影响,这一药理作用可能是通过加速鸟氨酸循环实现的。     

Elevation of serum levels of beta-aminoisobutyric acid in uremic patients and the toxicity of the amino acid

A reliable method for the determination of beta-aminoisobutyric acid in serum was developed utilizing an automated amino acid analyzer. The serum concentrations of beta-aminoisobutyric acid were determined in 20 normal subjects and in 71 uremic patients. The mean serum level of beta-aminoisobutyric acid was markedly increased in the uremic patients to 0.856 +/- 0.910 (mean +/- SD) mg/100 ml as compared with a normal value of 0.026 +/- 0.027 mg/100 ml. The distribution of serum beta-aminoisobutyric acid level in uremic patients was wide-spread, and there was no correlation between the serum levels of the amino acid and those of urea nitrogen, creatinine and uric acid. The toxicity of beta-aminoisobutyric acid on mice with acute renal failure induced by uranyl acetate was investigated and compared with that of alpha-amino-n-butyric acid and gamma-amino-n-butyric acid. All mice given more than 4 g/kg body wt of beta-aminoisobutyric acid showed twitching and cramps, and some of them died. However, the control mice given an equivalent dose of alpha-amino-n-butyric acid or gamma-amino-n-butyric acid showed no change. These results suggest that beta-aminoisobutyric acid might be a factor influencing the development and progression of uremic toxemia.     

Determination of autofluorescence of red blood cells (RbCs) in uremic patients as a marker of oxidative damage

AIM: It is suggested that the red blood cells (RBCs) of uremic patients have increased oxidative damage. The activities of different antioxidant enzymes and the levels of several antioxidants or lipid peroxidation products in RBCs are usually determined to estimate the oxidative stress in uremia. The autofluorescence of RBCs as measured by flow cytometry is caused by the formation of conjugated Schiff base compounds from aldehydes derived from lipid peroxidation and amino groups of phospholipids or cell proteins, and has been proposed as a marker of oxidative stress. The aim of this study was to evaluate if this method is suitable for estimation of oxidative stress in the RBCs of patients with different degrees of renal insufficiency. PATIENTS AND METHODS: To determine the oxidative damage in RBCs in uremia, the autofluorescence of RBCs was measured by flow cytometry in the following 3 groups of patients: group A: 16 patients with chronic renal failure (CRF); group B: 16 hemodialysis (HD) patients; group C: 16 patients with a well-functioning renal allograft. Twenty-four healthy volunteers served as controls. The basal value of RBC autofluorescence and the autofluorescence of RBCs after oxidative damage by treatment with 0.1 mM hydrogen peroxide (H2O2)/0.7 mM sodium azide were determined. RESULTS: In basal RBC autofluorescence values, no differences were found between the 3 groups and the controls. However, there was a significant correlation between the increase of serum creatinine and RBC autofluorescence in the group of patients with CRF (r = 0.521; p = 0.038). After H2O2 treatment, the RBC autofluorescence rose markedly in all individuals. This increase in RBC autofluorescence was significantly higher in the patients with CRF (p = 0.003) and in the HD patients (p = 0.001) compared to the controls. In contrast, there was no difference in RBC autofluorescence between the patients with renal allograft and the controls after H2O2 treatment. CONCLUSIONS: In conclusion, flow cytometry is a useful tool for determining oxidative damage in RBCs. The RBCs of uremic patients are more susceptible to oxidative damage induced by H2O2, likely caused by diminished antioxidant defense in the RBC membrane. Successful renal transplantation leads to a normal autofluorescence response in the RBCs after H2O2 treatment.     

Low red cell arachidonic acid in hemolytic uremic syndrome

A defect in prostacyclin generation has been proposed in hemolytic uremic syndrome (HUS): prostacyclin is formed from arachidonic acid derived from phospholipid and low levels of some phospholipids have been described in HUS cell membranes. Therefore fatty acid content of the red blood cell membranes of 8 children with HUS was compared with 32 children with other renal disease, with 33 patients with non-renal disease and with 8 normal adults. Children with HUS consistently had lower proportions of arachidonic acid (2.7-8.4%) than all other groups (10.1-18.8%) and the mean arachidonic acid level in HUS was very significantly reduced (p less than 0.0001). These findings suggest a reduced availability of arachidonic acid for prostaglandin synthesis in HUS and are consistent with the proposition that arachidonic acid is lost through peroxidative change.     

Triiodothyronine stimulates growth of peripheral blood mononuclear cells in serum-free cultures in uremic patients.

