Ronald H. Birkhahn

Urinary excretion of 3-methylhistidine: An assessment of muscle protein catabolism in adult normal subjects and during malnutrition, sepsis, and skeletal trauma

The urinary excretion of 3-methylhistidine (3 MEH) has been shown to be a reliable index of muscle protein breakdown. It is decreased in protein-calorie malnutrition and increased during the hypercatabolic phase of sepsis and thermal trauma. Losses of 3 MEH after moderate to severe skeletal trauma in man and animals are reported as increased or unchanged. To clarify this response, 24 male and 6 female skeletal trauma patients were evaluated for 24 hr urinary losses of 3 MEH, nitrogen and creatinine. Eight of the 24 males also received a catabolic steroid for treatment of a head injury. In addition, 3 male and 1 female septic patients were similarly evaluated. Controls consisted of 10 volunteers on a meat free diet for 4 days and of 8 volunteers who were given only intravenous 5% dextrose in water for 3 days. The 3 MEH excretion for all control males was 3.6 mumole/Kg/day and for females was 2.8 Skeletal trauma produced a 280% increase for the males and a 225% increase for the females. Trauma with steroids caused a 325% increase. Sepsis induced a 227% increase in 3 MEH losses for males and 292% for females during the febrile episode. Creatinine excretion also increased significantly in response to trauma and sepsis but the magnitude of the increase was less than for 3 MEH. This was reflected in the 3 MEH to creatinine molar ratio increase from 0.018 for controls to 0.030-0.040 in sepsis and trauma. Patients with extensive body weight loss showed decreases in 3 MEH and creatinine excretion and a molar ratio similar to controls. The calculated contribution of muscle protein to whole body protein breakdown in the trauma and septic groups showed a twofold increase compared to the control group. The data indicate that the increased muscle protein catabolic response following stress of skeletal trauma and sepsis provides an insight on the origin of the large urinary nitrogen losses following such insults.

A comparison of the effects of skeletal trauma and surgery on the ketosis of starvation in man.

The increase of plasma ketone bodies (acetoacetic acid and beta-hydroxybutyric acid) is related to the efficient protein-sparing adaptation during a total fast by healthy man. This study investigated the response to a total fast during the postinjury state. Twenty patients with skeletal or soft-tissue trauma received 3 days of carbohydrate-free intake and then 3 more days of carbohydrate intake. Control subjects were ten postoperative patients and two healthy volunteers who received similar nutritional treatment. The trauma patients lost nearly 20 gm of nitrogen/day, which was twice control, and had a resting energy expenditure of 27.07 kcal/kg, 21.4% greater than controls. Trauma was found related to an elevation in plasma glucose and to inhibit the rise in plasma ketone bodies and free fatty acids. In contrast, indirect calorimetry showed that fat contributed 63% of the nonprotein energy on the third day of fasting and injury. These data indicate that fat is utilized by the trauma patient but that fatty metabolism is abnormal compared to starvation in healthy or mildly stressed patients.

Inhibition of TPN-associated intestinal mucosal atrophy with monoacetoacetin

Total parenteral nutrition (TPN) is associated with intestinal mucosal atrophy. Acetoacetate is oxidized in preference to glucose by both enterocytes and colonocytes and is not present in TPN. The purpose of this study was to determine whether replacement of a portion of glucose calories with monoacetoacetin, the glycerol ester of acetoacetate, could inhibit TPN-associated intestinal atrophy. Male Sprague-Dawley rats (200-250 g) underwent superior vena caval cannulation and were assigned to receive chow ad libitium (CHOW), TPN with 0.86 M monoacetoacetin (ACAC), or TPN with 0.86 M glycerol to control for the glycerol component of monoacetoacetin (GLYC). Nitrogen balance was measured over 7 days after which time the animals were weighed and sacrificed. Jejunal and colonic segments were harvested and the mucosal weight, protein, RNA, and DNA contents measured. All groups showed comparable weight gain. Cumulative nitrogen balance was positive for both TPN groups. Significant decreases in mucosal parameters occurred in both TPN groups compared to the CHOW group, but atrophy was significantly inhibited in both jejunum and colon of the ACAC group compared to the GLYC group. Thus, the substitution of monoacetoacetin for glucose calories in parenteral nutrition solutions inhibited TPN-related atrophy of intestinal mucosa while maintaining normal growth.

