Donald A. Plumley

Prophylactic glutamine protects the intestinal mucosa from radiation injury.

Glutamine may be an essential dietary component, especially for the support of intestinal mucosal growth and function. This study evaluated the effects of a glutamine‐enriched elemental diet, administered before whole‐abdominal radiation on gut glutamine metabolism, mucosal morphometrics, and bacterial translocation. Rats were randomized to receive a nutritionally complete elemental diet that was glutamine‐enriched or glutamine‐free for 4 days. The animals were then subjected to a single dose of 1000 cGy x‐radiation to the abdomen. After irradiation, all animals received the glutamine‐free diet. Four days later the animals underwent laparotomy for sampling of arterial and portal venous blood, culture of mesenteric lymph nodes, and removal of the small intestine for microscopic examination. There was no difference in arterial glutamine or gut glutamine extraction between the two groups, but body weight loss was significantly diminished in the glutamine‐fed rats. Rats receiving the glutamine‐enriched elemental diet before radiation had a significant increase in jejunal villous number, villous height, and number of metaphase mitoses per crypt. Scanning electron microscopy confirmed the presence of an intact gut epithelium in eight of eight rats receiving prophylactic glutamine compared to one of eight animals in the glutamine‐free group. Three of eight rats fed glutamine had culture positive mesenteric lymph nodes compared with five of seven rats receiving the glutamine‐free diet. Glutamine exerts a protective effect on the small bowel mucosa by supporting crypt cell proliferation which may accelerate healing of the acutely radiated bowel.

The effects of sepsis and endotoxemia on gut glutamine metabolism.

The effects of sepsis on gut glutamine (GLN) metabolism were studied to gain further insight into the regulation of the altered glutamine metabolism that characterizes critical illnesses. Studies were done in laboratory rats and in hospitalized patients. The human studies were done in seven healthy surgical patients (controls) and six septic patients who underwent laparotomy. Radial artery and portal vein samples were obtained during operation and were analyzed for GLN and oxygen content. Despite no reduction in arterial glutamine concentration in the septic patients, gut glutamine extraction was diminished by 75% (12.0% +/- 1.6% in controls vs. 2.8% +/- 0.8% in septic patients, p less than 0.01). Similarly gut oxygen extraction was diminished by nearly 50% in the septic patients (p less than 0.05). To further investigate these abnormalities, endotoxin (10 mg/kg intraperitoneally) or saline (controls) was administered to adult rats 12 hours before cannulation of the carotid artery and portal vein. The arterial GLN concentration was increased by 13% in the endotoxin-treated animals (p less than 0.05) but gut glutamine uptake was diminished by 46% (526 +/- 82 nmol/100 g BW/minute in controls vs. 282 +/- 45 in endotoxin, p less than 0.01). Simultaneously gut glutaminase activity was diminished by 30% (p less than 0.01) and intestinal glutamate release fell by two thirds. Blood cultures were negative in control animals (0 of 20), but were positive in 25% of endotoxemic animals (6 of 24) for gram-negative rods (p = 0.019). Sepsis and endotoxemia impair gut glutamine metabolism. This impairment may be etiologic in the breakdown of the gut mucosal barrier and in the development of bacterial translocation.

Glutamine-enriched diets support muscle glutamine metabolism without stimulating tumor growth

