The amino acid transporter asc-1 is not involved in cystinuria

BACKGROUND: The human amino acid transporter asc-1 (SLC7A10) exhibits substrate selectivity for small neutral amino acids, including cysteine, is expressed in kidney, is located close to the cystinuria B gene and presents sequence variants (e.g., E112D) in some cystinuria patients. We have cloned human asc-1, assessed its transport characteristics, localized its expression in kidney, searched for mutations in cystinuria patients, and tested the transport function of variant E112D. METHODS: We used an EST-based homology cloning strategy. Transport characteristics of asc-1 were assessed by coexpression with 4F2hc in Xenopus oocytes and HeLa cells. Localization of asc-1 mRNA in kidney was assessed by in situ hybridization. Exons and intron-exon boundaries were polymerase chain reaction (PCR)-amplified from blood cell DNA and mutational screening was performed by single-stranded conformational polymorphism (SSCP). RESULTS: Asc-1 reaches the plasma membrane in HeLa cells, unlike in oocytes, most probably by interaction with endogenous 4F2hc and presents similar transport characteristics to those in oocytes coexpressing asc-1/4F2hc. Asc-1 mediates a substantial efflux of alanine in a facilitated diffusion mode of transport. Expression of asc-1 mRNA localized to Henle's loop, distal tubules, and collecting ducts. Finally, SLC7A10 polymorphisms were identified in cystinuria probands and the SLC7A10 sequence variant E112D showed full transport activity. CONCLUSION: The lack of expression of asc-1 in the proximal tubule indicates that it plays no role in the bulk of renal reabsorption of amino acids. No mutations causing cystinuria have been found in SLC7A10. The facilitated diffusion mode of transport and the expression in distal nephron suggest a role for asc-1 in osmotic adaptation.     

Renal polyamine excretion, tubular amino acid reabsorption and molecular genetics in cystinuria

Cystinuria is an autosomal recessive disorder of the tubular and intestinal resorption of cystine, ornithine, lysine and arginine leading to nephrolithiasis. Three cystinuria types can be distinguished by the mode of inheritance (true recessive or intermediate) and by the pattern of the intestinal amino acid transport. In the present study phenotypes were assessed by the urinary excretion of amino acids related to creatinine, the percentage tubular amino acid reabsorption and the urinary excretion of polyamines as a possible indicator of the intestinal transport defect. However, our thorough phenotyping did not reveal more than two cystinuria types. Genotypes were examined in linkage analyses and single-strand conformation polymorphism-based mutation identification. The SLC3A1 mutations M467T and T216M were disease causing in our homozygous patients of type I cystinuria. We can show the association of type I cystinuria with SLC3A1 and of non-type I cystinuria with a yet unidentified gene on chromosome 19q13.1. Our phenotype and genotype analyses provide evidence for only two types of cystinuria in the investigated patient cohort. Received: 18 February 1999 / Revised: 16 August 1999 / Accepted: 17 August 1999     

Long-term study of tiopronin in patients with cystinuria

Thirty two patients with cystinuria were enrolled in this long-term study and 16 patients were treated with tiopronin for 24 weeks. Tiopronin reduced daily urinary cystine excretion from 901.48 mg (before treatment) to 488.60 mg (on the average of 12th week and 24th week after tiopronin administration) successfully. Tiopronin therapy was tolerated well, but side effects were observed in 13 events in 6 patients. Thus tiopronin was expected to be effective in preventing cystine stone formation and tolerated well.     

Metabolic effects of amino acid solutions in severely burned patients: with emphasis on sulfur amino acid metabolism and protein breakdown

The metabolic effects of TPN containing high amounts of amino acids (18 gm N) was evaluated with respect to sulfur amino acid metabolism and protein breakdown in nine severely burned patients. The results were compared to corresponding data from burned patients receiving more moderate amounts of amino acids (9.2 gm N) or isocaloric amounts of carbohydrate and fat. Significantly increased urinary excretion and intracellular muscle tissue concentrations of methionine were found in patients receiving the concentrated amino acid solution, probably reflecting a combined effect of the injury and an increased load of the compound. These patients also showed a reduced oxidation of sulfur amino acids to inorganic sulfate, which may reflect an increased protein synthesis during treatment. A significantly decreased urinary excretion of 3-methyl-histidine and mercaptolactate was found in patients receiving the concentrated amino acid solution, probably reflecting a decreased breakdown of body protein. The findings favor the hypothesis of an anabolic effect of the new amino acid solution in burned patients during the early catabolic phase, but also emphasize the importance of monitoring the amounts of amino acids, e.g., methionine, given.     

