Neuroendocrine responses to hypertonic saline/dextran resuscitation following hemorrhage.

The neuroendocrine responses to resuscitation with 7.5% hypertonic saline/6% Dextran-70 (HSD) following hemorrhagic hypotension were evaluated in conscious swine. Following hemorrhage (37.5 ml/kg/60 min) animals received 4 ml/kg of HSD (n = 6) or 0.9% saline (n = 8). Administration of normal saline did not alter cardiovascular function nor attenuate an increase in hormones. HSD rapidly improved cardiovascular function and acutely decreased ACTH, plasma renin activity (PRA), cortisol, norepinephrine (NE), epinephrine (E), aldosterone, and lysine vasopressin levels (LVP). The initial decreased in ACTH, cortisol, and aldosterone levels was due primarily to hemodilution associated with the expansion of plasma volume. The reductions in NE, E, LVP, and PRA were greater than those attributed to hemodilution alone. Values for LVP, NE, and E remained at values below those at the end of hemorrhage, but greater than basal levels, while PRA returned to values similar to these at the end of hemorrhage. The decrease in LVP, NE, and E following HSD resuscitation for the treatment of hemorrhagic hypotension may result from and contribute to the rectification of cardiovascular and metabolic function.

Blood gas and acid-base status of conscious pigs subjected to fixed-volume hemorrhage and resuscitated with hypertonic saline dextran.

Conscious, chronically instrumented pigs were subjected to a progressive, fixed-volume hemorrhage (37.5 ml/kg over 1 h) and subsequent resuscitation with 7.5% hemorrhage (37.5 ml/kg over 1 h) and subsequent resuscitation with 7.5% NaCl/6% Dextran 70 (4 ml/kg). Hemorrhage led to increases in arterial PO2, HbO2, plasma lactate, base deficit, and mixed venous PCO2. It led to decreases in arterial PCO2, plasma bicarbonate, and buffer base, as well as mixed venous PO2, HbO2, and pH. These effects were attributable to reduced O2 delivery, lactacidemia, hyperventilation, and hemodilution. Resuscitation with hypertonic saline/dextran produced a transient increase in arterial PCO2 and base deficit and a transient decrease in pH, effects that were attributable to a transfer of venous blood attributes to the arterial circulation. Resuscitation also produced an immediate decrease in arterial buffer base, an effect attributable to hemodilution. Subsequently, over 4 h, most cardiopulmonary and metabolic variables gradually reverted toward control levels, thereby ameliorating the deleterious blood gas and acid-base disturbances produced by severe hemorrhage.

Inhibition of methemoglobin and metmyoglobin reduction by cobalt.

Because cobalt compounds tend to form stable complexes, there has been continued interest in the use of the salts and chelates of cobalt in cyanide poisoning, and continued uncertainty about the precise nature of their protective effects. We have found that cobalt ions inhibit the enzymatic reduction of both methemoglobin and metmyoglobin. Virtually total inhibition of methemoglobin and metmyoglobin reductase activity occurred with the addition of 2.5 mM cobalt acetate to the assay system. Both enzymes were inhibited by lower levels of cobalt in a dose-dependent manner. The similarity in susceptibility of cobalt inhibition is further evidence that the enzymes which reduce methemoglobin and metmyoglobin are functionally comparable. The inhibition of methemoglobin reductase activity may be, in part, responsible for the therapeutic effectiveness of cobalt salts and chelates in cyanide poisoning.

Influence of dietary iron deficiency on hemoglobin, myoglobin, their respective reductases, and skeletal muscle mitochondrial respiration.

Male weanling rats were fed a control diet (46 ppm iron) or an iron-deficient diet (11 ppm iron) for 7 wk to determine the influence of iron deficiency on heme proteins and skeletal muscle mitochondrial respiration. At the end of 7 wk, the hemoglobin in the blood of the iron deficient rats was 35% less and skeletal muscle myoglobin was 20 to 37% less than in the control animals. The concentration of myoglobin in the heart was not appreciably diminished by iron deficiency. Cytochrome c concentration was 20% less in the heart and 35% less in the mixed-fiber gastrocnemius in the iron-deficient animals. Iron deficiency did not influence the activity of metmyoglobin reductase in either heart or skeletal muscle. There was about 30% more methemoglobin reductase activity in the red blood cells of the iron-deficient animals, which resulted in methemoglobin levels that were so low as to be virtually unmeasurable. In the iron-deficient rats, skeletal muscle mitochondrial respiration with either pyruvate-malate or palmitylcarnitine as substrate was 17 to 20% less than in the control animals. This study demonstrates that dietary iron deficiency of sufficient severity to reduce blood Hb and skeletal muscle myoglobin or cytochrome c also results in an impaired skeletal muscle oxidative capacity. The study also illustrates the preferential utilization of iron, not only between tissues, but within tissues, and tissue specific adaptive responses to iron deficiency.