The effect of triiodothyronine (T3) on the responses to mitogens and on the production of prostaglandin E2 and interleukin 2 were studied in serum-free cultures of peripheral blood mononuclear cells (PBMC) in 20 patients undergoing hemodialysis and in 30 control subjects. T3 increased the growth of PMBC induced by phytohemagglutinin and pokeweed mitogen in both groups. PBMC reached growth maximum at 0.5 nM T3 when stimulated by phytohemagglutinin in both groups. At higher concentrations of T3 the effect declined in the control group, but the response of uremic PBMC was constant. The response to T3 of pokeweed mitogen stimulated PBMC was lower in the uremic patients. The production of prostaglandin E2 by PBMC was higher in the uremic patients than in the controls. T3 had no effect on prostaglandin E2 production. Indomethacin alone and in combination with T3 had a stimulatory effect on cell growth in the patient group. T3 had no effect on the release of interleukin 2 by PBMC. An additive effect of interleukin 2 and T3 was observed in cultures stimulated by suboptimal concentrations of the mitogens. In conclusion, the impaired growth of PBMC in serum-free cultures from uremic patients was enhanced, however, not normalized, by external addition of T3, inhibition of prostaglandin E2 synthesis, and addition of interleukin 2.     

Glucose uptake and amino acid metabolism in perfused hearts from tumor-bearing rats

The purpose of this study was to evaluate whether heart glucose metabolism can account for elevated heart oxygen consumption in a tumor-bearing host. This is the first report of altered metabolism in perfused hearts from tumor-bearing animals. Glucose, glycerol, lactate, and amino acid metabolism was examined under steady-state conditions in isolated perfused hearts from sarcoma-bearing rats and compared to the metabolism in hearts from starved (96 hr) and fed control rats. Heart dry weight was reduced by 10% in tumor-bearing rats and by 30% in starved rats when compared to freely fed control animals. Cardiac glucose uptake was decreased in tumor-bearing rats (206 +/- 33 mumoles/hr/g dry wt) compared to both starved (298 +/- 18) and fed control rats (293 +/- 25). Hearts from both fed and starved controls released lactate and glycerol at significant rates during perfusion which was not evident in hearts from tumor-bearing rats. The release of individual amino acids from working hearts during perfusion was different among the animal groups with a severe depression of both glutamine and alanine release in tumor-bearing rats. In starved rats alanine release was normal although glutamine release was depressed by more than 50%. The net release of all amino acids was lowest in hearts from tumor-bearing rats, intermediate in the starved animals, and highest in the control animals, while the nonmetabolized amino acids (phenylalanine, tyrosine, methionine) were released at increased rates only from tumor-host hearts, indicating an increased net breakdown of some cardiac proteins in tumor-bearing animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenic salvage quantified by uptake of heat-damaged radiolabeled red blood cells. Experimental and clinical studies

We evaluated a noninvasive radionuclide technique to quantify splenic trapping function, which is a key step in the disposition of blood-borne particulates such as poorly opsonized encapsulated microorganisms implicated in hyposplenic fulminant sepsis. Using computerized external gamma imaging, the percentage of splenic uptake of heat-damaged radiolabeled red blood cells was determined in adult Sprague-Dawley rats with eutopic (partial splenectomy) or ectopic (single or multiple autotransplantation) remnants or whole spleens, and in 14 patients with either an intact spleen or splenic remnants after treatment for trauma or hypersplenism. The masses of both eutopic and ectopic remnants correlated directly with the percentage of heat-damaged red blood cell uptake, but the percentage of uptake per gram was higher in eutopic remnants, paralleling more vigorous compensatory growth. In patients, the percentage of heat-damaged red blood cell uptake by remnant spleens was similar to that seen in the rats and, in addition, was supernormal in those with congestive splenomegaly. This noninvasive technique both provides a vivid biplanar image and quantifies blood-borne particle trapping, which is a key splenic function. A heat-damaged red blood cell uptake of less than 15 percent after splenic salvage suggests marginal splenic performance and continued vulnerability to overwhelming sepsis.     