The effect of major thermal injury on plasma ketone body levels.

Eleven patients with more than 30% total body surface burns were studied during 3 days of starvation and three more days of unrestricted feeding following their injury. All patients developed marked protein mobilization as demonstrated by 3rd day urine nitrogen excretion of 17.1 g daily compared to control excretion of 11.8 g N daily. As a group, the patients failed to mount the expected ketonemic response during their initial period of starvation. Whereas normal fasted controls achieved plasma ketone body levels of 727 +/- 81 mumol/liter, the burn patients responded with an average level of 385 +/- 77 mumol/liter (p less than 0.01).

Parenteral Monoacetoacetin and Liver Regeneration Interaction After Partial Hepatectomy in the Rat

Parenteral nutrients can be used to manipulate cell proliferation after partial hepatectomy. The relationship among macronutrients--glucose, monoacetoacetin, amino acids--and liver regeneration after partial hepatectomy was investigated. Male rats were anesthetized, received a 70% hepatectomy, and received a low-dose infusion of (1) glucose or (2) monoacetoacetin and a high-dose infusion of (3) glucose, (4) glycerol-glucose, or (5) monoacetoacetin-glucose beginning 6 hours after surgery. The five nonprotein nutrient combinations were infused with and without amino acids. Rats were killed 48 hours after partial hepatectomy, and the label and mitotic indices were determined. Each of the five treatments had a higher label index with amino acids present than with amino acids absent. Low-dose glucose and monoacetoacetin as well as high-dose glucose and glucose-glycerol had higher mitotic indices with amino acids than without amino acids. High-dose monoacetoacetin-glucose was associated with a greater mitotic index than was any other nonprotein substrate treatment, and this response was independent of amino acids being present or absent. In summary, (1) amino acids were needed for maximal cell proliferation rate; (2) the absence of amino acids and not the presence of glucose resulted in reduction of the label and mitotic indices for regenerating liver; (3) high-dose monoacetoacetin increased mitosis with or without amino acids; and (4) monoacetoacetin activity was dose dependent. The results indicate that the best nutrient for treatment of patients with liver injury is acetoacetate. The second best nutrient would be the combination of high-dose glucose and amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of ketosis and liver regeneration in the rat.

Monoacetoacetin, the monoglyceride of acetoacetate, was investigated as a nutritional support for the regenerating liver. Following partial hepatectomy, rats were either fed an oral diet ad libitum or administered by total parenteral feeding glucose alone, monoacetoacetin-glucose mixture, or lipid emulsion-glucose for the nonprotein calories. Five rats from each treatment were killed at 6-hr intervals beginning 12 hr after partial hepatectomy and ending at 72 hr. The number of cells synthesizing DNA and the number of cells in mitosis were compared. Rats fed orally or infused with glucose alone or with lipid emulsion had similar parameters throughout. Rats infused with monoacetoacetin had approximately double the number of cells in mitosis and DNA synthesis compared to the other treatments. This stimulation by monoacetoacetin persisted 72 hr. It was concluded from the data that acetoacetate was the agent responsible for increased DNA synthesis and mitosis, but the mechanism for the stimulation was not identified.