Glutamine is a principal fuel utilized by rapidly growing tumors. Advanced malignant disease results in muscle glutamine depletion and weight loss. Concern exists about providing dietary glutamine to the host with cancer since it may stimulate tumor growth. This study examined the effects of oral glutamine on muscle glutamine metabolism and tumor growth. Twenty-four rats with large sarcomas were pair fed a glutamine-enriched or glutamine-free elemental diet. Diets were isonitrogenous and isocaloric. After 6 days of feeding, the animals were anesthetized and arterial glutamine, hindquarter glutamine flux, muscle glutamine content, tumor weight, tumor DNA content, tumor glutaminase activity, and number of metaphase mitoses/high power field (HPF) in the tumor were determined. There was no difference in arterial glutamine between the two groups, but provision of a glutamine-enriched diet increased muscle glutamine content by 60% (2.31 +/- 0.21 mumole/g tissue vs 1.44 +/- 0.22 mumol/g tissue, P less than 0.05), which supported muscle glutamine release. There were no differences among tumor DNA content, tumor glutaminase activity, or tumor weight and there was no difference histologically in the number of metaphase mitoses/HPF. Glutamine-enriched oral diets may replete host glutamine stores and support muscle glutamine metabolism without stimulating tumor growth.

Lung Glutamine Metabolism

The role of the lungs in maintaining amino acid homeostasis has only recently been studied. The data suggest that the lungs play a key role in glutamine flow in both normal and catabolic states. Further studies are necessary to clarify the regulation of lung glutamine at the whole organ and cellular level.

Growth hormone regulates amino acid transport in human and rat liver.

Human growth hormone (GH) has been shown to improve nitrogen balance in surgical patients and to decrease urea production. This has been thought to be due primarily to an increase in protein synthesis in skeletal muscle. Little attention has focused on the liver as a possible site where GH may modulate amino acid uptake and thereby divert nitrogen away from urea-genesis. The authors hypothesized that GH regulates amino acid transport in hepatocytes at the plasma membrane level. They studied hepatic amino acid transport in 20 healthy surgical patients that received saline, low-dose GH (0.1 mg/kg/day), or high-dose (0.2 mg/kg/day) GH for 3 days before operation. At operation, a 5- to 10-g wedge biopsy of the liver was obtained, and hepatocyte plasma membrane vesicles were prepared by Percoll density gradient centrifugation. Vesicle transport of [3H]-MeAIB, a highly selective system A substrate, and [3H]-glutamine, a selective system N substrate, was measured, employing a rapid mixing/filtration technique. Hepatocyte plasma membrane vesicles were also prepared from 14 rats treated with saline or one of three different GH treatment regimens: (A) 12 hours after chronic GH treatment (6 mg/kg every 12 hours x 4 doses); (B) 4 hours after acute (1 dose) GH treatment; and (C) 4 hours after chronic GH treatment. In human liver vesicles, low-dose GH resulted in a 13% decrease in system A activity (p = not significant), whereas high-dose GH caused a marked 79% decrease (6.7 +/- 1.7 pmol/mg protein/10 seconds in control patients versus 1.4 +/- 0.7 in GH, p less than 0.05). System N was unaffected. Kinetic analysis of MeAIB transport by vesicles from high-dose GH patients showed the reduction in transport to be due to a 63% decrease in the Vmax (maximal transport velocity) with no alteration in the transport Km (carrier affinity). Vesicles from rats treated chronically with GH using a protocol similar to that used for human subjects exhibited decreased system A transport activity (10.4 +/- 0.4 pmol/mg pro/10 seconds in controls versus 7.5 +/- 0.2 in GH, p less than 0.05) secondary to a 59% reduction in the transport Vmax. Chronic growth hormone treatment decreases the activity of system A in both human and rat hepatocytes. This may be one mechanism by which GH diminishes hepatic urea-genesis and spares amino acids for peripheral protein synthesis.

Role of the lungs in maintaining amino acid homeostasis.

