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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 258-265
Copyright © 2004 by The Endocrine Society

Simultaneous and Continuous 24-Hour Plasma and Cerebrospinal Fluid Leptin Measurements: Dissociation of Concentrations in Central and Peripheral Compartments

Ma-Li Wong, Julio Licinio, Bulent O. Yildiz, Christos S. Mantzoros, Paolo Prolo, Mitchel Kling and Philip W. Gold

Center for Pharmacogenomics and Interdepartmental Clinical Pharmacology Center (M.-L.W., J.L., B.O.Y.), Neuropsychiatric Institute and David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California 90095; Division of Endocrinology (C.S.M.), Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; Clinical Neuroendocrinology Branch (P.P., M.K., P.W.G.), National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892; and Department of Psychiatry (M.K.), Baltimore Veterans Affairs Medical Center and University of Maryland School of Medicine, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Julio Licinio, M.D., Director, Center for Pharmacogenomics; and Interdepartmental Clinical Pharmacology Center, 3357A Gonda Center for Research on Genetics and Neuroscience, David Geffen School of Medicine at University of California, Los Angeles, 695 Charles Young Drive South, Los Angeles, California 90095-1761. E-mail: licinio{at}ucla.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The entrance of leptin into the central nervous system is of physiological relevance to the regulation of food intake, energy balance, and neuroendocrine function. To our knowledge, the relation between plasma and lumbar cerebrospinal fluid (CSF) leptin has not been examined across the 24-h period. To evaluate the relation between plasma and CSF leptin across the 24-h period, we studied simultaneous and continuous plasma and CSF leptin in nine subjects. We measured plasma and lumbar CSF leptin every 30 min for 24 h. All subjects had diurnal periodicity in 24-h plasma leptin levels, but not in CSF levels, as assessed by analysis of covariance and cosinor analysis. Plasma leptin had a significant 24-h pattern (P = 0.001), but CSF leptin did not. A 25-fold range of plasma leptin concentrations was reflected by a less-than-2-fold range in lumbar CSF leptin concentrations. Nocturnal increases in plasma leptin concentrations were not accompanied by commensurate changes in CSF values. It appears that leptin enters the brain by a mechanism that is highly saturable at physiological leptin concentrations and that the dynamics of plasma leptin is not accompanied by similar dynamics in CSF leptin measured at the lumbar spinal level. Therefore, it is not possible to use plasma leptin levels to predict lumbar CSF leptin concentrations. Although we acknowledge that lumbar CSF concentrations do not fully reflect the dynamics of leptin in the brain, we suggest that the nocturnal saturability of leptin transport into the central nervous system might help explain the lack of responsiveness to the central physiologic effects of leptin in states of hyperleptinemia, such as obesity.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LEPTIN, THE PRODUCT of the ob gene, is mainly synthesized by adipose tissue in proportion to percentage body fat and is released into circulation acting both in the brain and the periphery (1). Once released into the bloodstream, leptin must gain access to specific regions of the brain to exert its central effects. The entrance of leptin into the central nervous system represents a crucial step for the regulation of food intake and energy balance and may possibly occur by more than one pathway (1). The studies by Banks et al. (2), Schwartz et al. (3), and Caro et al. (4) suggest a saturable leptin transport system, providing evidence for impaired transport of leptin across blood-brain barrier (BBB) in obesity. Single time point leptin levels in the cerebrospinal fluid (CSF) correlate with plasma leptin levels in a nonlinear manner. In group comparisons, as plasma leptin levels become higher, the corresponding increase in CSF levels becomes smaller (3, 4). A circadian rhythm in the transport system of leptin at the BBB and partial saturation of this system at physiological leptin plasma concentrations have been reported in mice (5).