The influence of exercise and diet on myoglobin and metmyoglobin reductase in the rat.

This study was designed to evaluate the influence of exercise and diet on selected aspects of heme protein metabolism in the rat. Two levels of treadmill exercise and three levels of dietary restriction were imposed on growing male rats over a 12-week experimental period. Neither training nor diet had any effect on erythrocyte NADH-MetHB reductase. The group undergoing the highest level of treadmill exercise had a significantly lower HB concentration. The activity of NADH-MetMB reductase was increased in the group undergoing the highest level of training and decreased in the groups whose diet was restricted by 25% and 35%. These changes were seen only in the soleus muscle. Other muscles, including the heart, psoas, and quadriceps were unaffected by either exercise of diet. Both levels of exercise were effective in increasing muscle MB concentration, but only in the quadriceps and soleus muscles. These data illustrate the adaptive nature of muscle MB and NADH-MetMB reductase. They also illustrate the different adaptive patterns of these two components of muscle.

Metmyoglobin reductase. Identification and purification of a reduced nicotinamide adenine dinucleotide-dependent enzyme from bovine heart which reduces metmyoglobin.

Beef heart muscle has been found to contain an enzyme which will rapidly and directly reduce metmyoglobin in vitro. Reduction rates are far greater than any previously reported for nonspecific or nonenzymatic systems. The enzyme is NADH-dependent and requires the presence of ferrocyanide ion for in vitro assay. The artificial electron carriers, dichlorophenolindophenol and methylene blue, are not required. Nonenzymatic reduction of metmyoglobin, which has previously been reported, was not encountered under the assay conditions described herein. Demonstration of enzymatic activity is dependent on a suitable myoglobin substrate, NADH, and ferrocyanide. An equimolar amount of cytochrome b5 was more effective than ferrocyanide in the enzymatic reduction of metmyoglobin. The methods for preparation of beef heart myoglobin and for purification of the enzyme are presented. The enzyme has been purified over 2000-fold. The enzyme has a pH optimum about 6.5 and a Km of 5.0 x 10(-5) M, and is unaffected by the absence of O2. Sodium dodecyl sulfate-gel electrophoresis revealed a molecular weight around 30,000. Purified enzyme does not react with lipoamide. The reaction is markedly influenced by the composition of the buffering milieu. Enzyme activity is inhibited by p-chloromercuriphenyl sulfonic acid, quinacrine dihydrochloride, and N-ethyl-maleimide. Activity was slightly stimulated by FMN. The characteristics of the enzymatic activity and the assay system are similar to those reported by Hegesh et al. (J. Lab. Clin. Med. 72, 339-344, 1968) for erythrocyte methemoglobin reductase.

Clinical implications of lactose-positive breast secretions in nonpuerperal females.

Inappropriate milk-like secretion from the breasts is not infrequently encountered in patients. Usually these secretions have been identified as milk on the basis of their characteristic appearance, and only rarely has their identity as milk been proven by appropriate analysis. Since milk is chemically defined as a lactose-containing mammary secretion, the presence of lactose in a breast secretion identifies it as milk irrespective of its appearance or the presence of other constituents. Lactose can be readily identified by thin-layer chromatographic methods, which despite their inherent speed and sensitivity have not been widely utilized. A method using commercially prepared cellulose layers and a sandwich developing apparatus is presented in detail. This method utilizes current advances in chromatographic techniques and permits rapid and accurate identification of lactose in breast secretions. Such secretions from 10 nonpuerperal women were screened for the presence of lactose. The milk-like secretions from 8 contained varying amounts of lactose, which was not detectable in the secretions from the other 2. There was no correlation between the appearance of the secretion and the presence of lactose.

PIP: A thin-layer chromatographic (TLC) method for confirming the presence of lactose in any breast secretion characterized as milk is presented. The chromatography apparatus and technique are described in detail. Refinements in TLC were achieved with the use of improved sorbent materials and the incorporation of the S chamber (sandwich chamber). Breast secretions from 10 nonpuerperal women were analyzed by TLC for the presence of lactose. Varying amounts of lactose were present in the milklike secretions of 8 patients, while no detectable amounts were present in the remaining two. Patients with lactose-positive breast secretions should be examined to determine the cause of galactorrhea.