Melatonin and corticosterone regulation: Feeding time or the light:Dark cycle?

Under ad libitum feeding, male rats exhibit a marked rhythm of plasma and pineal melatonin; levels are low during the day and high at night. Restricting food availability to a 2 hour period during the light or dark does not markedly influence the melatonin rhythm, both groups having a crest in circulating melatonin during the dark. In contrast, plasma corticosterone levels are influenced by both the light-dark cycle and feeding. Animals fed early in the light period exhibit a biomodal corticosterone secretory pattern, with high steroid levels immediately prior to feeding and again just before lights-out, animals fed early in the dark have a single crest, just before food presentation. These data provide evidence for the dissociation of melatonin and corticosterone secretory patterns, providing support for the hypothesis that multiple regulators control neuroendocrine rhythmicity.     

Influence of bombesin, CCK, secretin and CRF on corticosterone concentration in the rat

The ingestion of food increases adrenoglucocorticoid secretion in humans and rats and influences the circadian periodicity of ACTH and corticosterone in rats fed on restricted schedules. The purpose of this study was to determine the influence of the brain-gut polypeptides CCK33 (10 U/kg), bombesin (10 micrograms/kg) and secretin (10 U/kg) on corticosterone concentrations in fed rats. The responses were compared to that of CRF (1 micrograms/kg). All experiments were begun at 10 a.m., 3 hours after the lights came on. The rats were given single, IP injections of peptide or vehicle (1 ml/kg) then sacrificed 0, 5, 10, 15, 30 or 60 minutes later. Corticosterone was measured fluorometrically. The control injection (vehicle) alone caused a mild stress response with corticosterone levels peaking between 10 and 15 minutes after the injection then returning to baseline. Both CCK33 and bombesin significantly increased corticosterone to approximately 2.5-fold above the control level in a fashion similar to that of CRF. In all three instances corticosterone levels peaked at 30 minutes post-injection. Secretin had no effect on corticosterone secretion. None of the peptides tested stimulated in vitro corticosterone output from isolated adrenal cells. These findings indicate that both CCK and bombesin cause pituitary-adrenal activation which may be related to the response of this system to food ingestion.     

Effects of fasting on aldosterone secretion in ovariectomized rats

The present study was designed to assess the effect of fasting on aldosterone secretion in ovariectomized (Ovx) rats. Ovx rats were divided into fed (allowed access to food ad libitum) and fasted (deprived of food for 24 hours) groups. The trunk blood of fed and fasted rats was collected after decapitation. In the in vitro study, adrenal zona glomerulosa (ZG) cells from fed or fasted rats were incubated with angiotensin II (Ang II, 10(-6) M), adrenocorticotropic hormone (ACTH, 10(-9) M), or forskolin (an activator of adenylyl cyclase, 10(-6) M) at 37 degrees C for 30 min. The levels of aldosterone in medium and plasma extracts were measured by radioimmunoassay. Results showed that the levels of plasma aldosterone in fasted rats were lower than those in fed rats. There were no significant differences in basal and Ang II-stimulated aldosterone secretion between fed and fasted groups. The increment of aldosterone induced by ACTH in fasted group was significantly less than that in fed group. Administration of forskolin led to a significant increase in aldosterone secretion in both fed and fasted groups. Fasted group had a decreased aldosterone secretion in response to forskolin as compared with fed group. In summary, these results suggest that fasting decreases aldosterone secretion in Ovx rats through a mechanism in part involving a reduction of aldosterone production in response to ACTH, a decreased activity of adenylyl cyclase, and/or an inhibition of post-cAMP pathway in ZG cells.     

Plasma Ghrelin Concentrations Were Altered with Oestrous Cycle Stage and Increasing Age in Reproductively Competent Wistar Females