We have previously shown that human plasma leptin levels have significant ultradian and diurnal variations, which might be an important requirement for the biological effectiveness of the hormone (6). However, it has not been established what the effects of increases in endogenous plasma leptin are on the CSF compartment. It is still unknown whether there is a dynamic relation between plasma and CSF leptin levels in humans across the 24-h period. To evaluate the relation between plasma and CSF leptin concentrations, we studied simultaneous and continuous plasma and CSF leptin in healthy subjects and subjects with a diagnosis of major depression over 24 h. This type of invasive physiological study could only be done in a small number of volunteers hospitalized at the National Institutes of Health (NIH) Clinical Center.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Clinical research protocol

Using an Institutional Review Board-approved clinical research protocol, we studied nine subjects after informed consent was obtained. Of the nine subjects studied, four were normal volunteers [age, 38.4 ± 2.9 yr (range, 32.9–45.5); body mass index (BMI), 22.6 ± 2.2 kg/m2 (range, 19.9–29.0)], and five had a diagnosis of major depression melancholic type [age, 38.1 ± 4.8 yr (range, 28.2–52.1); BMI, 24.4 ± 3.1 kg/m2 (range, 17.3–35.2)], who were drug-free for at least 2 wk, participated in this study; there were no significant age or BMI differences between patients and controls (see Table 1Go). Healthy volunteers were examined by a clinician and had no physical illness by history and physical examination and no psychiatric illness by both clinical and structured interviews. Women were studied during the follicular phase of the menstrual cycle. Before actual sampling of CSF and plasma, all subjects were adapted to the hospital setting for at least one night. The diagnosis of major depression was made using structured interviews and followed both the criteria of the American Psychiatric Association [Diagnostic and Statistical Manual of Mental Disorders, 3rd edition, revised (DSM-III-R)] and the World Health Organization (7, 8). A minimum Hamilton Rating Scale for Depression of 15 or more was required for inclusion in this study (9).


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  		TABLE 1. Clinical characteristics of the subjects

 
Serial CSF and plasma sampling was conducted as previously described (10). All patients and volunteers were on a low monoamine diet for at least 3 d before CSF sampling. CSF sampling began, at 0900–1000 h, with the introduction of a standard 20-gauge epidural catheter through an 18-gauge Touhy spinal needle into the subarachnoid space at the L3/4 or L4/5 level. The catheter was attached to a constant infusion pump, and 6 ml/h was exfused at a constant rate. Sampling lasted for 30 h, at which time the catheter was removed; during CSF sampling, the subject remained flat in bed but was free to lie in any position (i.e. prone, supine, or decubitus). Subjects were kept awake in a lighted room from 0700 h until 2300 h. The room was kept dark from 2300 until 0700 h, during which time subjects slept. Even though they had been acclimated to the room for at least 2 d before the study, the insertion of a spinal catheter is an invasive procedure, and their sleep during the study period was more fragmented than usual. They had a light, regular, isocaloric diet divided into four meals: breakfast (0830 h), lunch (1230 h), dinner (1800 h), and evening snack (2100 h). The CSF collection procedure has previously been demonstrated to be a safe and reliable method to continuously sample CSF (11, 12, 13, 14). Samples (1 ml each) were removed from the refrigerator at 30-min intervals and placed on dry ice. Previous studies had shown human peptide hormones to be stable in CSF under these conditions (10). Blood was drawn through an indwelling iv catheter at 30-min intervals into EDTA-coated, prechilled tubes, and the plasma was separated and frozen for measurement of plasma leptin. Subjects remained at bed rest during the study and until at least the morning after withdrawal of the catheters. The 24-h time period starting at 1100 h was used for this study.

Assays

Total human leptin was measured by RIA, as described previously (6, 15). The sensitivity of the assay was 0.2 ng/ml. The RIA for CSF samples was performed by the same method, except that CSF samples (200 µl) were concentrated to a final vol of 50 µl using a Savant lyophilizer (Farmingdale, NY), and the CSF concentrate and antileptin antiserum were incubated at 4 C for 24 h before the addition of tracer leptin. The sensitivity of this modified RIA was 38 pg/ml (15).