Splanchnic amino acid and glucose metabolism during amino acid infusion in dogs

With the organ-balance technique, we studied amino acid and glucose metabolism by hepatic and extrahepatic splanchnic tissues in awake dogs in the postabsorptive state and during a 3-h intravenous amino acid infusion. Dogs received a high (1.4 g/kg body wt, n = 5) or low (0.7 g/kg body wt, n = 8) dose of amino acids. In four of the latter dogs, the dose was delivered into a mesenteric vein. During the basal period there was a net removal of gluconeogenic amino acids (particularly alanine), but not branched-chain amino acids, and a net production of glucose by the liver in all dogs. During this time there was a net removal of glucose and production of alanine by the extrahepatic splanchnic tissues. During either high- or low-dose amino acid infusion, net hepatic glucose release increased; despite this, arterial plasma glucose declined due to an increase in tissue glucose uptake at extrasplanchnic sites. The net amount of glucogenic amino acids removed by the liver during high-dose (9.1 +/- 1.0 mmol.kg-1.3 h-1) and low-dose (4.8 +/- 0.6 mmol.kg-1.3 h-1) infusion equaled or exceeded the infused load of these amino acids. In addition, the liver contributed to the net disposal of branched-chain amino acids during high-dose (536 +/- 147 mumol.kg-1.3 h-1) and low-dose (341 +/- 70 mumol.kg-1.3 h-1) infusion. During high-dose infusion, extrahepatic splanchnic tissues participated in the net removal of branched-chain amino acids (436 +/- 162 mumol.kg-1.3 h-1) but not glucogenic amino acids, and net alanine production continued (410 +/- 91 mumol.kg-1.3 h-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Whole-body nitrogen and tyrosine metabolism in surgical patients receiving branched-chain amino acid solutions

Fifteen patients undergoing gastric bypass surgery for morbid obesity received preoperatively a standard crystalline amino acid solution containing 15.6% branched-chain amino acids. During the first five postoperative days, the patients were randomized to receive one of three amino acid solutions (9.0 to 10.3 g of nitrogen per day) of different branched-chain amino acid content. The branched-chain amino acid concentrations of the mixtures were 15.6%, 50%, and 100% by weight with five patients in each group. Whole-body amino acid appearance and oxidation were estimated using a continuous intravenous infusion of L-(U-14C)-tyrosine preoperatively and on the third postoperative day. Daily postoperative nitrogen balance, fractional albumin renewal rate, and whole-body tyrosine appearance, oxidation, and incorporation into protein were not significantly different among the three groups. This study suggests that an adequate nitrogen intake of a balanced amino acid mixture, as well as a solution enriched with branched-chain amino acids, maintains protein homeostasis and supports protein synthesis similarly in well-nourished patients following major abdominal surgery. A diet containing only branched-chain amino acids in isomolar ratios was as effective at maintaining protein retention and whole-body protein synthesis and albumin renewal postoperatively when compared with a standard amino acid formula.     

Short-term effects of glucocorticoid therapy on biochemical markers of bone metabolism in Japanese patients: a prospective study

Glucocorticoid (GC) therapy induces rapid bone loss, but the early changes in calcium and bone metabolism in patients treated with GC have not been clarified. To investigate the changes in calcium and bone metabolism during the early stage of GC therapy, we analyzed various biochemical markers of bone metabolism. The serum levels of calcium (Ca), phosphorus, parathyroid hormone (PTH), osteocalcin (OC), bone alkaline phosphatase (BAP), and type I collagen cross-linked N-telopeptide (NTx), as well as the urinary levels of Ca, creatinine, and NTx, were measured on days 0, 3, 7, and 28 of GC therapy. The subjects were divided into the following four groups: 9 patients receiving pulse therapy (P), 18 patients receiving prednisolone (PSL) at doses ≥40 mg/day (H), 9 patients receiving PSL at doses ≥20 mg/day (M), and 11 patients receiving PSL at doses ≤10 mg/day (S). The serum OC level showed a marked decrease on day 3 of GC therapy (−41.2% ± 6.6%, P < 0.01), while the BAP level decreased gradually. Both serum and urinary NTx levels significantly increased on day 7 of GC therapy (9.9% ± 4.5%, P < 0.05, and 42.2% ± 10.6%, P < 0.01, respectively). Urinary Ca excretion was increased on day 3 of GC therapy and continued to increase until 4 weeks, while intact PTH showed an increase on day 3 and then remained constant until 4 weeks. In groups P and H, there were significant early changes in OC, BAP, NTx, and intact PTH levels, as well as urinary Ca excretion. Even a PSL dose of <10 mg/day caused a decrease in the serum OC level. In conclusion, the biochemical markers of Ca and bone metabolism showed different kinetics depending on the dose of GC, and it is important for patients on high-dose GC therapy to receive prophylaxis for bone loss from the start of GC treatment.     