Changes in appetite occur during the ovarian cycle in female mammals. Research on appetite-regulatory gastrointestinal peptides in females is limited, because reproductive changes in steroid hormones present additional experimental factors to control for. This study aimed to explore possible changes in the orexigenic (appetite-stimulating) gastrointestinal peptide hormone ghrelin during the rodent oestrous cycle. Fed and fasted plasma and stomach tissue samples were taken from female Wistar rats (32–44 weeks of age) at each stage of the oestrous cycle for total ghrelin quantification using radioimmunoassay. Sampling occurred during the dark phase when most eating takes place in rats. Statistical analysis was by paired-samples t-test, one-way ANOVA on normally distributed data, with Tukey post-hoc tests, or Kruskal-Wallis if not. GLM univariate analysis was used to assess main effects and interactions in ghrelin concentrations in the fed or fasted state and during different stages of the ovarian cycle, with age as a covariate. No consistent fed to fasted ghrelin increases were measured in matched plasma samples from the same animals, contrary to expectations. Total ghrelin concentrations did not significantly change between cycle stages with ANOVA, in either fed or fasted plasma or in stomach tissue. This was despite significantly decreased fasted stomach contents at oestrus (P = 0.028), suggesting decreased food intake. There was however a significant interaction in ghrelin plasma concentrations between fed and fasted proestrus rats and a direct effect of age with rats over 37 weeks old having lower circulating concentrations of ghrelin in both fed and fasted states. The biological implications of altered ghrelin plasma concentrations from 37 weeks of age are as yet unknown, but warrant further investigation. Exploring peripheral ghrelin regulatory factor changes with increasing age in reproductively competent females may bring to light potential effects on offspring development and nutritional metabolic programming.     

Circadian rhythm of corticosterone in diabetic rats

We proposed that the circadian rhythm of corticosterone in diabetic rats would have a different pattern than that in non-diabetic control rats. To test this hypothesis, 20 male Sprague-Dawley rats were given ad libitum access to a stock diet and housed individually in a light and temperature controlled room. Ten rats were made diabetic by two subcutaneous injections of streptozotocin. Ten rats which were not injected served as controls. Thirteen days after induction of the diabetes, tail blood samples were taken every 4 h for 24 h. Plasma corticosterone levels were significantly higher in diabetic rats than in control rats at 3 time points during the light cycle; however, concentrations were similar during the dark cycle. We speculate that diabetes may cause alterations in the steroid feedback mechanism to the hypothalamus and/or pituitary, resulting in an abnormal circadian rhythm of plasma corticosterone.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

Attenuated blood corticosterone rhythm in rats with jejunal resection

Circadian rhythmic changes in blood corticosterone concentration were studied in rats after resection of the jejunum or the ileum. The rats with ideal resection showed a normal corticosterone rhythm, with a peak at the beginning of the dark period when they were fed ad libitum, and the phase of the rhythm shifted when the feeding time was restricted to a specific time of day during the light period. The rats with jejunal resection also showed a similar corticosterone rhythm, but its amplitude was lower compared to that of the rats with ideal resection. There were no differences in body weight and the circadian rhythm of blood urea concentration between two groups of rats. We conclude that the jejunum is an important site where the sense of food is received as an entraining signal for the corticosterone rhythm.     

Entrainment of circadian rhythms of cholesterol‐LDL,corticosterone and motor activity by feeding schedules in rats

Abstract

The daily variations of locomotor activity, plasma and adrenal corticosterone levels and cholesterol‐LDL were studied in male Wistar rats with food ad libitum and feeding restricted to the first 4 hours of the light phase in LD 12:12..

Under LD 12:12 (light on from 9:00 to 21:00h) rats with food ad libitum were eating and moving during the dark period and the locomotor activity clearly showed a biphasic pattern with three harmonic components. Plasma and adrenal corticosterone levels increased during the light period and reached a maximum value just before the dark period whereas the acrophase of cholesterol‐LDL is found at the beginning of the light phase.

The acrophases of activity, plasma and adrenal corticosterone levels in the restricted feeding schedule rats occurred in the first three hours of lighting and the cholesterol‐LDL acrophase at the beginning of the dark phase.

These results confirm a previous report that the shift of feeding to the light phase seems to cause a concomitant phase‐shift in all the variables measured.     

Effect of food synchronization on the circadian rhythm of bone marrow and thymus lipids in rats

Young male Wistar rats reared under standard laboratory conditions with a 12:12 h light:dark regimen were fed ad libitum or were allowed access to food for only 2 h in the first half of the light or the dark part of the day. In rats fed ad libitum, marked circadian oscillation of the bone marrow triacylglycerol and phospholipid concentration and oscillation of the same fractions in the thymus were found. The restricted feeding time raised the triacylglycerol concentration in the bone marrow, but did not noticeably affect the time course of the circadian curves. The mean lipid values in the thymus of animals with a shortened feeding time did not alter, but the acrophase of the two basic fractions shifted.     

