Dietary citrulline does not modify rat colon tumor response to chemotherapy, but failed to improve nutritional status.

During cancer therapy many patients experience significant malnutrition, leading to decreased tolerance to chemotherapy and decreased survival. Dietary citrulline supplementation improves nutritional status in situations such as short bowel syndrome and aging, and is of potential interest in oncology. However, a mandatory prerequisite is to test this amino acid for interaction with tumor growth and chemotherapy response. Dietary citrulline (Cit; 2%), or an isonitrogenous mix of non-essential amino acids (control), was given to Ward colon tumor-bearing rats the day before chemotherapy initiation. Chemotherapy included 2 cycles, one week apart, each consisting of one injection of CPT-11 (50 mg/kg) and of 5-fluorouracil (50 mg/kg) the day after. Body weight, food intake and tumor volume were measured daily. The day after the last injection, rats were killed, muscles (EDL, gastrocnemius), intestinal mucosa, tumor, spleen and liver were weighed. Muscle and intestinal mucosa protein content were measured. Phosphorylated 4E-BP1 was measured in muscle and tumor as a surrogate for biosynthetic activation. FRAPS (Ferric Reducing Ability of Plasma) and thiols in plasma, muscle and tumor were evaluated and plasma amino acids and haptoglobin were measured. Numerous parameters did not differ by diet overall: a) response of tumor mass to treatment, b) tumor antioxidants and phosphorylated 4E-BP1 levels, c) relative body weight and relative food intake, d) weight of EDL, gastrocnemius, intestinal mucosa, spleen and liver and e) plasma haptoglobin concentrations. Moreover, plasma citrulline concentration was not correlated to relative body weight, only cumulated food intake and plasma haptoglobin concentrations were correlated to relative body weight. Citrulline does not alter the tumor response to CPT-11/5FU based therapy but, has no effect on nutritional status, which could be due to the anorexia and the low amount of citrulline and protein ingested.

Effect of age, stress and protein supply on plasma amino acids during continuous enteral nutrition; a pragmatic study in rats.

BACKGROUND & AIMS: As life expectancy increases, an increasing older population may require surgery with perioperative nutritional management. While little is known about the combined effect of age and stress on amino acid metabolism during enteral nutrition, we hypothesized that blood amino acid bioavailability may be influenced not only by the characteristics of the ingested protein but also by intestinal ageing and splanchnic sequestration of amino acids. Plasma amino acid kinetics were thus evaluated in aged and adult rats receiving continuous enteral nutrition before and after standardized surgical stress. METHODS: Sixteen 5-month-old and sixteen 21-month-old male rats were used. After a gastrostomy, the insertion of a jugular vein catheter and a one-week recovery, the animals were enterally fed with commercially available formulas containing whole milk proteins or a whey hydrolysate for 24 h before (healthy state) and 18 h after a standardized laparotomy (surgical stress). Data were analyzed by 3-factor ANOVA. RESULTS: In all rats, enteral nutrition was associated with a marked increase in plasma alanine, threonine, lysine and proline (+50 to +150 µmol/L; p < 0.001), and a decrease in glycine (≈-80 µmol/L; p < 0.01). For most amino acids, their availability depended first on the amino acid composition of each protein and second on surgical stress. Aging was only associated with higher tyrosine and threonine availability (p < 0.001). There was only limited statistical interaction between age and surgical stress. CONCLUSION: In rats, plasma amino acid availability during continuous enteral nutrition is determined by the nature of the protein source and the occurrence of stress. The effects of aging on plasma amino acid availability seem very limited. Commonly used formulas therefore appear to be as suitable for elderly patients as for adult patients.

Effect of aging on the availability of amino acids from an immune-enhancing diet (IED) after a surgical stress in rats.