Fat metabolism during total parenteral nutrition: a biochemical and ultra-structural study

Background : Before the introduction of Intralipid in 1961, the incidence of adverse reactions to intravenous fat emulsions was quite common. Concurrent with hyperlipidaemia, multiple organ dysfunction could occur, but the most prominent symptoms were high fever and anaemia, and the reaction was called “the fat overloading syndrome”. Although the reports are quite rare today the reaction does still appear, and we have observed that some patients develop severe fever during or after incidental retention of exogenous fat in the blood during total parenteral nutrition (TPN). The pathophysiological background to these adverse reactions, as well as the final destiny and the effects of the retained exogenous fat particles, are largely unknown. These questions initiated the present study, which focuses on two factors of importance for the clearance of infused fat from the circulation: the lipoprotein lipase (LPL) system and the reticluloendo-thelial system. Methods : An introductory analysis of the clinical course of a head-injured patient suffering from fat intolerance during TPN implied that observed adverse reactions could be due to influence on macrophages by retained exogenous fat particles. Results : During infusion of lipid emulsions, cholesterol and phospholipids accumulated in non-high-density lipoproteins, while high-density lipoproteins were enriched in triglycerides. Triglycerides also accumulated in hepatic tissue, and the activity of hepatic lipase decreased. Although lipoprotein lipase activity in the heart was up-regulated and serum levels of triglycerides were normal, suggesting that the total elimination rate of triglycerides was not impaired, ultra-structural investigations indicated that Kupffer cells were involved in clearing exogenous lipids from the blood during the infusion of long-chain triglycerides. Furthermore, the Kupffer cells were activated, according to morphological criteria. When high doses of fat were given intravenously, the Kupffer cells were grossly distended by fat vacuoles, and serum levels of lactate dehydrogenase increased. During treatment with fat emulsions containing medium-chain triglycerides and long-chain triglycerides in equal amounts, the alterations in lipid transport were less pronounced, and signs of Kupffer cell activation were weaker than during infusion with long-chain triglycerides only. Conclusion : The experimental studies showed that infusion of exogenous fat particles interfered with the regulation of lipid transport, and there were signs of increased demands on various aspects of lipid metabolism. Although the capacity of the LPL was sufficient in most situations in healthy rats, the Kupffer cells seemed to be involved in the clearance of exogenous lipids. The present investigations identified one factor, LCT fat emulsion, which was necessary for the appearance of fat vacuoles in the Kupffer cells during TPN. To some extent, the experimental findings may be related to clinical observations of a head-injured patient who developed signs of acute, severe macrophage activation with haemophago-cytosis during and after accumulation of exogenous fat in the blood after treatment with intravenous fat emulsions. The condition was considered to be a consequence of impaired lipid elimination and altered macrophage function during clearance of fat from the blood, a consideration supported by the morphological changes of rat Kupffer cells after TPN in the present study.     

Experimental study on glucose turnover and amino acid metabolism after total pancreatectomy

Glucose turnover and plasma amino acid profile were measured in total pancreatectomized dogs to clarify the mechanism of hyperaminoacidemia after total pancreatectomy. Twenty-one male mongrel dogs underwent total gastrectomy and pancreatectomy and were divided into two groups. I(-) group (n = 11) was treated without insulin and I(+) group (n = 10) received insulin postoperatively. Another 15 dogs of S group received only splenectomy. Glucose appearance rate (Ra), plasma amino acid profile and blood sugar level (BS) were measured before operation, and on the 1st and 3rd postoperative days. Significantly higher Ra and BS were observed in I(-) group than in S group postoperatively and either decreased or unaltered by the glucagon infusion. On the other hand, Ra and BS in I(+) group were maintained at the same levels as S group and increased significantly by the glucagon infusion. Hyperaminoacidemia was observed after total pancreatectomy regardless of insulin treatment. Branched chain amino acids (BCAA) were significantly elevated in I(-) group but not in I(+) group. Glucagon infusion decreased almost all amino acids levels except BCAA in both groups. These results suggest that mechanism of hyperaminoacidemia observed in totally pancreatectomized dogs is the reduced amino acids uptake by the liver related to glucagon deficiency.     

