Improved methodology to assay carnitine and levels of free and total carnitine in human plasma

Carnitine, once known as vitamin Bt, is intrinsic to human tissue and is biochemically established as being acylated with fatty acids by Acyl-CoA to give Acyl-carnitines which then are transported to the inner mitochondrial membrane by a translocase. Carnitine is of increasing clinical interest and importance, and endomyocardial deficiencies of carnitine have been reported for patients in heart failure. Consequently, a reproducible and accurate analysis of human tissue specimens for levels of free carnitine and Acyl-carnitine to guide and to support continuing clinical studies of disease states is needed. We have devised an analytical method which utilizes 5,5'-dithiobis-2-nitro-benzoate and demonstrated recovery, reproducibility and precision. Hydrolysis of a specimen at 90 degrees C for 15 min, and control of pH below 6.0 are critical steps. The mean levels of free carnitine and total carnitine in 17 ordinary subjects were 50.6 +/- 9.7 nmol./ml and 62.6 +/- 11.7 nmol./ml. respectively.     

Effect of exogenous carnitine on carnitine homeostasis in the rat

The interaction of exogenous carnitine with whole body carnitine homeostasis was characterized in the rat. Carnitine was administered in pharmacologic doses (0-33.3 mumols/100 g body weight) by bolus, intravenous injection, and plasma, urine, liver, skeletal muscle and heart content of carnitine and acylcarnitines quantitated over a 48 h period. Pre-injection urinary carnitine excretion was circadian as excretion rates were increased 2-fold during the lights-off cycle as compared with the lights-on cycle. Following carnitine administration, there was an increase in urinary total carnitine excretion which accounted for approx. 60% of the administered carnitine at doses above 8.3 mumols/100 g body weight. Urinary acylcarnitine excretion was increased following carnitine administration in a dose-dependent fashion. During the 24 h following administration of 16.7 mumols [14C]carnitine/100 g body weight, urinary carnitine specific activity averaged only 72 +/- 4% of the injection solution specific activity. This dilution of the [14C]carnitine specific activity suggests that endogenous carnitine contributed to the increased net urinary carnitine excretion following carnitine administration. 5 min after administration of 16.7 mumol carnitine/100 g body weight approx. 80% of the injected carnitine was in the extracellular fluid compartment and 5% in the liver. Plasma, liver and soleus total carnitine contents were increased 6 h after administration of 16.7 mumols carnitine/100 g body weight. 6 h post-administration, 37% of the dose was recovered in the urine, 12% remained in the extracellular compartment, 9% was in the liver and 22% was distributed in the skeletal muscle. In liver and plasma, short chain acylcarnitine content was increased 5 min and 6 h post injection as compared with controls. Plasma, liver, skeletal muscle and heart carnitine contents were not different from control levels 48 h after carnitine administration. The results demonstrate that single, bolus administration of carnitine is effective in increasing urinary acylcarnitine elimination. While liver carnitine content is doubled for at least 6 h following carnitine administration, skeletal muscle and heart carnitine pools are only modestly perturbed following a single intravenous carnitine dose. The dilution of [14C]carnitine specific activity in the urine of treated animals suggests that tissue-blood carnitine or acylcarnitine exchange systems contribute to overall carnitine homeostasis following carnitine administration.     

Effect of exercise on plasma interferon levels

The effect of exercise on plasma interferon activity was studied on eight male subjects before and after exercise on a bicycle ergometer for 1 h at 70% of their maximal O2 consumption (VO2 max). Acid-labile interferon, alpha-type according to immunological characterization, rose significantly from a preexercise value of 3 +/- 1 to 7 +/- 2 IU/ml postexercise. Negligible changes were recorded for plasma protein, lipid, and glucose concentrations, whereas blood lactate slightly increased only at the end of exercise. According to hematocrit and plasma protein values before and after exercise, hemoconcentration did not occur. These data provide evidence that plasma interferon activity increased following a bout of submaximal exercise.     

Effect of glucose on plasma glucagon and free fatty acids during prolonged exercise

The effects of glucose ingestion on the changes in blood glucose, FFA, insulin and glucagon levels induced by a prolonged exercise at about 50% of maximal oxygen uptake were investigated. Healthy volunteers were submitted to the following procedures: 1. a control test at rest consisting of the ingestion of 100 g glucose, 2. an exercise test without, or 3. with ingestion of 100 g of glucose. Exercise without glucose induced a progressive decrease in blood glucose and plasma insulin; plasma glucagon rose significantly from the 60th min onward (+45 pg/ml), the maximal increase being recorded during the 4th h of exercise (+135 pg/ml); plasma FFA rose significantly from the 60th min onward and reached their maximal values during the 4th h of exercise (2177 +/- 144 muEq/l, m +/- SE). Exercise with glucose ingestion blunted almost completely the normal insulin response to glucose. Under these conditions, exercise did not increase plasma glucagon before the 210th min; similarly, the exercise-induced increase in plasma FFA was markedly delayed and reduced by about 60%. It is suggested that glucose availability reduces exercise-induced glucagon secretion and, possibly consequently, FFA mobilization.     