BACKGROUND & AIMS: Dietary amino acid (AA) requirements increase after a surgical stress while the systemic AA availability from the diet decreases with age, due to splanchnic sequestration. While immune-enhancing diets (IEDs) have been recommended for the nutritional management of surgical patients, the systemic bioavailability of their AA supply has not been evaluated in elderly surgical patients. This was determined in surgically-stressed IED-fed aged rats. METHODS: Thirty-four 5-month- or 21-month-old male Sprague-Dawley rats were used. After a gastrostomy and placement of a jugular vein catheter and a one-week recovery period, the animals underwent two 24 h-enteral feedings with an arginine-enriched IED (Impact®, Nestlé Health Science) before (healthy state) and 18 h after a standardized laparotomy, used as a model of surgical stress. During enteral nutrition, blood samples were repeatedly collected to measure plasma AA bioavailability (incremental areas under the curve) at 2, 5 and 24 h. Surgical stress was evaluated from urinary catecholamines and plasma protein profile. RESULTS: Whatever the age or stress situation, IED feeding was associated with decreased plasma glycine and increased alanine, proline and arginine. Aging was mainly associated with a delayed plasma AA accumulation in the first hours after the initiation of enteral nutrition. Stress was associated with higher plasma arginine increase and lower histidine, methionine, phenylalanine and tyrosine accumulation. Age and stress interactions seem limited. CONCLUSIONS: AA bioavailability from an arginine-enriched IED seems to be maintained whatever age and stress situation. Aging appears to be mainly associated with a delay in plasma AA accumulation probably related to age-associated splanchnic sequestration of AAs. Additional effects of surgical stress per se seem limited.

Malnutrition with hypoaminoacidemia in a 22-year-old pregnant patient masking a likely ornithine transcarbamylase deficiency.

BACKGROUND: Symptoms and clinical presentations of OTC deficiency vary widely according to the remaining activity of the enzyme. Three factors determine the residual enzyme activity. First, as the OTC gene is carried on the X chromosome, a complete inactivation of this enzyme in a newborn boy results an acute ammonia intoxication. Second, the female mosaicism due to lyonization (differential randomized X-inactivation) leads to differential OTC expression in hepatocytes. Third, the degree of severity depends on the mutation and the level of remaining activity it leaves to the protein. Published cases of OTC deficiency during pregnancy are scant. Most often, diagnosis of the metabolic disease is made before pregnancy or during the post-partum period. METHODS: We report the case of a 22-year-old woman's successful pregnancy with a moderate form of ornithine transcarbamylase (OTC) deficiency, unsuspected before pregnancy, biochemically consistent with plasma aminoacidogram and orotic acid analysis, and initially masked by malnutrition. RESULTS - CONCLUSION: Although maternal ammonia was subnormal and the neonate was safe, an OTC deficiency was revealed by stress factors such as the pregnancy itself and infection, and associated with uncontrollable maternal vomiting and psychiatric syndrome. However, this metabolic disease, revealed by aminoacidogram and urine orotic acid analysis, fortunately did not prevent a successful pregnancy. Even if infrequent, this situation deserves to be highlighted.

Short-term Effects of Diet and Activity Changes on Inflammation and Insulin Resistance.

The present study focused on rapid responses of inflammation markers and insulin resistance to dietary restriction and exercise in inactive patients. 13 obese women were included during a 5-day time frame during which decreases in food intake (-1 378±298 kcal) were associated with 2 exercise sessions (80 and 40 min). Circulating inflammatory biomarkers, insulin resistance index and muscle soreness were measured in fasted conditions. Fasting plasma concentrations of CRP and insulin resistance index decreased over the period (respectively, p=0.02 and p=0.01), concentrations of IL-6 and TNF-α appeared unchanged (p>0.05). Changes in IL-6 (enhanced) and TNF-α (reduced) concentrations following the prolonged exercise differed compared to days with 40 min exercise and days without exercise (p<0.05). Muscle soreness appeared higher after the 80 min than after the 40-min exercise (p=0.01), and were related with IL-6 and CRP concentration changes. A 5-day period combining exercise and diet reduced the insulin-resistance index and the CRP fasting concentrations. The 80-min exercise enhanced IL-6 and lowered TNF-α concentration changes while days without exercise unaffected these cytokines. These exercise effects on cytokines may have benefited to the insulin resistance index. The duration and number of the exercise sessions appeared sufficient for inactive subjects to initiate health benefits without inducing negative effects on inflammation and muscle soreness.

Onset of exercise and diet program in obese women: metabolic and anorexigenic responses related to weight loss and physical capacities.