Basic principles and clinical applications of biochemical markers of bone metabolism: biochemical and technical aspects.

The interest in and the need for effective measures to be used in the screening, diagnosis, and follow-up of disorders of connective tissue, bone, and mineral metabolism has markedly grown. Next to clinical and imaging techniques, indices of bone turnover have come to play an important role in the assessment of metabolic bone disease. In osteoporosis, recent research has shown that bone markers may also be used to predict future bone loss and hip fractures (in larger cohorts of older patients), identify individuals at risk for osteoporosis, select therapy, and predict and monitor the therapeutic response in individual patients. The development of new markers of bone metabolism has greatly enriched the spectrum of serum and urine analytes used in the assessment of skeletal pathologies. Besides total alkaline phosphatase, other markers such as bone-specific alkaline phosphatase, osteocalcin, or the collagen propeptides are being used to measure bone formation. Bone resorption, previously assessed only by the measurement of urinary calcium and hydroxyproline, may now be detected more precisely by a number of new serum and urine markers. Among these, the pyridinium crosslinks and the telopeptides of collagen type I are presently considered the most specific markers of bone resorption. More recently, bone sialoprotein has also been suggested as a marker of bone resorption in serum. Tartrate-resistant acid phosphatase is now measurable by immunoassay. This article surveys the biochemistry and relevant technical aspects of the currently available markers of bone metabolism.     

An experimental and clinical study of energy-protein metabolism and host defense-repair mechanism in postoperative period--a significance of administration of branched chain amino acid

The aim of this study is to evaluate in vivo the effect of branched chain amino acid (BCAA). Experimentally, hepatic energy production and protein synthetic rate were measured in gastrectomized rat which was infused BCAA postoperatively. Clinically, following indices were examined in prospectively randomized patients who underwent abdominal operation and were administered with conventional total parenteral nutrition keeping Calorie/N ratio about 150, including nitrogen balance, urinary 3-methylhistidine, retinol binding protein, B lymphocyte percentage and lymphocyte blastogenesis by phytohemagglutinin. Furthermore, plasma BCAA with their keto-analog level, Factor XIII and opsonic activity were determined in another group of patients who received full strength load of BCAA immediately after subtotal or total gastrectomy, in a controlled prospective randomized double-blinded manner. Results obtained from above mentioned measurements exhibited significant improvement by the administration of BCAA. From these findings, it is suggested that BCAA sustains energy-protein metabolism, supports immunocompetence and promotes wound healing under moderately stressed condition where catabolic response is physiologically compensated.     

The effect of fuel source on amino acid metabolism in critically ill patients.

The ideal energy substrate for critically ill patients receiving total parenteral nutrition (TPN) remains controversial. While glucose has been proved to have nitrogen sparing properties in postoperative patients, critically ill patients tolerate glucose loads poorly and fat appears to be an obligatory fuel in sepsis. Furthermore, it is not yet certain whether the changes in whole body protein metabolism induced by critical illness are influenced by the nature of the TPN provided. This study was conducted on patients admitted to a surgical intensive care unit (SICU) who fulfilled the criteria of requiring TPN and mechanical ventilation for at least four days. Patients were randomized to receive either glucose (G) or equicaloric proportions of glucose and lipid (GF) as an intravenous energy source. TPN was commenced early, within 24-48 hr of trauma or surgery and admission to the ICU. Nonprotein calorie intake was 125% of calculated basal energy expenditure. Nitrogen balance was calculated from 24-hr urinary urea excretion. Protein synthesis, turnover, and catabolism were measured on Day 4 of the study using an established radiolabeled C14-leucine technique. Degree of sepsis and illness were calculated using published scores. Fifty patients entered the trial but 32 were excluded by Day 4. Of the 18 patients completing an initial four day study, eight went on to complete a second study on the alternative regimen--a total of 26 studies (14 G, 12 GF). Net protein synthesis was achieved in 18 studies (12 G, 6 FG) and positive nitrogen balance by Day 4 in 22 studies. Four patients on the G regimen were withdrawn due to glucose intolerance while none of the patients on GF developed glucose intolerance or hyperlipidaemia. Both whole body protein synthesis and catabolism correlated significantly with degree of sepsis. The type of TPN fuel used, G and GF, did not appear to influence whole body protein dynamics, both regimens achieving greatly improved whole body protein kinetics.     