The relative contributions of skeletal muscle and the pulmonary bed in maintaining amino acid homeostasis were studied. Inasmuch as more than 60% of whole blood amino acid nitrogen is transported as glutamine and alanine, the flux of these two amino acids across the lungs (n = 20) and hindquarter (n = 20) was determined in the postabsorptive adult rat. Both skeletal muscle and the lungs released net amounts of glutamine and alanine in the postabsorptive state. Blood flow to the hindquarter was approximately 16% of cardiac output (3.8 +/- 0.3 cc/100 g BW/min), while pulmonary blood flow (cardiac output) was 23.7 +/- 1.7 cc/100 g BW/min. Thus, despite a lower glutamine concentration difference across the lungs (-32 +/- 6 mumol/liter) compared with the hindquarter (-59 +/- 10 mumol/liter (p less than 0.01), the lungs released significantly more glutamine (741 +/- 142 nmol/100 g BW/min) than the hindquarter (208 +/- 39 nmol/100 g BW/min) (p less than 0.01) because of the significantly higher pulmonary blood flow. Similarly, the concentration difference for alanine across the lungs was less than that of the hindquarter (-24 +/- 8 mumol/liter vs -60 +/- 12 mumol/liter, p less than 0.01) but the lungs released significantly more alanine than the hindquarter (553 +/- 159 nmol/100 g BW/min vs 221 +/- 41 nmol/100 g BW, p less than 0.01. Compositional studies demonstrated that the hindquarter comprises 40% of total body muscle mass in the rat; thus both total skeletal muscle mass and the lungs contribute approximately equally to the maintenance of blood glutamine and alanine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Lung glutamine flux following open heart surgery

Despite the attenuated skeletal muscle proteolysis that occurs following hypothermic anesthesia and open heart surgery, blood amino acid levels are maintained, suggesting enhanced amino acid release by another organ. To investigate the role of the lung in this response, we determined the release of glutamine (Gln) and alanine by the lung, since these two amino acids transport two-thirds of circulating amino acid nitrogen. Three groups of patients were studied: (a) preoperative non-stressed controls; (b) postoperative general surgical patients; and (c) postoperative cardiac surgical patients studied on Postoperative Day 1 following open heart surgery requiring cardiopulmonary bypass and hypothermic anesthesia. In preoperative controls the lung was an organ of glutamine and alanine balance. These exchange rates were unaffected by the stress of an abdominal surgical procedure despite a mild increase in pulmonary blood flow. However, lung Gln release in the cardiac surgical patients was significantly increased (-0.6 +/- 1.2 mumole/kg/min in controls vs -6.5 +/- 1.3 mumole/kg/min in postoperative hearts, P less than 0.05) and was due exclusively to an increase in the pulmonary artery-systemic arterial concentration difference. Alanine release by the lungs was also increased in the postoperative cardiac surgical patients. The mechanism by which this augmented pulmonary glutamine release occurs following open heart surgery is unclear, but the lungs appear to play a central role in maintaining amino acid homeostasis. This metabolic role of the lungs following hypothermic anesthesia and cardiopulmonary bypass has not been previously described.

Trajectory of Metabolic Derangement in Infants with Necrotizing Enterocolitis Should Drive Timing and Technique of Surgical Intervention

BACKGROUND Seven clinical metrics of metabolic derangement (MD7) have improved the timing of surgical intervention in infants with necrotizing enterocolitis (NEC). We compared surgical NEC outcomes based on MD7 at our center (unit S) with a similar center (unit B) that based its intervention on abdominal radiograph. STUDY DESIGN Premature infants undergoing surgical care for NEC were evaluated. MD7 included positive blood culture, acidosis, bandemia, hyponatremia, thrombocytopenia, hypotension, and neutropenia. Surgical recommendations were stratified as observation or intervention. Good outcomes included full enteric feeding by discharge and poor outcomes were death or dependence on parenteral nutrition. For unit S and unit B, the frequency, median, and mode of MD7 component per case were determined for observation and intervention. Mann-Whitney U test and Wilcoxon matched pairs were used to compare positive MD7 frequency for observation with intervention. Institutional mortality was compared and metabolic severity of unit cohorts was evaluated by incidence of MD7 in each. RESULTS From March 2005 to July 2008, forty-one infants at unit S underwent 62 surgical evaluations. Observation was elected in 38 (median 1 MD7 per case, mode 0). Operative intervention occurred in 24 (median 4 MD7 per case, mode 4). Proportional MD7 difference between observation and intervention was significant (p = 0.018, U = 6). From February 2007 to December 2008, sixty-five unit B infants received 81 evaluations, recommending 37 observations (median 2 MD7 per case, mode 2), and 44 interventions (median 3 MD7 per case, mode 3). MD7 proportions between observation and intervention were not significant (p = 0.318, U = 16). Poor outcomes rates for unit S and unit B infants were 24% and 66%, respectively (p = 0.0001). Severity of MD7 did not differ between institutions (p = 0.53, U = 19). CONCLUSIONS These data demonstrate variability in surgical approach to NEC. The MD7 panel describes the trajectory of metabolic derangement, defines more timely surgical intervention, and demonstrates that waiting for free air is too late.

