Metabolism of 3H- and 14C-labeled glutamate, proline, and alanine in normal and adrenalectomized rats using different sites of tracer administration and sampling

Alanine, glutamate and proline labeled with 14C and 3H were infused into fasted normal and adrenalectomized rats. Alanine was administered by the A-V mode (arterial administration-venous sampling), and glutamate and proline by both the A-V and V-A (venous administration-arterial sampling) modes. The kinetics of 14C alanine and 14C glutamate differed markedly from those of the tritium-labeled compounds, but there was little difference in the kinetics of 3H and 14C proline. The replacement rate calculated from the A-V mode for glutamate was about half that obtained in the V-A mode, but there was little difference with proline. The masses of the amino acids (total content of amino acids in the body) were calculated from the washout curves of the tritium-labeled compounds after the infusion of tracer was terminated. The masses for the normal rats were 407 mumol/kg for alanine, 578 mumol/kg for glutamate and 296 mumol/kg for proline. The so-called distribution spaces calculated conventionally from total masses and the amino acid concentrations in plasma are much greater than the volume of the body, reflecting the fact that amino acid concentrations in tissues greatly exceed those in plasma. Adrenalectomy markedly affected the kinetics of the three amino acids, and their replacement rates were greatly reduced. The proline and glutamate masses were reduced by at least one half, while that of alanine was unchanged. Adrenalectomy markedly reduced the conversion of proline to glutamate. The hydrocortisone regimen used in this study restored the metabolism of alanine and glutamate to normal, but had no effect on that of proline.     

Amino acid and glucose metabolism in the postabsorptive state and following amino acid ingestion in the dog

Amino acid and glucose metabolism was studied in nine awake 18-hour fasted dogs with chronic portal, arterial, and hepatic venous catheters before and for three hours after oral ingestion of amino acids. The meal was composed of a crystalline mixture of free amino acid, containing neither carbohydrate nor lipid. Following the amino acid meal, plasma glucose concentration declined slowly and this occurred despite a rise in hepatic glucose release. Portal plasma insulin rose transiently (30 +/- 7 to 50 +/- 11 microU/mL, P less than 0.05) while the increase in portal glucagon was more striking and persisted throughout the study (162 +/- 40 to 412 +/- 166 pg/mL). Over the three hours following amino acid ingestion, the entire ingested load of glycine, serine, phenylalanine, proline, and threonine was recovered in portal blood as was 80% of the ingested branched chain amino acids (BCAA). The subsequent uptake of these glucogenic amino acids by the liver was equivalent to the amount ingested, while hepatic removal of BCAA could account for disposal of 44% of the BCAA absorbed; the remainder was released by the splanchnic bed. During this time, ongoing gut production of alanine was observed and the liver removed 1,740 +/- 170 mumol/kg of alanine, which was twofold greater than combined gut output of absorbed and synthesized alanine. In the postcibal state, the total net flux of alanine and five other glucogenic amino acids from peripheral to splanchnic tissues (1,480 mumol/kg 3 h) exceeded the net movement of branched chain amino acids from splanchnic to peripheral tissues (590 mumol/kg/3 h).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum levels of short-chain fatty acids in cirrhosis and hepatic coma

Short-chain fatty acids cause reversible coma in animals and may contribute to the pathogenesis of the hepatic coma in humans. The concentrations of short-chain fatty acids in peripheral venous blood were significantly elevated in 15 patients with hepatic encephalopathy caused by cirrhosis (362 +/- 83 mumol/L; mean +/- S.E.M.) compared with 17 cirrhotic patients without encephalopathy (178 +/- 57 mumol/L) and 11 normal individuals (60 +/- 8 mumol/L). However, no correlation between the depth of coma and the level of short-chain fatty acids was found after repetitive measurements in the coma group. Compared with normal individuals, all short-chain fatty acids, except valerate, were elevated in patients with hepatic encephalopathy, whereas only the concentrations of isobutyrate and isovalerate were significantly elevated in cirrhotic patients without encephalopathy. The concentrations of short-chain fatty acids in 21 nonencephalopathic cirrhotic patients who underwent catheterization were equally distributed in the aorta (187 +/- 56 mumol/L), the hepatic vein (212 +/- 75 mumol/L), the azygos vein (140 +/- 37 mumol/L) and the renal vein (135 +/- 43 mumol/L) compared with peripheral venous blood (178 +/- 57 mumol/L). This study does not support the idea that short-chain fatty acids are of major importance in the pathogenesis of hepatic coma in patients with cirrhosis.     