Cosinor analysis

The presence of sinusoidal trends was tested with single and population cosinor as well as multiple components analysis (16), using ChronoLab (University of Vigo, Vigo, Spain) (17). This procedure fits the data to a cosine function of a fixed anticipated period and calculates an estimate of the midline estimating statistic of rhythm, which represents the mean value of the rhythmic function, amplitude (which is the difference between the peak and the midline estimating statistic of rhythm of the fitted curve), acrophase [the time lag expressed in radians from a reference tie point (in our case, midnight) of the top point in the fitted curve], and telophase (the time lag from the reference point of the lowest point in a fitted curve, which occurs exactly 12-h after the acrophase). The probability of rhythm was tested by an F test, according to the null hypothesis of zero amplitude. Tests for normality and independence of residuals and homogeneity of their variance were also used.

Prediction of CSF leptin concentrations

In a previous study, Schwartz et al. (3) proposed a nonlinear, saturable mechanism by which plasma leptin enters CSF, according to the equation: y = 0.0047x + 0.0404Loge x + 0.0486. That equation was derived from a multiple linear regression model. We applied that equation to our data and obtained, for each individual, estimated 24-h CSF levels. Actual and estimated CSF levels were compared, and the accuracy of the formula and its error rate were calculated, according to the following formulas: accuracy = (calculated/actual)·100; error = absolute value of (100 - accuracy). Accuracy reflects the percent variation between predicted and actual measurements, 100 indicating that predicted and actual values were the same. Because, within a time series, underestimations and overestimations of values might cancel each other out, the error rate was calculated as the absolute value of the difference between predicted and actual CSF leptin concentrations.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
All subjects exhibited diurnal periodicity in 24-h plasma leptin levels, but not in CSF levels, as assessed by both analysis of covariance (ANCOVA) and cosinor analysis (Fig. 1GoGo). Table 1Go shows the demographics of each subject. All data for the nine subjects are presented individually (see Figs. 1–3GoGoGo and Tables 1–4GoGoGoGo). Because there was no significant effect of diagnosis on plasma or CSF leptin concentrations (see Figs. 4–5GoGo), we group all subjects.



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  		FIG. 1. Raw plasma and CSF leptin concentrations (first and second columns, respectively), and plasma:CSF leptin ratio in nine subjects.

 


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  		FIG. 1A. Continued.

 


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  		FIG. 2. Linear regression between 24-h average plasma leptin concentrations and the error between predicted and actual leptin concentrations when the equation of Schwartz et al. (3 ) was used.

 


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  		FIG. 3. Linear regressions between (A) 24-h average plasma and CSF leptin concentrations, (B) 24-h average plasma leptin concentrations and plasma:CSF leptin ratio, and (C) 24-h average CSF leptin concentrations and plasma:CSF leptin ratio. NS, Not significant.

 

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  		TABLE 2. Comparison of individual 24-h averages of plasma and CSF leptin concentrations1

 

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  		TABLE 3. Cosinor analysis of plasma leptin concentrations

 

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  		TABLE 4. Cosinor analysis of CSF leptin concentrations

 


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  		FIG. 4. Plasma (A) and CSF (B) leptin for the nine subjects are expressed as percent variability and aggregated in 4-h intervals. ANCOVA shows a significant time effect in (A) but not in (B). Twenty-four-hour single cosinor analysis of plasma (C) and CSF (D) leptin variability expressed as polar graphs, in which the radius represents the amplitude and the angle depicts the acrophase. As it is apparent from amplitude values, only plasma leptin (C) shows a statistically significant fitted period (P < 0.001).

 


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  		FIG. 5. The plasma:CSF ratio for the nine subjects is expressed as percent variability and aggregated in 4-h intervals. There is a significant time effect.