Perturbations of energy balance induce compensatory processes that may alter expected weight loss. In obese patients, our aim was to investigate the relationships that occurred between fasting plasma concentrations of anorexigenic peptides and metabolic parameters, appetite, physical capacity, and weight loss in the 5 first days of a program associating exercise and caloric reduction. Thirteen obese women were monitored from day 1 to day 5 with 2 exercise sessions in day 2 and day 4. We measured, in a fasted state, changes in body weight, hunger ratings, and plasma concentrations of fatty acids, triglycerides, leptin, insulin, amylin, peptide YY, and insulin-resistance index. Physical performance was assessed by a 6-min walking test. The program resulted in significantly reduced body weight (0.75±0.4 kg; p=0.001), of plasma concentrations of triglycerides, insulin, amylin, peptide YY, and the insulin-resistance index, and also increased fatty acids (p<0.05). Hunger ratings were increased (p<0.05). Program-induced changes in fatty acids, leptin, and insulin concentrations were related to physical performance (r(2)=0.45, 0.59, and 0.52; p<0.05, respectively) and to weight loss (r(2)=0.65, 0.57, 0.55; p<0.05, respectively). Five days of diet and exercise induced weight loss, improved lipid profile, and decreased insulin resistance while hunger ratings increased. Subjects with higher physical capacity lost more weight, presented higher increases in fatty acids and lower changes of leptin and insulin concentrations suggesting a better metabolic flexibility. To reduce the compensatory responses that can occur with energy imbalances, our study supports to account for individual activity level before prescribing weight-loss program associating diet and exercise.

Long-term intermittent glutamine supplementation repairs intestinal damage (structure and functional mass) with advanced age: assessment with plasma citrulline in a rodent model.

OBJECTIVE: Glutamine is the preferred fuel for the rat small intestine and promotes the growth of intestinal mucosa, especially in the event of gut injury. Quantitatively, glutamine is one important precursor for intestinal citrulline release. The aim of this study was to determine whether the effect of glutamine on the increase in intestinal villus height is correlated with an increase in both gut mass and citrulline plasma level in very old rats. METHODS: We intermittently supplemented very old (27-mo) female rats with oral glutamine (20% of diet protein). Intestinal histomorphometric analysis of the small bowel was performed. Amino acids, in particular citrulline, were measured in the plasma, liver and jejunum. Markers of renal (creatinine, urea) and liver (alanine aminotransferase [ALT]) and aspartate aminotransferase (AST) functions were measured to evaluate renal and liver functions in relation to aging and to glutamine supplementation. Liver glutathione was also determined to evaluate cellular redox state. RESULTS: Glutamine supplementation maintains the body weight of very old rats, not by limiting sarcopenia but rather by increasing the organ mass of the splanchnic area. Total intestine mass was significantly higher in glutamine-supplemented rats than in controls (15%). Measurement of villus height and crypt depth demonstrated that the difference between villus and crypt was significantly improved in glutamine pre-treated rats compared to controls (~ 11%). Plasma citrulline also increased by 15% in glutamine-supplemented rats compared to controls. CONCLUSION: Citrulline appears as a biomarker of enterocyte mass in villous atrophy associated with advanced age. Non-invasive measurement of this metabolite may be useful in following the state of the gastrointestinal tract in very old people, whose numbers are increasing worldwide and the care of whom is a major public health issue. The gut may contribute to the malnutrition caused by malabsorption frequently observed in the elderly.

Biocrude yield and productivity from the hydrothermal liquefaction of marine and freshwater green macroalgae.

Six species of marine and freshwater green macroalgae were cultivated in outdoor tanks and subsequently converted to biocrude through hydrothermal liquefaction (HTL) in a batch reactor. The influence of the biochemical composition of biomass on biocrude yield and composition was assessed. The freshwater macroalgae Oedogonium afforded the highest biocrude yield of all six species at 26.2%, dry weight (dw). Derbesia (19.7%dw) produced the highest biocrude yield for the marine species followed by Ulva (18.7%dw). In contrast to significantly different yields across species, the biocrudes elemental profiles were remarkably similar with higher heating values of 33-34MJkg(-1). Biocrude productivity was highest for marine Derbesia (2.4gm(-2)d(-1)) and Ulva (2.1gm(-2)d(-1)), and for freshwater Oedogonium (1.3gm(-2)d(-1)). These species were therefore identified as suitable feedstocks for scale-up and further HTL studies based on biocrude productivity, as a function of biomass productivity and the yield of biomass conversion to biocrude.