Oral supplementation with gamma-linolenic acid extracted from Mucor circinelloides improves the deformability of red blood cells in hemodialysis patients

BACKGROUND: The development of abnormalities in red blood cell (RBC) deformability in patients undergoing hemodialysis remains a major problem, because it is related to peripheral microcirculation, oxygen supply, and various complications in such patients. gamma-Linolenic acid (GLA; 18:3n-6), one of the polyunsaturated fatty acids and a precursor of prostaglandin E(1), is reported to have a favorable effect on the deformability of circulating blood cells in diabetic patients. METHODS: In order to clarify the efficacy of GLA on RBC deformability in 7 patients undergoing maintenance hemodialysis, we examined in a pilot study the changes in the deformability of RBC and the changes in the phospholipid fatty acid composition in both plasma and RBC membrane before and after high-dose oral supplementation with GLA derived from Mucor circinelloides for 12 weeks. RESULTS: Before supplementation, the micropore passage time of RBC suspension, which is an indicator of RBC deformability, in these patients was markedly longer than that in healthy control subjects. After administering GLA, the prolonged passage time of the patients both rapidly and steadily decreased and nearly reached control levels. Light microscopic observations of RBCs using Giemsa stain revealed a decreased number of poikilocytes after supplementation. An analysis of the fatty acid composition before treatment and 8 weeks after starting the treatment showed the dihomo-gamma-linolenic acid (DGLA; 20:3n-6) level in the plasma to have increased (p < 0.05), while the arachidonic acid (AA; 20:4n-6) concentration in the RBC membrane decreased (p < 0.05). The level of DGLA in the RBC membrane, the level of GLA, and the ratio of GLA + DGLA/AA in plasma and RBC membrane did not change significantly; however, these all tended to increase. CONCLUSION: The results of this pilot study indicate that the oral supplementation of GLA extracted from M. circinelloides improves the poor RBC deformability in hemodialysis patients, partly by inducing changes in the composition of fatty acids in plasma and RBC membrane.     

Plasma free, sulfo- and glucuro-conjugated catecholamines in uremic patients

The metabolism of catecholamines (CA) in non-selected patients with chronic renal failure and under hemodialysis (CRFh) was studied by measuring the concentration of plasma free, sulfo- and glucuroconjugated CA, dopamine (DA), norepinephrine (NE), and epinephrine (EPI). Our data demonstrate a statistically significant increase of free DA and free NE concentration in CRFh, while that of free EPI was not different from controls. However a careful scrutiny of 35 individual data suggests that sub-groups of patients with either high normal or low plasma-free NE concentration could exist; this likely heterogeneity could be a good explanation for conflicting conclusions provided by previous reports. Suspecting that conjugated CA might be altered in CRFh, plasma sulfo- and glucuro-conjugated DA, NE and EPI were also measured. We have found a predictable and highly significant increase of sulfo-conjugated CA; plasma concentration of glucuro-conjugated DA and NE in CRFh was not different from controls, while that of glucuro-conjugated EPI was significantly increased. The physiological meaning, if any, of these new observations on conjugated CA cannot be assessed at the moment. The effects of hemodialysis were also investigated. Measurements on the arterial and on the venous line showed highly significant differences for tyrosine, free and sulfo-conjugated CA, and a lack of difference for glucuro-conjugated CA. Thus tyrosine, free and sulfo-conjugated CA were eliminated by the artificial kidney, but not glucuro-conjugated amines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Branched chain amino acid uptake and muscle free amino acid concentrations predict postoperative muscle nitrogen balance.

Amino acid solutions rich in branched chain amino acids (BCAA) are commonly utilized both clinically and in experimental protocols in an attempt to reduce skeletal muscle and whole body protein catabolism. To investigate the effectiveness of BCAA infusion, amino acid formulas containing varying concentrations of BCAA were given during operation in this study to three groups of dogs undergoing a standard laparotomy and retroperitoneal dissection. A fourth group was given saline alone. With the use of previously described hindquarter flux techniques, individual and total amino acid nitrogen exchange rates were measured and utilized in estimating skeletal muscle protein catabolism. Intracellular free amino acid concentrations were measured in percutaneous muscle biopsy samples. Although there was no relationship with the rate of BCAA infusion, there was a significant correlation between the rate of BCAA uptake by muscle and diminished total nitrogen release from hindquarter skeletal muscle after operation. There was also a significant relationship between muscle nitrogen balance and the postoperative change in the muscle concentration of either total amino acids or the single amino acid glutamine. When combined in a single equation, BCAA uptake and the change in muscle free amino acid concentration predict skeletal muscle nitrogen release with an r = 0.86. Thus, the rate of BCAA uptake and the free glutamine or total amino acid concentration in muscle appear to be independent predictors of muscle nitrogen balance. The nitrogen-sparing effect of BCAA in skeletal muscle is unrelated to infusion concentration or rate of infusion.