Isoleucine and Valine Oxidation following Skeletal Trauma in Rats

Nitrogen losses in the urine are derived from amino acid oxidation, and the increased loss of urinary nitrogen during stress indicates accelerated amino acid oxidation. This study compared isoleucine and valine oxidation by traumatized rats with that by pair-fed control rats. Seventy rats received bilateral hind limb fractures and were fed an oral liquid diet ad libitum, and 70 healthy rats were pair-fed with the trauma group. Daily food intake, body weight, and 24-hr urinary nitrogen were monitored for each animal, and isoleucine and valine oxidation were measured on days 1 through 7 postinjury using five rats from each group for each amino acid. Amino acid oxidation was determined from the percentage of dose appearing in the breath during 4 hours following a single injection of C-14 labeled amino acid. Anesthesia had a pronounced effect on all parameters on day 1, and its effects were dissipated by day 2. Skeletal trauma produced elevated urinary nitrogen losses that lasted for 5 days and peaked on day 3. Valine and isoleucine oxidation were increased for 5 days, and the peak increase occurred on day 3 post-trauma. These data show that isoleucine and valine oxidation parallel excessive urinary nitrogen excretion after skeletal trauma and that isoleucine and valine, like leucine, contribute to the increased urinary losses after trauma.

Potential of the monoglyceride and triglyceride of dl-3-hydroxybutyrate for parenteral nutrition: Synthesis and preliminary biological testing in the rat

Esters of short-chain organic acids have shown some promise as potential nutrients for parenteral feeding. Most glycerols are water insoluble but those of the ketone bodies have some water solubility. Of interest is that the triacylglycerol of 3-hydroxybutyrate has water solubility while the triacylglycerol of acetoacetate does not. The mono- and triacylglycerol of DL-3-hydroxybutrate were synthesized and tested for toxicity and nutritional value as parenteral nutrients. Both compounds have an estimated energy density of 19.7 kJ/g (4.7 kcal/g) and are water soluble. The compounds were infused into rats for 7 d at a rate to provide 113 kJ/d and were accompanied by a low-energy oral diet. Control, pair-fed rats were infused with isocaloric glucose or 0.9% saline. Nitrogen intake, output, and balance, body weight changes, and liver size were compared. The two glycerols of 3-hydroxybutyrate supported similar nitrogen retention, body weight changes, and liver size as found in the pair-fed control animals infused with glucose. Rats infused with saline retained less nitrogen, had decreased body weight, and had smaller livers. The data demonstrated that the glycerols of DL-3-hydroxybutyrate are not toxic, provide metabolic energy when infused intravenously, and could be used as nutrients for parenteral feeding.

The influence of ketosis on the metabolic response to skeletal trauma

Intravenous glucose and ketone body feeding were compared for their potential in altering urinary nitrogen losses by the traumatized rat. Eighteen male rats were traumatized by bilateral femoral fracture. The rats were fed totally by vein for 3 days prior and 3 days after injury and the infusion rate was held constant over the 6 days of infusion. Group GT rats were fed glucose as the source of nonprotein energy while group MT rats were fed a mixture of 72% monoacetoacetin (the monoglyceride of acetoacetate)-28% glucose for the nonprotein energy. Total urinary nitrogen excretion on a 24-hr basis was measured for each of the 6 days of intravenous feeding. On the third day post-trauma, each rat was evaluated for leucine kinetics using a continuous infusion of L-[1-14C]leucine and measurement of breath and plasma specific activities. Rats from group MT were hyperketonemic and normoglycemic and rats from group GT were normoketonemic and hyperglycemic. Urinary nitrogen losses, leucine oxidation, and leucine turnover were similar for the two groups. We conclude that ketone bodies are as good an intravenous source of energy as is glucose, and the ketone bodies do not cause hyperglycemia.

Evaluation of free fatty acid kinetics during TPN feeding of healthy rats

Free fatty acid (FFA) kinetics were evaluated in TPN-fed healthy rats using a single fatty acid tracer. Rats were divided into three groups according to the nonprotein energetic substrate infused: glucose (A), monoacetoacetin-glucose (B), and lipid emulsion-glucose (C). Fat kinetics were measured by continuous infusion of [1-14C]palmitate. Total FFA and individual FFA concentrations were measured and turnover and oxidation were determined for the total pool of FFA and for palmitate. Groups A and B were similar in all parameters. Group C had increased total and individual FFA concentrations. Group C appeared to have decreased total plasma FFA turnover and unchanged oxidation compared to groups A and B. Palmitate appeared to have a 400% increase in oxidation and a 50% increase in turnover for rats in Group C when compared to Groups A and B. It is concluded that a single tracer does not accurately reflect plasma FFA during TPN using lipid emulsion.