Amino acids and keto acids in the treatment of chronic renal failure

Clinical trials using very low protein supply supplemented with amino acid or keto acid preparations have shown the possibility of slowing the rate of decline of renal function in patients with advanced chronic renal failure together with preservation of nutritional status. The possible mechanisms by which this effect is obtained are reviewed. The potential advantages of keto acids upon amino acids, the optimal composition of the preparations used, and the best time to start treatment in chronic uremics are discussed. Projection of recent data indicate that a prolongation of renal autonomy of about 4 years instead of a spontaneous duration of 15 months until dialysis can be expected using keto acid treatment if started when plasma creatinine reaches 500 mumol/l.     

Some aspects of homocysteine metabolism in hemodialysis patients

Homocysteine (Hcy) is a non-protein forming sulfur amino acid, synthesised from methionine (Met), whose metabolism is at the junction of two metabolic pathways: remethylation and transsulfuration. Increased Hcy serum concentration is a well established independent risk factor of cardiovascular diseases and a known feature of end stage renal disease. Hcy plasma level is influenced by folate, vitamin B6 and genetic factors. Mutation C677T in gene encoding methylenetetrahydrofolate reductase (MTHFR), an enzyme involved in Hcy remethylation has been associated with elevated Hcy in homozygous carriers (TT genotype). Several amino acids take part in metabolism of Hcy. There are abnormalities of concentration of the non essential and essential of amino acids in serum of patients treated with hemodialysis (HD). It is possible that these abnormalities of amino acids can change the Hcy metabolism. The aim of this study was the evaluation of some aspects of Hcy metabolism. We examined the MTHFR gene polymorphism and its relationship with plasma Hcy concentration. The plasma levels of total amino acids and amino acids connected with Hcy metabolism: methionine (Met), seryne (Ser), cysteine (Cyst) and tauryne (Tau) were evaluated in hemodialysis patients. The study was conducted in 71 (35 male, 36 female) patients, mean age 56.2 +/- 12.4 years. They were dialysed for a mean duration of 87.7 +/- 84.7 months (range 2-302). The control group (CG) in which Hcy and amino acids levels were examined consisted of 12 healthy subjects. Serum (EDTA) Hcy levels were measured by EIA-Hcy ELISA kit. The MTHFR gene polymorphism was evaluated by means of the polymerase chain reaction (PCR). The amino acids were measured by chromatography in amino acid analyser AAA 400. Mean concentration of Hcy was significantly higher in patients than in CG (31.1 +/- 9.1 vs 11.9 +/- 2.9 mumol/L; p < 0.01). Genotype frequencies in patients were: 42.8% for CC, 48.5% for CT and 8.7% for TT. Mean concentration of Hcy were similar in above genotype groups: 31.2 +/- 9.4; 30.7 +/- 10.7; 32.8 +/- 5.1 mumol/L, respectively. We did not find any correlation between Hcy level and the mutation in gene coding for MTHFR in our study group of patients. Mean total amino acid concentrations were significantly lower in plasma patients than in CG: 3624.48 +/- 140.32 vs 4454.45 +/- 774.91 mumol/L; p < 0.05. Mean plasma level of Tau was significantly lower in patients than in CG: 93.01 +/- 43.73 vs 286.75 +/- 57.02 mumol/l; p < 0.01. Also mean plasma level of Ser was significantly lower in patients than in CG; 125.71 +/- 24.25 vs 233.61 +/- 44.55 mumol/L; p < 0.01. Mean concentration of Cys were significantly higher in hemodialysis patients than in CG: 100.82 +/- 43.53 vs 31.31 +/- 21.31 mumol/L; p < 0.01. Mean Met concentrations were not significantly different between two studied groups. We found significant positive correlation between plasma Hcy levels and plasma Cys level (r = 0491; p < 0.05). Also there was a significant positive correlation between plasma Hcy level and duration of hemodialysis (r = 5411; p < 0.05). We concluded that in our studied population of hemodialysis patients there was no significant association between mutation in the gene coding for MTHFR and hyperhomocysteinemia and hypercysteinemia. There are abnormalities of plasma level of amino acids which are take part in Hcy metabolism in hemodialysis patients.     