 
The range for average 24-h plasma leptin concentrations was far greater than for lumbar CSF leptin concentrations (Table 2Go). The range for plasma leptin concentrations was 1.01–25.32 ng/ml, whereas that for CSF leptin concentrations was 0.0563–0.0940 ng/ml. Thus, a 25-fold range of plasma leptin concentrations was reflected by a less-than-2-fold range in lumbar CSF concentration. These data provide further support to the hypothesis that leptin enters the brain by a saturable transport mechanism. The formula proposed by Schwartz et al. (3) has a reasonable error of under 25% for leptin values under 2.5 ng/ml. However, as leptin concentrations increase, so does the error rate. We found a highly significant linear correlation between the plasma leptin concentrations and the error rate between predicted and actual leptin concentrations when that equation was used (r = 0.92, P < 0.0005): the higher the average 24-h plasma leptin concentrations, the higher the error (Fig. 2Go). Thus, the level of saturability of leptin transport into CSF appears to be higher than previously estimated.

Figure 3Go shows that there was no correlation between 24-h average plasma and CSF leptin concentrations (A). There was a highly significant correlation between plasma (B) but not CSF, leptin concentrations, and plasma:CSF ratio, indicating that that ratio is reflective of plasma but not CSF leptin concentrations. This further supports the concept of a highly saturable transport mechanism for leptin to enter the brain, even within the physiologic nocturnal rise of leptin.

Visual inspection of the raw data of plasma and CSF leptin concentrations and plasma:CSF ratio (Fig. 1GoGo) shows that, whereas plasma leptin has an expected pattern of circadian variability, CSF leptin does not. The plasma:CSF ratio also has a circadian variability. Cosinor analysis, which represents a linear reduction of sinusoidal regression, was performed by ChronoLab freeshare software for the Macintosh (http://www.tsc.uvigo.sp). It showed a very significant probability of circadian rhythm (P < 0.001), with a mean acrophase at 2319 h for leptin plasma values, whereas no rhythmicity was apparent for CSF levels (Tables 3Go and 4Go and Fig. 4Go). By using ANCOVA analysis, we did not find any association between plasma and CSF leptin levels across all subjects and time, nor were the 24-h concentrations of leptin in this small group of subjects correlated to the BMI; although, as previously reported, mean plasma leptin concentrations were higher in women than in men (mean ± SEM, 10.97 ± 9.03 vs. 3.25 ± 3.59 ng/ml). Figures 4Go and 5Go and Tables 3Go and 4Go show those results in more detail. Plasma leptin has, in each subject, a highly significant probability of rhythm (Table 3Go and Fig. 4CGo). In contrast, CSF leptin does not show a significant probability of rhythm in our subjects (Table 4Go and Fig. 4DGo). When the data were expressed as percent individual variability and averaged for all subjects in 4-h intervals, starting at 1100 h, it becomes apparent that plasma leptin concentrations have a clear and statistically significant 24-h pattern (P = 0.001), with a nadir of leptin levels during the 1100- to 1500-h period and highest levels during the 2300- to 0300-h period (Fig. 4AGo). Moreover, ANCOVA showed that plasma leptin levels during the 1100- to 1500-h period were lower than any other period, whereas no significant variation emerged for CSF values (Fig. 4BGo).

The ratio of plasma:CSF leptin also showed a significant circadian variation similar to that of plasma leptin (Fig. 5Go). Thus, in the evening, the proportion of plasma leptin required to maintain a certain CSF level is increased. This would indicate that, within subjects, the nocturnal rise of leptin is already within the overall saturable range, in terms of plasma-CSF leptin transport.