Effect of citrulline on muscle functions during moderate dietary restriction in healthy adult rats.

Low calorie diets are designed to reduce body weight and fat mass, but they also lead to a detrimental loss of lean body mass, which is an important problem for overweight people trying to lose weight. In this context, a specific dietary intervention that preserves muscle mass in people following a slimming regime would be of great benefit. Leucine (LEU) and Citrulline (CIT) are known to stimulate muscle protein synthesis (MPS) in post-prandial and post-absorptive state, respectively. This makes them interesting bioactive components to test in the context of dietary restriction. We tested the concept of combining LEU and CIT in adult female rats. We postulated that the sequential administration of LEU (mixed in chow) and CIT (given in drinking water before a rest period) could be beneficial for preservation of muscle function during food restriction. Sixty female rats (22 weeks old) were randomized into six groups: one group fed ad libitum with a standard diet (C) and five food-restricted groups (60 % of spontaneous intake for 2 weeks) receiving a standard diet (R group), a CIT-supplemented diet (0.2 or 1 g/kg/day, CIT0.2 group and CIT1 group, respectively), a LEU-supplemented diet (1.0 g/kg/day) or a CIT + LEU-supplemented diet (CIT + LEU 1.0 g/kg/day each). At the end of the experiment, body composition, muscle contractile properties and muscle protein synthesis (MPS) rate were studied in the tibialis anterior muscle. Dietary restriction tended to decrease MPS (R: 2.5 ± 0.2 vs. C: 3.4 ± 0.4 %/day, p = 0.06) and decrease muscle strength (R: 3,045 ± 663 vs. C: 5,650 ± 661 A.U., p = 0.03). Only CIT administration (1 g/kg) was able to restore MPS (CIT1: 3.4 ± 0.3 vs. R: 2.5 ± 0.2 %/day, p = 0.05) and increase muscle maximum tetanic force (CIT1: 441 ± 15 vs. R: 392 ± 22 g, p = 0.05) and muscle strength (CIT1: 4,259 ± 478 vs. R: 3,045 ± 663 A.U., p = 0.05). LEU had no effect and CIT + LEU supplementation had few effects, limited to adipose mass and fatigue force. The results of this study highlight the ability of CIT alone to preserve muscle function during dietary restriction. Surprisingly, LEU antagonized some effects of CIT. The mechanisms involved in this antagonistic effect warrant further study.

Markers of intestinal injury are associated with endotoxemia in successfully resuscitated patients.

AIMS: Gut dysfunction is suspected to play a major role in the pathophysiology of post-resuscitation disease through an increase in intestinal permeability and endotoxin release. However this dysfunction often remains occult and is poorly investigated. The aim of this pilot study was to explore intestinal failure biomarkers in post-cardiac arrest patients and to correlate them with endotoxemia. METHODS: Following resuscitation after cardiac arrest, 21 patients were prospectively studied. Urinary intestinal fatty acid-binding protein (IFABP), which marks intestinal permeability, plasma citrulline, which reflects the functional enterocyte mass, and whole blood endotoxin were measured at admission, days 1-3 and 6. We explored the kinetics of release and the relationship between IFABP, citrulline and endotoxin values. RESULTS: IFABP was extremely high at admission and normalized at D3 (6668 pg/mL vs 39 pg/mL, p=0.01). Lowest median of citrulline (N=20-40 µmol/L) was attained at D2 (11 µmol/L at D2 vs 24 µmol/L at admission, p=0.01) and tended to normalize at D6 (21 µmol/L). During ICU stay, 86% of patients presented a detectable endotoxemia. Highest endotoxin level was positively correlated with highest IFABP level (R(2)=0.31, p=0.01) and was inversely correlated with lowest plasma citrulline levels (R(2)=0.55, p<0.001). Endotoxin levels increased between admission and D2 in patients with post-resuscitation shock, whereas it decreases in patients with no shock (median +0.33 EU vs -0.19 EU, p=0.03). Highest endotoxin level was positively correlated with D3 SOFA score (R(2)=0.45, p=0.004). CONCLUSION: Biomarkers of intestinal injury are altered after cardiac arrest and are associated with endotoxemia. This could worsen post-resuscitation shock and organ failure.