Splanchnic metabolism of amino acids in healthy subjects: effect of 60 hours of fasting

Brief starvation is accompanied by decreased circulating levels of most amino acids, which has been attributed to an increased splanchnic uptake of amino acids, primarily alanine, for gluconeogenesis. However, quantitative data on splanchnic exchange of amino acids and gluconeogenic precursors is lacking. Consequently, arterial concentrations and splanchnic exchange of whole blood amino acids, ketone bodies, glucose, and gluconeogenic precursors were measured in 16 prolonged fasted (60 to 64 hours) and 15 overnight fasted (12 to 14 hours) healthy, nonobese subjects. After the 60-hour fast net splanchnic glucose production decreased by 41% to 0.31 +/- 0.02 mumol/L (P less than .001), whereas the splanchnic uptake of gluconeogenic precursors increased and could account for the total glucose output. Net splanchnic uptake of taurine, threonine, serine, glycine, lysine, histidine, and arginine rose significantly in response to fasting (P less than .05 to .01) due to increased splanchnic fractional extraction. Although the splanchnic fractional extraction of alanine was augmented by 40% (P less than .001), net splanchnic uptake was not influenced by fasting. Total net splanchnic uptake of amino acids increased by 68%, from 231 +/- 44 mumol/min in the postabsorptive state to 388 +/- 63 mumol/min (mean +/- SEM) (P less than .05) in the 60-hour fasted state. However, only one half of this rise was accounted for by gluconeogenic amino acids.     

Effect of leucine on amino acid and glucose metabolism in humans.

Leucine has been reported to be an important regulator of protein metabolism. We investigated the effect of intravenous infusion of L-leucine versus saline on amino acid metabolism in eight healthy human subjects. Plasma concentrations of amino acids were measured and protein turnover was estimated using L-(1-13C)lysine and L-(3,3,3,-2H3)leucine as tracers. Glucose kinetics were measured using D-(6,6-2H2)glucose as a tracer. Leucine infusion increased the plasma leucine concentration from 103 +/- 8 to 377 +/- 35 mumol/L (P less than .01). Plasma concentrations of essential amino acids, including threonine, methionine, isoleucine, valine, tyrosine, and phenylalanine were significantly decreased by leucine infusion. Leucine infusion did not change lysine flux significantly (108 +/- 4 during saline v 101 +/- 4 mumol/kg/h-1 during leucine infusion), but decreased lysine oxidation (13.2 +/- 0.9 v 10.7 +/- 1 mumol/kg/h, P less than .05) and endogenous leucine flux (from 128 +/- 4 to 113 +/- 7 mumol/kg/h, P less than .05) when plasma (2H3) ketoisocaproate (KIC) was used for calculation. During leucine infusion, the (2H3) KIC to (2H3) leucine plasma enrichment ratio increased from 0.76 +/- 0.02 to 0.88 +/- 0.01 (P less than .001), while estimation of leucine flux using plasma (2H3) leucine showed no change in endogenous leucine flux. Leucine infusion decreased hepatic glucose production and metabolic clearance of glucose, but did not change plasma concentrations of glucose, insulin, C-peptide, glucagon, epinephrine, norepinephrine, or free fatty acids. We conclude that leucine spares glucose and lysine catabolism and decreases plasma concentrations of essential amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation and release of isoferritins during incubation in vitro of human peripheral blood mononuclear cells