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
Our results show that, whereas plasma leptin concentrations varied 25-fold among our subjects, CSF leptin concentrations exhibited only a 2-fold range of variation. Thus, high plasma leptin concentrations were not accompanied by proportionately high CSF values. This is consistent with the findings of Caro et al. (4), using single time point measurements. Human and rodent studies indicate that the ability of leptin to cross the BBB is impaired because of saturation of leptin transport system at physiological concentrations (18). Our findings expand considerably those earlier observations by demonstrating that, within subjects, as leptin levels increase at night (exhibiting a typical diurnal rhythm), CSF leptin concentrations do not. This indicated to us that, within each individual, there might be saturability of leptin entry into the brain at the daytime concentrations, and that, as plasma leptin concentration rise at night, CSF concentrations do not rise in a commensurate manner and therefore do not exhibit the marked increase that would be needed to characterize a circadian rhythm. Such a hypothesis should be tested by future studies of plasma-to-CSF dynamics in appropriate animal models. It appears that leptin enters the brain by a mechanism that is highly saturable at physiologic diurnal leptin concentrations and that the marked dynamics of plasma leptin concentrations are not accompanied by similar dynamics in CSF leptin concentration. The data might help explain why individuals with relatively high leptin levels do not undergo weight loss that would be expected as the result of the central effects of leptin.

A previous study has proposed a multiple linear regression model, defined by the equation (3). We show here that such a model was not able to predict CSF leptin concentrations. The accuracy of that model was 204.3 ± 42.5%, and its error rate was 113.7 ± 40.0%, ranging from 12.6 to 404.1%. The error rate increased in direct proportion to plasma leptin concentrations, indicating that the saturability of the plasma:brain leptin transport system is far greater than previously estimated.

In previous studies (1, 2), we have found highly significant association between the nocturnal rise of leptin and the decrement of pituitary-adrenal function at night and the change of LH pulsatility in women from high frequency/low amplitude to low frequency/high amplitude in the mid-to-late-follicular phase of the menstrual cycle. If CSF leptin concentration does not go up at night, how could these associations be explained? It is possible that the endocrine effects of leptin occur in very discrete areas of the brain (such as specific hypothalamic nuclei) as well as in peripheral glands (such as the anterior pituitary, the adrenal, and the ovary). We measured leptin in CSF collected at the spinal level. This represents an integration of the total rate of leptin entry into the brain. It is possible that a putative active transport mechanism might make leptin available to specific brain areas, such as the hypothalamus, in a pattern that more closely reflects its dynamics in the circulation. Banks et al. (2, 19) showed in mice that leptin radioactively labeled with 125I crossed BBB by a saturable mechanism and transported into the whole brain, but uptake at the hypothalamic region was especially high. A high uptake into the hypothalamus has been confirmed in rats (20). Pan et al. (5) have shown in mice that the rate of leptin’s transport from blood to brain has a diurnal rhythm. There was no correlation between the rhythms of influx rate and blood concentrations of leptin in this study. Differences in regional uptake and saturation provide a mechanism by which leptin can control events mediated at the arcuate nucleus, and other regions of the central nervous system with different regional thresholds for optimal function (19) and CSF concentrations of leptin may not correlate with actual hypothalamic leptin levels. The testing of the hypothesis that the BBB in specific hypothalamic nuclei might have a more efficient transport system for leptin, in comparison with other brain regions in humans, should be accomplished in future studies aimed to measure leptin receptor isoforms in vascular beds of specific brain regions. Such work, if done in humans, would face a high threshold of technical and ethical limitations.

This study was itself very limited in its scope because of the high level of intervention required and because of the invasive nature of continuous CSF collection. For that reason, our number of subjects is limited. A detailed analysis by subgroups, including diagnosis, gender, ethnicity, and adiposity, would be highly speculative because of the small subject numbers that would be contained in each cell. Previous data from our group and others indicate that sample sizes of at least 15 subjects per group are needed to detect differences at an {alpha} of 0.05 or greater. Statistically meaningful relationships between physiological variables are not likely to be observed with group sizes less than 10. Using Bartko’s nomogram (21, 22), assuming a large effect size (standardized difference of 1.0), a detection power of .8 requires a sample size of 15 subjects per group. Even though we did not see an effect of diagnosis, gender, ethnicity, or adiposity, we must emphasize here that this study was not powered to examine the effects of those variables. We therefore felt it would be more informative to focus on the issue of the dissociation of 24-h plasma and CSF leptin concentrations, which occurred in all subjects, and is relevant to the understanding of the dynamics of leptin in humans.