Effect of social isolation on 24-h pattern of stress hormones and leptin in rats

This work analyzes the effect of social isolation of growing male rats on 24-h changes of plasma prolactin, growth hormone, ACTH and leptin, and on plasma and adrenal corticosterone concentrations. At 35 days of life, rats were either individually caged or kept in groups (6-8 animals per cage) under a 12:12 h light/dark schedule (lights on at 08:00 h). A significant arrest of body weight gain regardless of unchanged daily food intake was found in isolated rats after 2 weeks of isolation. On the 4th week, rats were killed at 6 time intervals during a 24-h cycle, beginning at 09:00 h. In isolated rats the 24-h pattern of all parameters tested became distorted, as assessed by Cosinor analysis. When analyzed as a main factor in a factorial analysis of variance, isolation decreased plasma prolactin and growth hormone, increased plasma leptin and corticosterone while decreased adrenal corticosterone. Plasma corticosterone levels correlated significantly with plasma ACTH and with adrenal corticosterone levels in group-caged rats only. These changes can be attributed to an effect of mild stress on the endogenous clock that modulates the circadian hormone release.     

C-terminal fragments of ACTH stimulate feeding in fasted rats.

Peptides derived from pro-opiomelanocortin, including alpha-MSH and ACTH, play important roles in the regulation of feeding. We investigated the central effect of ACTH 1-39 (ACTH) and peptides derived from the N-terminus (ACTH 1-10, Acetyl-ACTH 1-13-amide [alpha-MSH]) and C-terminus (ACTH 18-39 and ACTH 22-39) of this peptide on feeding in 16 hour-fasted or rats fed ad libitum. As expected, ACTH reduced feeding in fed and previously fasted rats, although this anorectic effect was more pronounced in fasted rats. The N-terminal-derived peptide alpha-MSH, but not ACTH 1-10, reduced cumulative food intake over 2 h after its injection intracerebroventricularly (icv) in 16 h-fasted, but not in fed rats. In contrast, the C-terminal fragments produced a long-lasting increase in feeding in fasted, but not in fed rats. The anorectic effects of N-terminal fragments of ACTH are recognised to be mediated via melanocortin MC4 receptors. However, the orexigenic effects of the C-terminal fragments do not appear to be conducted via MC4 receptors, since neither ACTH 18-39 nor ACTH 22-39 stimulated cAMP accumulation nor inhibited the ACTH-stimulated cAMP accumulation in HEK-293 cells transfected with the recombinant MC4 receptor.     

Changes of salivary amylase in serum and parotid gland during pharmacological and physiological stimulation.

Although serum amylase level is an important diagnostic factor in certain salivary and pancreatic diseases, little information is available regarding the mechanism by which parotid amylase reaches the circulatory system. The present study was carried out to investigate the relationship between parotid isoamylase concentrations in blood serum and in parotid tissue in response to various stimuli. Wistar rats were fed with standard laboratory rodent chow; water was supplied ad libitum. In the first experiment, after a 16-h fasting, rats received either 5 mg/kg pilocarpine or saline (control). In the second study, after fasting, half of the rats were fed for 1 h, the other half received no food. In the third experiment, the changes in serum and tissue enzyme levels were monitored in freely fed animals during the peak-food intake phase, the first 2 h of the dark period. Amylase concentration was determined by using starch as a substrate. Pancreatic and parotid isoamylase levels in serum were separated by gel-electrophoresis utilizing differences in ionic properties of the isoenzymes. As expected, pilocarpine strongly stimulated tissue amylase discharge and serum amylase elevation. Similar, but less pronounced changes were observed not only during refeeding of fasted animals, but also in nonfasted rats during their peak-feeding period. Our data suggest that pharmacological stimulation, such as with pilocarpine or feeding in fasted state, as well as a mild stimulation of parotid function by spontaneous food intake during nonfasted state results in a decrease in parotid tissue amylase activity and a proportional increase in serum levels of parotid isoamylase.     

The effect of chronic food and water restriction on open-field behaviour and serum corticosterone levels in rats