Ferritin concentration has been measured in peripheral blood mononuclear cells and in the incubation medium following in vitro culture. Antibodies to both heart and spleen ferritin were used. Mononuclear cells cultured in medium containing about 12 mumol Fe/l accumulate ferritin rapidly with an increase in the heart:spleen ferritin ratio from 3:1 to about 10:1. Higher concentrations of iron (100 mumol/l) produce an even greater effect. The accumulation of ferritin is prevented by the addition of desferrioxamine (2 mmol/l) to the incubation medium. Accumulation of ferritin appears to take place largely in monocytes. Phagocytosis of red blood cells also causes rapid accumulation of ferritin but without any change in the heart:spleen ratio. Small amounts of both spleen and heart type ferritin are released during incubation in an iron containing medium and following phagocytosis of red blood cells. Some concanavalin A binding ferritin is also released suggesting that phagocytic cells may be a source of the concanavalin A binding ferritin found in normal plasma.     

Urinary excretion of carnitine in multiply injured patients on different regimens of total parenteral nutrition

Carnitine derives from intake of preformed exogenous carnitine and synthesis from lysine and methionine, but is absent in parenteral fluids. Urinary excretions of carnitine and its derivatives was measured in 30 patients 2-8 days after severe multiple injuries and compared with controls. The patients received five different isocaloric parenteral nutritional regimens;group 1 glucose and fat, group 2 glucose, fat and amino acids, group 3 glucose and insulin, group 4 glucose and amino acids, and group 5 branched-chain amino acids. The mean total carnitine excretion in healthy men was 420 mumol/24 h +/- 57 (SEM), and in women 266 mumol/24 h +/- 29, 41% of which was free carnitine. Mean excretion of total carnitine during days 2-8 after trauma for the five groups was: 900 +/- 100, 1169 +/- 112, 1251 +/- 102, 1023 +/- 117, and 668 +/- 128 mumol/24 h, being significantly higher in groups 1-4 than in healthy men. The free carnitine fraction in the patients was significantly higher than in controlled healthy subjects. Total carnitine excretion was unaffected by different nutritional regimens in the very first days. During days 6-8, group 5, receiving branched-chain amino acids had lower excretion of total carnitine (compared to groups 2-4) and free carnitine (compared to groups 3-4). Groups 3 and 4 excreted a higher percentage as free carnitine compared to the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splanchnic, renal, and muscle clearance of alanylglutamine in man and organ fluxes of alanine and glutamine when infused in free and peptide forms

The present study was designed to investigate organ metabolism of intravenously (IV) infused (100 mumol.h-1.kg-1) alanylglutamine and its amino acid constituents in a group of healthy subjects. The dipeptide clearance (mumol/min) by kidney (51 +/- 3) was significantly (P less than .01) greater than the clearance by either splanchnic organs (19 +/- 6) or skeletal muscle (21 +/- 8). Infusion of alanylglutamine significantly (P less than .01) increased arterial plasma concentrations of free alanine (260 +/- 31 v 330 +/- 38 mumol/L) and free glutamine (620 +/- 66 v 764 +/- 65 mumol/L) when compared with the baseline period. Concurrently, splanchnic uptake of alanine and glutamine increased and muscle release of alanine ceased. However, muscle release of glutamine remained unaffected. Renal balances of alanine and glutamine changed from neutral to negative (net release) and from positive (net uptake) to neutral, respectively. Infusion of a corresponding mixture of alanine and glutamine had similar effects on arterial plasma concentrations and splanchnic and muscle balances of alanine and glutamine, but had no effect on renal balances of these amino acids. From these studies in man, we conclude that kidney predominates over other organs in clearance of alanylglutamine from plasma and that this may account for the different effect of infusion of alanine and glutamine in free and peptide forms on renal fluxes of these amino acids.     