Effect of acute exercise on plasma neurotensin levels

Neurotensin (NT) levels were examined in five aerobically untrained females aged 20-36 engaged in acute graded exercise testing. In addition to radioimmunoassay measurements, high pressure liquid chromatography was performed to further characterize plasma NT-like immunoreactivity (NTLI). Epinephrine (E), norepinephrine (NE), and lactate (L) responses were also determined. Exercise testing consisted of one hour of treadmill running subdivided into three 20-minute segments representing 50, 60, and 70%, respectively, of the previously determined maximal aerobic capacity. Mock testing established baseline values for each subject. Three components of NTLI were evaluated: NT(1-13), NT(1-8), and NT(1-11). Resting NT(1-13) concentrations averaged 5.8 +/- 4.2 fmol/ml, while mean NT(1-8) values were 13.0 +/- 5.2 fmol/ml, and NT(1-11) averaged 5.8 +/- 3.2 fmol/ml. Peak exercise values were: for NT(1-13), 5.4 +/- 2.0 fmol/ml, for NT(1-8), 13.5 +/- 2.8 fmol/ml, and for NT(1-11), 5.9 +/- 0.5 fmol/ml. Analysis of variance with repeated measures detected no changes in these levels with exercise. Four-fold increases in E (36 +/- 3 pg/ml to 121 +/- 51 pg/ml), NE (340 +/- 95 pg/ml to 1431 +/- 319 pg/ml), and L (0.8 +/- 0.1 mM to 4.3 +/- 1.7 mM) confirmed the stress of exercise on the body in general, and the sympatho-adrenal system in particular. While other research has associated peripheral NT metabolite elevations with stressful stimuli in laboratory animals, the results of the present study suggest either that NT is not released from the human adrenal medulla during exercise, or that peripheral sampling precludes detection of any increases in NT from the adrenal medulla with currently available radioimmunoassay systems.     

Failure of exogenous insulin to inhibit insulin secretion in man

In order to explore whether or not the negative feedback mechanism of insulin per se on insulin secretion exists in man, changes in plasma C-peptide immunoreactivity (CPR), as an index of pancreatic B cells secretory function, were studied in 6 nonobese healthy volunteers in the presence of high circulating levels of exogenous insulin. 10% glucose was infused concurrently so as to maintain blood sugar at the basal level. The insulin-glucose infusion was maintained for 120 minutes, achieving mean plasma levels of 140-180 mu1/ml. After this period, the insulin infusion was continued at the same rate for an additional 10 minutes while the glucose was omitted. Despite the elevated level of circulating insulin, no significant change in plasma CPR concentration was observed so long as the blood sugar was maintained at the basal levels. Following cessation of the glucose infusion, the plasma CPR levels declined with a decrease in blood sugar level. Under the conditions of the present study, no inhibitory effect of exogenous insulin on the secretory function of the B cells was noticed.     

Comparison of the effect of semisynthetic human insulin and porcine insulin on glucose kinetics, plasma free fatty acid and amino acid levels in man

The effect of semisynthetic human insulin on hepatic glucose output, peripheral glucose clearance, plasma levels of C-Peptide, free fatty acids and amino acids was compared with purified pork insulin using the glucose clamp technique. 8 normal overnight-fasted subjects received intravenous infusions of either human or porcine insulin at 20 mU/m2.min(-1) during 120 min achieving plasma insulin levels of approximately equal to 50 mU/l. Plasma glucose levels were maintained at euglycaemia by variable rates of glucose infusion. Hepatic glucose production measured by continuous infusion of 3-(3) H-glucose was similarly suppressed by both insulins to rates near zero. The metabolic clearance rate of glucose increased during infusion of human insulin by 120%, C-peptide levels decreased by 41% and plasma FFA concentrations fell by 74%. The respective changes during infusion of pork insulin were similar, 118%, 48% and 72%. Both insulins decreased the plasma levels of branched-chain amino acids, tyrosine, phenylalanine, methionine, serine and histidine similarly. Thus, the results demonstrate that semisynthetic human and porcine insulin are aequipotent with respect to suppression of hepatic glucose output, stimulation of glucose clearance, inhibition of insulin secretion, lipolysis and proteolysis.     

Influence of carnitine supplementation on muscle substrate and carnitine metabolism during exercise

We examined 1) the effect of L-carnitine supplementation on free fatty acid (FFA) utilization during exercise and 2) exercise-induced alterations in plasma levels and skeletal muscle exchange of carnitine. Seven moderately trained human male subjects serving as their own controls participated in two bicycle exercise sessions (120 min, 50% of VO2max). The second exercise was preceded by 5 days of oral carnitine supplementation (CS; 5 g daily). Despite a doubling of plasma carnitine levels, with CS, there were no effects on exercise-induced changes in arterial levels and turnover of FFA, the relation between leg FFA inflow and FFA uptake, or the leg exchange of other substrates. Heart rate during exercise after CS decreased 7-8%, but O2 uptake was unchanged. Exercise before CS induced a fall from 33.4 +/- 1.6 to 30.8 +/- 1.0 (SE) mumol/l in free plasma carnitine despite a release (2.5 +/- 0.9 mumol/min) from the leg. Simultaneously, acylated plasma carnitine rose from 5.0 +/- 1.0 to 14.2 +/- 1.4 mumol/l, with no evidence of leg release. Consequently, total plasma carnitine increased. We concluded that in healthy subjects CS does not influence muscle substrate utilization either at rest or during prolonged exercise and that free carnitine released from muscle during exercise is presumably acylated in the liver and released to plasma.