In operant conditioning experiments, two methods are commonly used to motivate laboratory rats to perform designated tasks. The first is restricting food so that rats are forced to lose 20% of body weight within one week, followed by maintenance at 80% of the baseline weight for the remainder of the experiment. The second is restricting access to water to 15 min in each 24 h period. These methods are effective in motivating the animals. There is, however, little information available on the effects on performance in tests of behaviour that are not related to operant conditioning. In addition, it is not clear if these commonly used methods of food and water restriction will lead to physiological stress as indicated by an elevation of serum corticosterone. Male rats were either food-restricted to reduce and maintain their weight at 80% of baseline weight, or were restricted to 15 min access to water every 24 h. Activity in the open field was significantly greater in food-restricted rats than in water-restricted or control rats, but freezing behaviour was similar in all experimental groups. Food-restricted rats had a higher mean serum corticosterone level than water-restricted and control rats 37 days after the start of the experimental period. These data suggested that chronically restricting food and maintenance of body weight at 80% of baseline body weight led to significant behavioural changes and physiological stress. In contrast, water restriction did not lead to changes in behaviour or corticosterone levels. A second experiment was conducted to compare the effects of food restriction to 80% of baseline body weight, as described above, with a less stringent protocol in which test rats were initially reduced to 80% of baseline weight, but were then maintained at 80% of an ad libitum fed control rat's weight. Serum corticosterone levels and adrenal gland weights were measured after the initial week of forced weight loss and after maintenance for 21 days. Forced loss of 20% of body weight in the first week led to significantly increased serum corticosterone levels and adrenal gland weights compared to ad libitum fed controls. Serum corticosterone levels and adrenal gland weights in rats maintained at 80% of their initial body weight for 21 days remained higher than ad libitum fed control rats. However, rats maintained at 80% of an ad libitum fed control rat's weight did not differ from control rats in serum corticosterone levels or adrenal gland weights at the end of the 21-day study period. Adjustment of the feeding regimen in this manner eliminated physiological evidence of chronic stress.     

Influence of fasting on glycogen depletion in rats during exercise

We recently observed that a 24-h fasted group of rats could run longer than an ad libitum fed control group before becoming exhausted. Because of the demonstrated importance of glycogen levels and free fatty acid availability during endurance exercise, we have investigated several parameters of carbohydrate and lipid metabolism in exercised and nonexercised rats that were either fed ad libitum or fasted for 24 h. A 24-h fast depleted liver glycogen, lowered plasma glucose concentration, decreased muscle glycogen levels, and increased free fatty acid and beta-hydroxybutyrate concentrations in plasma. During exercise the fasted group had lower plasma glucose concentration, higher plasma concentration of free fatty acids and beta-hydroxybutyrate, and a lower muscle glycogen depletion rate than did the ad libitum fed group. Since fasted rats were able to continue running even when plasma glucose had dropped to levels lower than those of fed-exhausted rats, it seems unlikely that blood glucose level, per se, is a factor in causing exhaustion. These results suggest that fasting increases fatty acid utilization during exercise and the resulting "glycogen sparing" effect may result in increased endurance.     

Dietary supplementation with 2-deoxy-D-glucose improves cardiovascular and neuroendocrine stress adaptation in rats

Dietary restriction and physical exercise can enhance stress resistance and reduce the risk of cardiovascular disease. We investigated the effects of dietary supplementation with 2-deoxy-d-glucose (2-DG), a glucose analog that limits glucose availability at the cellular level, on cardiovascular and neuroendocrine responses to stress in rats. Young adult male Sprague-Dawley rats were implanted with telemetry probes to monitor blood pressure (BP), heart rate, body temperature, and body movements. These variables were measured at designated times during a 6-mo period in rats fed control and 2-DG-supplemented (0.4% 2-DG, fed ad libitum on a schedule of 2 days on the diet and 1 day off the diet) diets during unperturbed conditions and during and after immobilization stress or cold-water swim stress. Rats fed the 2-DG diet exhibited significant reductions in resting BP, attenuated BP responses during stress, and accelerated recovery to baseline after stress. Plasma concentrations of ACTH and corticosterone were elevated under nonstress conditions in rats fed the 2-DG diet and exhibited differential responses to single (enhanced response) and multiple (reduced response) stress sessions compared with rats fed control rat chow ad libitum. The 2-DG diet improved glucose metabolism, as indicated by decreased concentrations of blood glucose and insulin under nonstress conditions, but glucose and insulin responses to stress were maintained. We conclude that improvements in some cardiovascular risk factors and stress adaptation in rats maintained on a 2-DG-supplemented diet are associated with reduced neuroendocrine responses to the stressors.     