N-acetyl-L-tyrosine and N-acetyl-L-cysteine as tyrosine and cysteine precursors during intravenous infusion in humans

The usefulness of N-acetyl-L-tyrosine (NAT) and N-acetyl-L-cysteine (NAC) as tyrosine and cysteine precursors during intravenous infusion was investigated in humans. Plasma levels and urinary excretion of NAT, tyrosine, NAC, and total cysteine were determined, and the site of deacetylation was examined by measuring the splanchnic and renal balances. Eleven healthy volunteers were given 5 g of either NAT or NAC as a 4-hour intravenous infusion. Plasma levels of NAT and NAC increased rapidly, accompanied by a 25% increase in tyrosine levels and a 35% decrease in total cysteine. Urinary excretion of NAT and NAC in 4 hours accounted for 56% and 11% of the infused amount, respectively. No net production of tyrosine or cysteine was found from the splanchnic area, but from the kidneys there was a small release of both tyrosine (10 +/- 3 mumol/min) and cysteine (64 +/- 3 mumol/min). We conclude that under these conditions the usefulness of NAT and NAC as precursors for the corresponding amino acids in humans is not apparent.     

Free amino acid levels in muscle and liver of a patient with glucagonoma syndrome

This study set out to measure the amino acid concentrations in the femoral artery, femoral vein, hepatic vein, muscular and hepatic tissue, and urine of a patient with the glucagonoma syndrome. The total plasma amino acid concentration was severely reduced on admission (737 mumol/L, 26% of normal), with only a slight increase during intravenous administration of 200 g of amino acids per day. The total intracellular amino acid levels in the muscle were 86%, and those of the liver were 47% of the normal range. Only 0.62% of the amino acids administered were found in the urine. Arteriovenous amino acid concentration differences across the muscle and splanchnic tissue indicated the release of amino acids (mainly glutamine, glycine, and alanine) from the muscle and the absorption of amino acids by the splanchnic bed. This study shows that the infusion of a high amount of amino acids cannot increase the subnormal plasma AA levels of patients with the glucagonoma syndrome. The low total plasma AA levels are paralleled by decreased intracellular free amino acid levels in the muscle and liver.     

Plasma arginine in cancer of the gastrointestinal tract: effect of surgical treatment.

The concentration of 21 amino acids was measured in the venous plasma of 41 patients with cancer of the gastrointestinal tract who had lost weight, 12 patients who had lost a similar amount of weight from non-malignant, non-septic conditions (benign weight loss), 12 patients with cancer of the gastrointestinal tract who had not lost weight and 21 control patients. Sixteen patients with localised tumours were restudied eight and 12 weeks after successful removal of the primary growth. Six patients with pyloric stenosis (benign weight loss) were similarly studied for comparison after corrective surgery. The concentration of the amino acid arginine was significantly greater in patients with cancer both with weight loss (71.2 +/- 4.1 mumol/l mean +/- SEM) and without weight loss (66.8 +/- 4.1 mumol/l) when compared both with patients with benign weight loss (34.6 +/- 3.2 mumol/l) and with control patients (48.2 +/- 3.5 mumol/l) (p less than 0.05). In patients with cancer subjected to surgery the concentration of arginine (76.4 +/- 7.5 mumol/l) fell to normal levels eight weeks after operation and remained normal 12 weeks after surgery (48.6 +/- 4.4 mumol/l) (p less than 0.05). This was in contrast with the rise in plasma arginine in patients with pyloric stenosis after surgery, suggesting that the raised level of arginine was due to the presence of the primary tumour.     

Influence of beta blockade on branched chain amino acid concentrations in cirrhosis.