Splanchnic sequestration of amino acids in aged rats: in vivo and ex vivo experiments using a model of isolated perfused liver.

Splanchnic sequestration of amino acids (SSAA) is a process observed during aging that leads to decreased peripheral amino acid (AA) availability. The mechanisms underlying SSAA remain unknown. The aim of the present study was to determine whether a high-protein diet could increase nitrogen retention in aged rats by saturating SSAA and whether SSAA could be explained by dysregulation of hepatic nitrogen metabolism. Adult and aged male Sprague-Dawley rats were housed in individual metabolic cages and fed a normal-protein (17% protein) or high-protein diet (27%) for 2 wk. Nitrogen balance (NB) was calculated daily. On day 14, livers were isolated and perfused for 90 min to study AA and urea fluxes. NB was lower in aged rats fed a normal-protein diet than in adults, but a high-protein diet restored NB to adult levels. Isolated perfused livers from aged rats showed decreased urea production and arginine uptake, together with a release of alanine (vs. uptake in adult rats) and a hepatic accumulation of alanine. The in vivo data suggest that SSAA is a saturable process that responds to an increase in dietary protein content. The hepatic metabolism of AA in aged rats is greatly modified, and urea production decreases. This result refutes the hypothesis that SSAA is associated with an increase in AA disposal via urea production.

Dose-ranging effects of citrulline administration on plasma amino acids and hormonal patterns in healthy subjects: the Citrudose pharmacokinetic study.

Previous experimental studies have highlighted that citrulline (CIT) could be a promising pharmaconutrient. However, its pharmacokinetic characteristics and tolerance to loading have not been studied to date. The objective was to characterise the plasma kinetics of CIT in a multiple-dosing study design and to assess the effect of CIT intake on the concentrations of other plasma amino acids (AA). The effects of CIT loading on anabolic hormones were also determined. Eight fasting healthy males underwent four separate oral loading tests (2, 5, 10 or 15 g CIT) in random order. Blood was drawn ten times over an 8 h period for measurement of plasma AA, insulin and growth hormone (Gh). Urine samples were collected before CIT administration and over the next 24 h. None of the subjects experienced side effects whatever the CIT dose. Concerning AA, only CIT, ornithine (ORN) and arginine (ARG) plasma concentrations were affected (maximum concentration 146 (sem 8) to 303 (sem 11) micromol/l (ARG) and 81 (sem 4) to 179 (sem 10) micromol/l (ORN); time to reach maximum concentration 1.17 (sem 0.26) to 2.29 (sem 0.20) h (ARG) and 1.38 (sem 0.25) to 1.79 (sem 0.11) h (ORN) according to CIT dose). Even at high doses, urinary excretion of CIT remained low ( < 5 %). Plasma insulin and Gh were not affected by CIT administration. Short-term CIT administration is safe and well-tolerated. CIT is a potent precursor of ARG. However, at the highest doses, CIT accumulated in plasma while plasma ARG levels increased less than expected. This may be due to saturation of the renal conversion of CIT into ARG.

Plasma concentrations of arginine and related amino acids following traumatic brain injury: Proline as a promising biomarker of brain damage severity.