Endocrine function of neuropeptide Y knockout mice

Among its many proposed functions, neuropeptide Y (NPY) is thought to modulate the hypothalamic-pituitary axis. Specifically, increased hypothalamic NPY signaling may be critical in mediating the neuroendocrine response to fasting. To determine the consequences of NPY deficiency on endocrine physiology, multiple hormones were quantitated in wildtype and NPY-knockout mice under fed and fasted conditions. Serum concentrations of leptin, corticosterone, thyroxine, and testosterone were normal in NPY-knockout males fed ad libitum. A 48-hour fast resulted in a 50% reduction in leptin, a 60% reduction in thyroxine, a 75% reduction in testosterone, and a 12-fold increase in corticosterone in both wildtype and NPY-knockout mice. Fasting also increased the estrous cycle length by 3 days in both wildtype and NPY-deficient female mice. We conclude that NPY is not essential for appropriate function of the gonadotropic, thyrotropic, or corticotropic axes under ad lib fed conditions or in response to acute fasting.     

Effect of food restriction on cold adaptability of rats

To determine the role of the nutritional state in nonshivering thermogenesis during cold adaptation, cold adaptability was compared between cold-adapted (5 degrees C for 4-5 weeks) rats fed ad libitum and cold-adapted rats pair fed with warm controls having the same food intake. Cold-adapted pair-fed rats suffered a significant loss in body weight during cold exposure. However, brown adipose tissue (BAT) in both cold-adapted ad libitum fed and cold-adapted pair-fed rats was enlarged to the same extent as compared with that in control rats. Fat-free dry matter in BAT also increased in cold-adapted ad libitum fed and cold-adapted pair-fed rats to the same extent. Cold tolerance as assessed by the change in the colonic temperature at -5 degrees C was improved relative to control rats and was the same for cold-adapted ad libitum fed and cold-adapted pair-fed rats. Nonshivering thermogenesis as estimated by the noradrenaline-induced increase in oxygen consumption was significantly greater in the cold-exposed rats and there was no significant difference between cold-adapted ad libitum fed and cold-adapted pair-fed rats. These results suggest that an improved cold tolerance by means of nonshivering thermogenesis in brown adipose tissue is closely related to the low temperature itself but not the increased food intake which occurred in the cold.     

Short-term fasting affects locomotor activity,corticosterone, and corticosterone binding globulin in a migratory songbird

Unpredictable events such as severe storms lead to an increase in circulating corticosterone (CORT) in breeding birds. This increase is often accompanied by elevations in foraging and irruptive behavior. We were interested in determining if acute food restriction (such as might occur during inclement weather) is a sufficient cue to elicit an increase in locomotor activity, increase CORT secretion, and/or decrease circulating levels of corticosterone binding globulin (CBG) in white-crowned sparrows (Zonotrichia leucophrys gambelii). Male Z.l. gambelii were housed individually in environmental chambers on long days (LD 20:4) to simulate breeding season daylength. Birds were fed ad libitum, and on select days, food was removed 2 h after lights on (fasted treatment), or was removed and replaced (control). We analyzed CORT and CBG levels after 1, 2, 6, 22 (lights on), and 23 h under fasted and control conditions. We also measured activity during the 23-h experiment. Activity levels were increased under fasted conditions during the daytime relative to control conditions, but activity levels did not differ between treatments during the night. Fasting as little as 1, 2, and 6 h significantly increased total CORT levels above baseline (control), although after 22 h, total CORT levels under fasted conditions matched those under control conditions. Plasma CBG decreased after the 22-h fast, and remained low after the 23-h fast. This change was sufficient to significantly elevate free CORT levels in fasted birds relative to ad libitum food conditions, despite the lack of difference in total CORT levels.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Circadian rhythm of neurotensin levels in rat small intestine

The present studies were undertaken to determine whether a 24 h rhythm occurs in neurotensin (NT) levels in the small intestine of the rat and if so, whether the rhythm depends upon the 24 h cycles of light or feeding. A total of 145 male rats were sacrificed at 4 h intervals and the levels of neurotensin-like immunoreactivity (NTLI) in the middle 30 cm of small intestine were determined by radioimmunoassay with region specific antisera. There was a significant (P less than 0.05) 24 h rhythm in the levels of NTLI in groups of rats maintained under constant illumination or a 12:12 light:dark cycle and fasted for either 24 h or provided food ad libitum. Levels of NTLI ranged from 50 to 140 pm/g and were highest during the early morning (0400-0800 h) and lowest during the afternoon (1200-1600 h). The NTLI from samples taken at 0400 and 1600 h was subjected to high-performance liquid chromatography. The levels of chromatographically and immunochemically characterized NT were consistent with the levels of NTLI, evidence that the 24 h variation in NTLI most likely reflects changes in the intestinal content of NT and not other substances with similar immunochemical properties.