Leptin can also be synthesized by the brain and pituitary. This was first demonstrated by Morash et al. (23), who used RT-PCR to show that leptin mRNA is selectively transcribed in specific areas of rat brain and pituitary and in a rat glioblastoma cell line. They also used immunocytochemistry and Western blot to show evidence of protein expression in the corresponding tissues. Such expression was suppressed in the hypothalamus by fasting, which suggests a possible physiological role by leptin synthesized within the brain. Central and pituitary leptin expression is developmentally regulated in rats, increasing in cortex after birth (postnatal d 14–28) and having highest levels in pups on postnatal d 7–14 (24). Jin et al. (25) showed that leptin gene expression by RT-PCR and protein by immunohistochemistry was present in 20–25% of anterior pituitary cells and was expressed in most normal anterior pituitary cells, including ACTH (70% of ACTH cells), GH (21%), FSH (33%), LH (29%), TSH (32%), and folliculo-stellate cells (64%). In addition, leptin expression was detected by RT-PCR in some pituitary tumors, including ACTH, GH, null cells, and gonadotroph tumors as well as in normal pituitary. The findings indicate the possible existence of an autocrine/paracrine loop in the production and regulation of leptin in the pituitary. Despite these interesting results, it is believed that the principal source of leptin in adult humans is the peripheral white fat cell. Our results have to be interpreted with the caveat that what we measure in lumbar spine is the combination of leptin synthesized peripherally, in possible combination with a smaller amount of centrally secreted leptin.

An additional potential confounding variable is that leptin exists in bound and free forms. This work is completely focused on total leptin concentrations. Future studies should address the relative contributions of free and bound fractions to total leptin concentrations in plasma and CSF.

The role of leptin in human biology remains to be fully elucidated. Although genetically based deficiency of leptin is associated with a morbid obesity phenotype (26), most obese individuals have high plasma leptin concentrations. The reasons why such elevated plasma leptin concentrations do not lead to suppression of food intake, increase in energy expenditure, and normalization of body weight remain elusive. Early research in this field, using single time point measurements in the morning, suggested that obesity might be associated with decreased plasma-to-CSF leptin transport (4). Our findings lead us to expand this concept. We show that as leptin concentrations increase at night, the target organ of leptin’s effects, the brain, is not necessarily exposed to a commensurate increase in leptin concentrations. However, the interpretation of these results must be tempered by the fact that our data result from lumbar CSF measures that might not reflect the dynamics of leptin concentrations in the brain. Keeping this limitation in mind, we suggest that the high saturability of plasma-to-CSF leptin transport at night, within the physiologic range of leptin concentrations, might explain why increases in plasma leptin concentrations, such as those observed in obesity, may not lead to commensurate increased central effects of leptin on body weight and energy expenditure.


   Footnotes
 
This work was supported by NIH Grants RR017365, MH062777, RR000865 (to M.-L.W.), GM61394, HL04526, RR16996, HG002500, RR017611, DK063240, DK58851 (to J.L.), and DK58785 (to C.M.) and by awards from the Dana Foundation, National Alliance for Research on Schizophrenia and Depression, and Amgen, Inc.

Abbreviations: ANCOVA, Analysis of covariance; BBB, blood-brain barrier; BMI, body mass index; CSF, cerebrospinal fluid.

Received July 23, 2003.

Accepted September 29, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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U. Meier and A. M. Gressner
Endocrine Regulation of Energy Metabolism: Review of Pathobiochemical and Clinical Chemical Aspects of Leptin, Ghrelin, Adiponectin, and Resistin
Clin. Chem., September 1, 2004; 50(9): 1511 - 1525.
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