The aim of this study was to find a plasma biomarker, in relation with nitric oxide (NO), as a sign of brain damage severity following traumatic brain injury (TBI). We first investigated the post-traumatic evolution of the plasma concentrations of NO via the level of NO end-products metabolites (nitrite plus nitrate, NO(x)), that of l-arginine (Arg) and amino acids involved in its metabolism as well as the time course of neurological score in a rat model of lateral fluid percussion brain injury. First, the level of NO(x) was increased in plasma at 24 and 48 h post-TBI with a marked increase at 72 h. In contrast, this elevation was neither accompanied by a modification of plasma concentrations of Arg, nor of amino acids involved in NO and Arg metabolism, l-ornithine (Orn), l-glutamate (Glu), and l-glutamine (Gln). Second, TBI induced a fall of plasma l-proline (Pro) concentrations. The time course of post-TBI neurological deficit showed also a decrease of neurological score at 24, 48, and 72 h. Furthermore, there is a weak negative correlation between the neurological score and the plasma level of NO(x) (r=-0.305; P<0.05), while a marked positive correlation has been found between the neurological score and the plasma level of Pro (r=0.563; P<0.001). In conclusion, the plasma concentrations of Pro could be a promising marker of post-traumatic neurological deficit.

Effects of an immune-enhancing diet in endotoxemic rats.

OBJECTIVE: This work compared the nutritional efficiency of a recently available enteral formula enriched with arginine, omega-3 fatty acids, and antioxidants and supplied nitrogen as peptides (Crucial, Nestle Clinical Nutrition) with that of a standard polymeric formula (Sondalis HP, Nestle Clinical Nutrition) in endotoxemic rats. METHODS: Male Wistar rats (209 +/- 2 g) underwent catheter gastrostomy and received Sondalis HP until they recovered their preoperative weight. At that time (day 0), an endotoxemic shock was induced by an intraperitoneal injection of lipopolysaccharide (Escherichia coli, 8 mg/kg) and rats then received 290 kcal x kg(-1) x d(-1) and 3.29 g of nitrogen x kg(-1) x d(-1) in the form of Crucial (IED group, n = 7) or Sondalis HP (S group, n = 6) for 3 d. Another group underwent no treatment and was fed ad libitum (AL group). Rats were killed on day 3. Results are presented as mean +/- standard error of the mean (analysis of variance and Newman-Keuls test). RESULTS: The endotoxemic shock induced a weight loss in group S on days 1 and 2 and a weight gain in group IED (-3.5 +/- 1.3 g in group S versus +6.0 +/- 2.2 g in group IED, P < 0.05). In the same way, atrophy of extensor digitorum longus muscle was observed in group S, whereas wasting was limited in group IED (102 +/- 4 mg in group IED versus 90 +/- 3 mg in group S versus 119 +/- 3 mg in group AL, P < 0.05). Muscular atrophy was associated with muscular glutamine depletion and correlated with hyperphenylalaninemia (R = 0.60), with the latter being blunted in group IED (57 +/- 1 microM/L in group AL versus 77 +/- 4 microM/L in group S versus 66 +/- 2 microM/L in group IED, P < 0.05). No difference was observed between the experimental groups of endotoxemic rats with respect to nitrogen balance, urinary excretion of 3-methyl histidine, or total tissue protein content. CONCLUSION: Crucial counteracts injury-mediated weight loss, extensor digitorum longus muscle atrophy, and hyperphenylalaninemia in endotoxemic rats.

Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection.

OBJECTIVE: Arginine supplementation seems logical in situations where this amino acid becomes essential, for example after massive intestinal resection. Arginine is taken up and metabolised by the liver to a large extent and its supplementation is potentially unsafe. Citrulline is not captured by the liver and passes freely to the kidneys where it is metabolised to arginine, and so is a good candidate to generate arginine and thereby improve nutritional status. METHODS: Twenty four rats were assigned to four groups: citrulline, arginine, control, and sham. The sham group underwent transection and the three other groups resection of 80% of the small intestine. All rats were fed by enteral nutrition and its composition was as follows: supplementation with citrulline in the citrulline group, supplementation with arginine in the arginine group, and standard polymeric enteral nutrition in the control and sham groups. All groups received isonitrogenous nutrition and citrulline and arginine intakes were equimolar in their respective groups. After 10 days, the rats were sacrificed. RESULTS: Arginine concentration was higher (p<0.05) in plasma and muscle in the citrulline group than in the three other groups. Plasma levels of arginine were 110 (12), 79 (7), 167 (22), and 228 (13) mumol/l in the sham, control, arginine, and citrulline groups respectively. Arginine concentrations in the gastrocnemius were: 0.15 (0.02), 0.16 (0.02), 0.40 (0.05), and 0.94 (0.20) mumol/g, respectively. Citrulline preserved nitrogen balance in resected rats but not in arginine supplemented rats (mean J10: 2.27 (0.29), 1.67 (0.15), 1.98 (0.29), and 2.43 (0.41) g/24 hours in the sham, control, arginine, and citrulline groups, respectively). CONCLUSION: Supplementing the diet with citrulline is a very efficient means of increasing arginine levels and improving nitrogen balance after massive intestinal resection. The results of this study form a strong rationale for citrulline supplementation in these patients.