Cirrhosis of the liver is typically accompanied by low plasma levels of the three branched chain amino acids (BCAA). These patients also demonstrate increased concentrations of several hormones such as insulin, glucagon and catecholamines. Catecholamines have been shown to influence the plasma levels of amino acids in healthy subjects and diabetics. In the present study, amino acid concentrations were investigated before and up to 3 hours after beta blockade (Inderal, 40-80 mg, n = 10) or fasting (n = 8) in cirrhotic patients. In the basal state the patients had low levels of all three BCAA, as compared with healthy subjects. Norepinephrine was more than 3 times as high in the patients (3.65 +/- 0.6 vs. 0.84 +/- 0.08 nmol/l, p < 0.01) while epinephrine was only slightly raised (0.43 +/- 0.1 vs. 0.25 +/- 0.06 nmol/l, NS). Significant correlations were observed between the concentrations of norepinephrine and individual as well as the sum of the three BCAA (r = 0.43-0.62, p < 0.05-0.001), while no correlation was observed between the BCAAs and epinephrine or insulin. Three hours after beta blockade the concentrations of leucine (basal: 74 +/- 6, 180 min: 89 +/- 6 mumol/l, p < 0.05) and valine (basal: 110 +/- 10, 180 min: 132 +/- 11 mumol/l, p < 0.01) had increased significantly. A similar tendency was observed for isoleucine. No changes were observed after prolonged fasting. The results suggest that catecholamines, primarily norepinephrine, might contribute to the low levels of BCAA in cirrhotics.     

Bile acids in human breast cyst fluid: the identification of lithocholic acid

Breast cyst fluid (BCF) aspirated from 12 women with fibrocystic disease of the breast and sera obtained simultaneously were analyzed for bile acids. Analysis was performed by gas-liquid chromatography of the acetoxy methyl esters of the bile acids prepared after alkaline hydrolysis of the bile salts. An internal standard served to correct for methodological losses. Low levels of bile acids were found in serum samples, precluding overt hepatobiliary complications. Deoxycholic acid (17-160 mumol/L), chenodeoxycholic acid (18-305 mumol/L), and cholic acid (3-119 mumol/L) were detected in 11 of 12 samples of BCF. In 2 cases, chosen at random, the identities of the bile acids were verified by mass spectrometry. Lithocholic acid (9-23 mumol/L), a reported cocarcinogen, was detected in 6 of the 12 samples of BCF. This is the first report of the presence of lithocholic acid in BCF with confirmation by Mass spectrometry. There was no correlation between the levels of individual bile acids and those of potassium ion, Na+/K+, estriol-3-sulfate, or 16 alpha-hydroxyandrogen sulfates that had been quantified previously in these samples. There was borderline correlation between concentrations of total bile acids and K+ (P less than 0.06) and Na+/K+ (P less than 0.07). Yet to be elucidated are the mechanism of accumulation of bile acids in BCF and whether levels of particular bile acids in BCF may serve to identify that small subset of women with fibrocystic disease at risk for developing breast cancer.     

Effect of ursodeoxycholic acid treatment on intestinal absorption of triglycerides in man

The aim of the present study was to evaluate whether treatment with ursodeoxycholic acid (UDCA) may affect the absorption of dietary fat in man. Fifteen healthy subjects volunteered for the study. They were treated with UDCA in a daily dose of 15 mg/kg body weight for 4 weeks. Before and during treatment fat absorption was measured with a 14C-triolein breath test. In addition, fasting serum bile acids were measured in 11 of the subjects. The maximum specific activity of 14CO2 was not significantly changed during the treatment period. However, the cumulative output of 14CO2 during a 6-h period was decreased by about 25% (p less than 0.03). Several subjects with decreased outputs also lost 1-2 kg of body weight during the study period. UDCA treatment raised the serum level of this bile acid from 0.18 +/- 0.11 mumol/l to 5.98 +/- 1.08 mumol/l. The concentrations of the other bile acids were not significantly changed. It is suggested that UDCA treatment may in some patients be associated with an impaired fat absorption. Whether this effect is of any clinical importance remains to be elucidated.     