Influence of N-acetylcysteine on hepatic amino acid metabolism in patients undergoing orthotopic liver transplantation

Experimental treatment with the antioxidant and glutathione precursor N-acetylcysteine (NAC) has been performed in orthotopic liver transplantation (OLT) to reduce reperfusion injury. To investigate the effect of NAC on the hepatic and intestinal amino acid metabolism, intraoperative amino acid exchange rates were studied in liver transplant recipients with high dose NAC treatment (n = 10) and in control patients (n = 9). Treatment with NAC was found to cause a loss of amino acids and increased urea nitrogen release from the liver graft. The net balance of most amino acids was shifted to increased hepatic release or decreased hepatic uptake. The initial cumulative splanchnic release of all proteinogenic amino acids in the NAC treated group was significantly higher than in the control group. These findings are tentatively explained by an increased net protein catabolism in the liver. The increased hepatic urea and glutamine production rate of the NAC treated patients is expected to increase the energy and oxygen demand of the liver in this critical situation. Thus, NAC may have caused marked metabolic disturbances in the freshly implanted graft. The dosage of NAC should therefore be modified to avoid these disadvantages. Received: 19 April 2000 Revised: 27 February 2001 Accepted: 8 June 2001     

Comparison of amino acid oxidation and urea metabolism in haemodialysis patients during fasting and meal intake.

BACKGROUND: The PNA (protein equivalent of nitrogen appearance) is used to calculate protein intake from urea kinetics. One of the essential assumptions in the calculation of PNA is that urea accumulation in haemodialysis (HD) patients is equivalent to amino acid oxidation. However, urea is hydrolysed in the intestine and the resulting ammonia could be used metabolically. The magnitude and dependence on protein intake of this process are unknown in HD patients. METHODS: Seven HD patients were studied twice, 1 week apart, on a similar protocol. After an overnight fast, patients fasted in the morning and received meals in the afternoon. On one day, amino acid oxidation was measured by infusion of L-[1-(13)C]valine. Urea production, measured from the dilution of [(13)C]urea, and urea accumulation, calculated from the increase in plasma urea concentration multiplied by the urea dilution volume, were measured during the other day. PNA was calculated using standard equations. RESULTS: Amino acid oxidation and urea production were not significantly different during fasting. Urea accumulation during fasting was significantly lower than both amino acid oxidation and urea production. Urea accumulation during feeding remained significantly lower than amino acid oxidation. PNA was equal to the average of the urea accumulation values during fasting and feeding. CONCLUSION: We conclude that during fasting, urea accumulation is not associated with amino acid oxidation or urea production. During meal intake, amino acid oxidation, urea production and urea accumulation show acutely an almost identical increase. PNA represents the average of fasting and fed urea accumulation and is lower than average amino acid oxidation or urea production.     

Effects of an amino acid dialysate on leucine metabolism in continuous ambulatory peritoneal dialysis patients

BACKGROUND: Protein-energy malnutrition is frequent in continuous ambulatory peritoneal dialysis (CAPD) patients. The use of amino acids in the dialysate could improve the protein balance, especially if associated to a concomitant energy intake. METHODS: A 1.1% amino acid solution for peritoneal dialysis was administered to CAPD patients over 30 minutes during concomitant absorption of 600 ml water (control study) or of a 600 kcal meal/600 ml. Leucine metabolism was studied using the combination of intravenous [2H3] and intraperitoneal [13C] leucine. RESULTS: The rate of leucine appearance was stimulated by 56 and 53% (control and meal) at 45 minutes. The rates of leucine appearance and disappearance were lower from 180 to 300 minutes during the meal versus control study (P < 0.05). Proteolysis was unaffected during the control study and was inhibited by 25% during the meal study (P < 0.05). During the five-hour cycle dialysis with or without a meal, 80% of the leucine administered into the peritoneum was absorbed. Forty-one percent was retained in the splanchnic bed. Forty-three percent was used for protein synthesis, and 16% was oxidized. CONCLUSIONS: This amino acids solution is efficaciously utilized for protein synthesis in CAPD patients with no effect on protein breakdown. The concomitant ingestion of a carbohydrate-lipid meal inhibits protein breakdown and reinforces a positive effect of the amino acids solution on protein balance.