Effect of apoE/ATP-containing liposomes on hepatic energy state.

BACKGROUND/AIMS: ATP-containing liposomes partially prevent ATP depletion in the cold-stored liver. As hepatocytes can specifically bind apoE, we investigated whether the addition of apoE to large (200 nm) ATP-containing liposomes increases their uptake by the liver and further improves hepatic energy stores. METHODS: Livers from fasted male Hartley guinea-pigs (231 +/- 3 g) were perfused for 90 min under our standard conditions (Control, n = 6) or after a single bolus addition of plain liposomes (Lip, n = 6), ATP (5 micromol)-containing liposomes (ATP-Lip, n = 6) or apoE/ATP-containing liposomes (0.8 or 8mg apoE/g phospholipids; apoE1-Lip and apoE10-Lip, respectively, n = 6 in each group). Liposome uptake and its impact on energy and nitrogen metabolism were studied. RESULTS: At its highest concentration, apoE significantly increased liposome uptake (apoE10-Lip: - 9.17 +/- 0.69 vs apoE1-Lip: - 6.18 +/- 0.44 vs ATP-Lip: - 6.40 +/- 0.88 nmol min(-1) g(-1) P < 0.05). This was associated with a significant increase in intrahepatic ATP (apoE10-Lip: 1033 +/- 137 vs apoE1-Lip: 811 +/- 98 and ATP-Lip: 648 +/- 36 nmol g(-1); P < 0.05), which was restored to its level in non-perfused livers. Hepatic viability and nitrogen metabolism were not affected. CONCLUSIONS: Hepatic ATP content being a key factor in the maintenance of liver graft function, apoE/ATP-containing liposomes should be useful in liver preservation for transplantation.

Hepatic preservation, liposomally entrapped adenosine triphosphate and nitric oxide production: a study of energy state and protein metabolism in the cold-stored rat liver.

BACKGROUND: Liposomally entrapped adenosine triphosphate (ATP) has been demonstrated to improve energy state and function of the cold-stored liver. The increased nitrite release associated with liposome administration led us to investigate the interactions between liposome supply and nitric oxide (NO) production through the use of L-NAME, a non-selective inhibitor of NO synthesis. METHODS: Twenty-four livers from fasted rats were stored for 18 h at +4 degrees C in University of Wisconsin solution directly (control group) or after infusion with ATP-containing liposomes (Lip-ATP), L-NAME (L-NAME) or both (Lip-ATP-L-NAME). Metabolic fluxes, cell volume and energy state were studied during reperfusion. RESULTS: After storage, nitrite release was increased by 61% in the Lip-ATP group, markedly decreased in the Lip-ATP-L-NAME group and almost abolished in the L-NAME group. The ATP content was increased by 20% in the Lip-ATP group (P < 0.05 versus control) and on reperfusion this was associated with an increase in cell volume (17%; P < 0.05) and a decrease in branched-chain amino acid release (21%; P < 0.01). The simultaneous addition of L-NAME did not affect these results, but induced a large (6-fold) increase in glucose production, possibly related to the metabolism of glycerol supplied by the liposomes. In the L-NAME group, global amino acid release was 50% lower and was associated with a dramatic decrease in urea production while the energy state deteriorated rapidly. CONCLUSIONS: The improvement in energy state and anabolic cell swelling induced by ATP-containing liposomes seems to be independent of NO synthesis. On the other hand, inhibition of NO synthesis appears to exert a detrimental effect on the liver, presumably through the decrease in hepatic energy content.

Evaluation of a newly available biochemical analyzer: the Olympus AU 600.