Adaptation of pancreatic amino acid transport in rats after treatment with the synthetic protease inhibitor camostat mesilate

Exocrine pancreatic amino acid transport was studied following oral treatment of rats with the synthetic protease inhibitor camostat mesilate. Camostat (200 mg/kg/day) was administered via an orogastric tube for 10 days, and kinetics of amino acid transport were then examined in the isolated perfused pancreas using a dual-isotope dilution technique. Administration of camostat doubled the rate constant (Vmax/Kt) for L-phenylalanine transport via System L by increasing the Vmax from 12.3 +/- 0.4 (n = 12) to 19.6 +/- 2.2 mumol/min/g (n = 4). Although the Kt (35 +/- 2 mM, n = 4) and Vmax (47.7 +/- 1.6 mumol/min/g) for L-serine transport via System asc increased, the Vmax/Kt ratio decreased from a control value of 1.85 to 1.36. Increases in Kt (5 +/- 2 mM, n = 4) and Vmax (5.18 +/- 1.01 mumol/min/g) for L-lysine transport via System y+ were also detected but Vmax/Kt remained unchanged. In the presence of a low Na+ perfusate, influx of L-serine, L-phenylalanine, and L-lysine were inhibited between 36 and 50%. Treatment with camostat results in an increase in the rate and Na+-dependence of large neutral amino acid transport in the rat exocrine pancreas. Pancreatic growth induced by repeated oral administration of camostat appears to be accompanied by an enhanced uptake of precursor amino acids.     

Homocysteine content of plasma proteins in ischemic heart disease

It has been shown previously that accumulation of homocysteine produces atheromatous changes. The present study was done on 26 male survivors of myocardial infarction 2-3 months after the acute phase and 26 healthy males of the same age (30-60 years). The concentrations of homocysteine, its derivatives and other amino acids were determined in acid hydrolyzate of plasma and in deproteinized plasma. The plasma proteins of survivors of myocardial infarction were found to contain a high concentration of homocysteine. The average value was 958 +/- 84 mumol/l of plasma, which was about 25 times the quantity found in the control group. Large differences were also found in alpha-amino adipic acid and cystathionine concentrations. These substances were found in significantly higher concentrations in the plasma of the survivors compared to controls. The high positive correlation between homocysteine and alpha-amino adipic acid level (r = 0.83; P less than 0.001) suggests a common source of these 2 compounds in the analyzed samples. The levels of the other 15 measured amino acids were not significantly different in the 2 groups. The results support the homocysteine theory and suggest a method for more exact diagnosis of atherosclerosis.     

On the role of the purine nucleotide cycle in the isolated working rat heart

The purine nucleotide cycle catalyzes the net reaction: aspartate + GTP + H2O----fumarate + NH3 + GFDP + Pi and leads to regeneration of AMP. In skeletal muscle the cycle's rate of operation increases several fold in response to a corresponding increase in work load, which results in a net increase in citric acid cycle intermediates and in release of ammonia. The same may be expected in heart muscle which, like skeletal muscle, possesses the enzymes of the purine nucleotide cycle. Isolated working rat hearts were therefore perfused for 45 min at low or high work load (0.16 v. 0.42 kg m/min/g dry wt) with glucose (5 mM) as substrate. Release of ammonia into the perfusate as well as the tissue content of citric acid cycle intermediates (citrate, isocitrate, 2-oxoglutarate, malate, and oxaloacetate), amino acids (aspartate, glutamate and glutamine), adenine nucleotides and phosphocreatine were measured in freeze-clamped tissue. There was no significant change in the sum of the citric acid cycle intermediates (1.295 v. 1.313 mumol/g dry wt), in aspartate (13.21 v. 14.32 mumol/g dry wt), in glutamate (15.58 v. 15.67 mumol/g dry wt), ATP (19.60 v. 19.17 mumol/g dry wt), ADP (5.00 v. 4.11 mumol/g dry wt), AMP (1.45 v. 1.01 mumol/g dry wt) and phosphocreatine (22.58 v. 25.80 mumol/g dry wt) when low and high work load were compared. Ammonia release was 26 mumol/h/g dry wt and 22 mumol/h/g dry wt at low and high work load respectively. The results suggest that in rat heart the activity of the purine nucleotide cycle does not increase with an increase in work load.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA

A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library. A mouse beta- spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli. This clone was used to probe total RNA from various mouse tissues. Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb. Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA. All of these bands could be detected after hybridization under both stringent and nonstringent conditions. This suggests that erythroid beta-spectrin may also be expressed in the brain. No bands could be detected in kidney, liver, or spleen RNA. Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions. Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin. The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.