Cyclo-oxygenase blockade abrogates the endotoxin-induced increase in Na+-dependent hepatic amino acid transport

BACKGROUND Endotoxemia is characterized by a marked increase in the uptake of amino acids by the liver, but the regulation of this response has not been fully elucidated. In the current study, we investigated the potential role of prostaglandins as mediators of this response. We examined the in vivo effects of the anti-inflammatory agent ketorolac, a cyclo-oxygenase inhibitor that blocks prostaglandin synthesis, on hepatic amino acid transport activity in endotoxin-treated rats. METHODS We assayed the activities of the Na(+)-dependent transport systems A and N in hepatic plasma membrane vesicles prepared from endotoxemic rats that were pretreated with ketorolac or vehicle. Hepatic plasma membrane vesicles were prepared by differential centrifugation, and the transport of [3H]glutamine (system N) and [3H]2-methylamino-isobutyric acid (system A) was assayed. Hepatic plasma membrane vesicles were also prepared from normal rats that received prostaglandin E2, and glutamine and MeAIB transport were measured. RESULTS Endotoxin treatment resulted in a twofold to threefold increase in Na(+)-dependent amino acid transport activity in hepatic plasma membrane vesicles secondary to an increase in the transport Vmax, which was consistent with the appearance of increased numbers of corresponding transporter proteins in the hepatocyte plasma membrane. Pretreatment with ketorolac almost completely abrogated the endotoxin-induced increase in hepatic amino acid transport. Administration of prostaglandin E2 to normal rats resulted in a statistically significant increase in glutamine and alanine transport by hepatic plasma membrane vesicles prepared from these animals. CONCLUSIONS Prostaglandins play a key role in mediating the accelerated hepatic amino acid transport that occurs during endotoxemia.

Simple Method of Determining Pulmonary Blood Flow in the Anesthetized Rat

We have developed a simple, accurate, and relatively inexpensive method of measuring pulmonary blood flow in the anesthetized rat using a modification of the dye dilution technique. The method is attractive because it allows for the measurement of pulmonary blood flow and also the determination of pulmonary substrate flux. Using rats with a catheter in the carotid artery and a doubly cannulated right ventricle (RV), a constant infusion of [14C]P-aminohippurate ([14C]PAH) is begun via the distal RV catheter. After steady state is obtained the infusion of [14C]PAH is then stopped and blood is immediately withdrawn from the proximal RV cannula. This maneuver insures that [14C]PAH from the infusate is not sampled through the proximal RV catheter. Obtaining the blood from the proximal RV catheter within 5 s after clearing the infusion catheter insures that no dilution of [14C]PAH in the right ventricle (from recirculating blood) occurs. Catheter position is verified at autopsy. Calculations are performed to determine pulmonary blood flow. In 20 normal rats studied, the pulmonary blood flow was 24 +/- 1 mL/100 g body wt min-1 and in 12 endotoxin-treated rats (10 mg/kg body wt) the pulmonary blood flow was 32 +/- 2 mL/100 g body wt. These values are similar to values obtained with other methods used to measure total pulmonary blood flow.