The performance of the Olympus AU 600, a newly available open multiparametric analyzer, available for routine biochemical analysis of biological samples, was evaluated. The analytical and technical performance of the apparatus and the quality of the Olympus reagents were both examined in a single site study. Electrolyte concentrations were determined with patented ion-selective electrodes; substrate concentrations and enzyme activities were determined by spectrophotometric measurement after coloured reaction or UV detection-based-reactions. The protocol of the evaluation and the acceptability criteria were those recommended by the French Society for Clinical Biology. For the parameters studied, the upper limits of linearity were equal to or higher than those claimed by the manufacturer. The CV values for within-run and between-run precision were lower than the target values with few exceptions. The comparison study gave satisfactory results for most of the parameters. Only expected interferences occurred. In summary, the results obtained for the 25 parameters studied and the characteristics of the apparatus were satisfactory. The analyzer is rapid (800 to 1200 tests per hour) and easy to use. In addition, the analyzer complies with good analytical practice and its flexibility enables users to plan work according to local laboratory constraints.

Cold preservation injury in rat liver: effect of liposomally-entrapped adenosine triphosphate.

BACKGROUND/AIMS: Energy charge and capacity for adenosine triphosphate (ATP) synthesis have been demonstrated to play a major role in the maintenance of organ function after liver preservation for transplantation. The aim of this study was to evaluate whether a supply of liposomally-entrapped ATP during preservation could improve the energy state and metabolism of cold-stored rat liver. METHODS: In the first set of experiments, the uptake of ATP-containing liposomes and their effects on hepatic viability were determined in isolated perfused unstored rat liver. In the second set of experiments, rat livers were preserved for 18 h at 4 degrees C in UW solution in the presence of these liposomes, and effects on energy state, cell volume and metabolism were evaluated. In each part, data were compared with adequate control, unloaded liposome-treated, and free ATP-treated groups (n=6 in each group). RESULTS: In non-stored livers, ATP-containing liposomes were taken up by the liver; they did not alter hepatic viability and induced a decrease in energy substrate consumption (glucose and amino acids), and an improvement in intrahepatic ATP content (+23% vs. Control). Addition of liposomally-entrapped ATP during cold storage produced a significant attenuation of the decrease in hepatic ATP content (Lip ATP 2: 524+/-45 vs. Control 2: 364+/-106 nmol/g; p<0.05), and induced, during reperfusion, a decrease in proteolysis associated with an increase in cell volume compared with the other groups (Lip ATP 2: 633+/-63 vs. Control 2: 532+/-38, Unloaded Lip 2: 483+/-55 and Free ATP 2: 500+/-29 microl/g; p<0.01). CONCLUSIONS: These data indicate that liposomally-entrapped ATP represents an effective means to improve liver graft energy state and function. The decrease in protein degradation may be related to the modification of cell volume.

Deletion of hydroxyethylstarch from University of Wisconsin solution induces cell shrinkage and proteolysis during and after cold storage of rat liver.

Among the numerous components of the University of Wisconsin (UW) solution used for organ preservation, the usefulness of hydroxyethylstarch (HES), the colloido-osmotic support of this solution, is controversial. The aim of our study was to determine the influence of HES on hepatic metabolism and intracellular hydration state during hypothermic preservation and after reperfusion in a model of isolated perfused rat liver. Three groups of eight livers were perfused either immediately or after 18 hours of cold storage in a UW-based preservation solution with or without HES. Omission of HES results in 1) a stimulation of protein degradation shown by the marked increase in branched-chain amino acid (BCAA) release (211 +/- 55 vs. 87 +/- 28 nmol/min/g; P < .05, modified UW group vs. UW group), 2) an increase in oxygen consumption (81.7 +/- 4.8 vs. 61.5 +/- 5.0 micromol/h/g; P < .05), 3) a decrease in glucose production (2.3 +/- 0.6 vs. 5.0 +/- 0.6 micromol/min/g; P < .05), and 4) a reduction in intracellular volume (414 +/- 36 vs. 557 +/- 41 microL/g; P < .05). We conclude that HES plays an important role in liver preservation by limiting proteolysis, possibly through the observed preservation of cell volume.