1 Obesity Research Center, St. Luke's-Roosevelt Hospital, Columbia University College of Physicians and Surgeons, New York, New York 10025; and 2 Exercise Physiology Laboratory, School of Education, The Flinders University of South Australia, Adelaide 5000, Australia
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ABSTRACT |
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Although the study of human body composition is advancing rapidly, confusion still prevails regarding the molecular-level lipid component. Most molecular-level body composition models are presently based on the overall hypothesis that nontriglyceride lipids constitute an insignificant proportion of total body lipid. A single lipid or "fat" component consisting of triglycerides is thus assumed in most molecular-level body composition models. To test this hypothesis, the present study, carried out in adult rats, was designed to examine two questions: 1) What is the proportion of total lipids as triglycerides? and 2) Is this proportion constant or does it change with negative energy balance and weight loss produced by calorie restriction and increased exercise? Results indicated that with negative energy balance and weight loss there were progressive losses of total body triglyceride and lipid. The proportion of total lipids as triglyceride was 0.83 ± 0.08 (SD) in control animals, with reductions at 2 and 9 wk of energy restriction [0.82 ± 0.04 (P = NS vs. control) and 0.70 ± 0.15 (P = 0.05)] and at 9 wk for energy restriction plus exercise [0.67 ± 0.09 (P = 0.003)]. Nontriglyceride lipids comprised 2.8% of carcass weight at baseline and decreased to 2.2% by 9 wk of energy restriction and exercise (P = NS). Substantial differences were observed between body composition ratios expressed as percentages of the lipid-free body mass (LFM) and triglyceride-free body mass (TGFM); (e.g., total body water/LFM and TGFM in controls = 72.7 ± 0.7 and 70.4 ± 2.2, respectively; P = 0.02). These observations strongly support the existence and importance of nontriglyceride lipids as a body composition component that responds independently from storage triglycerides, with negative energy balance produced by food restriction and exercise.
total body fat; adipose tissue; animal models
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INTRODUCTION |
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LIPIDS represent a diverse group of chemical compounds that in most mammalian species comprise a large fraction of body weight (14). Lipids are usually classified according to physical or chemical properties, and the main recognized groups include triglycerides, phospholipids, sphingolipids, cholesterol, and waxes (8, 14). An alternative classification is to divide lipids into two groups, those that are essential for sustaining life and those that are nonessential (8, 14). Essential or "structural" lipids include phospholipids, sphingolipids, cholesterol, and other lipid species that are necessary for maintaining cellular integrity, cell membrane fluidity and function, and other indispensable processes. The nonessential lipids in rodents, humans, and other mammals are made up almost entirely of triglycerides (8, 14).
Lipids all share in common solubility in nonaqueous solvents such as diethyl ether, petroleum ether, acetone, and methanol (8). Nonpolar lipids such as triglycerides are usually bound in tissues by weak Van der Waals forces or hydrophobic bonds and can be extracted with ether and other nonpolar solvents (14). More polar lipids, such as phospholipids, may in part be bound to proteins by hydrogen bonds and electrostatic associations that require polar solvents such as acetone and methanol for disruption and extraction. The nature of tissue-lipid isolates is therefore directly related to the employed solvents and associated tissue preparation procedures.
Substantial confusion surrounds the concept of lipid as a human body composition component (14). Various terms are applied to the lipid-solvent extractable material observed in vivo, including "lipids," "fat," and "essential" and "nonessential lipids." Another common problem is that the term fat is often used in reference to two different but closely related components, lipids and adipose tissue.
Several widely used body composition methods rely on lipid-related assumptions, and the prevailing confusion creates problems ranging from inaccurate body composition models to incorrect terminology in research reports. For example, the classic molecular-level body composition model includes five components: fat, protein, water, mineral, and glycogen (16). It is not always clear in this model whether fat represents triglycerides or total lipid. If triglycerides are considered fat, then what provision is made in the model for nontriglyceride lipids?
In the classic studies by Pace and Rathbun (22, 25) of total body water and other components in guinea pigs, fat was the total petroleum ether-extractable material from whole carcass. The "fat-free body mass" in this study therefore contained some lipids such as sphingomyelin, a compound that is soluble in hot absolute alcohol and insoluble in ether, acetone, and water (14). The extraction method employed by Rathbun and Pace thus does not allow us to precisely ascertain whether the classic ratio of total body water to fat-free body mass of 0.732, compiled by the investigators for several species in addition to the guinea pig, is the ratio of total body water to triglyceride or lipid-free mass (28).
As another example of the prevailing confusion, the classic underwater weighing method is based on the two-compartment molecular-level model that consists of fat and fat-free body mass. This method assumes that fat has a density of 0.9000 g/ml, which is based on ethyl ether-extractable material from adipose tissue (10, 21). The material obtained from adipose tissue extraction with ethyl ether as a solvent is almost entirely triglyceride, and, accordingly, nontriglyceride lipids are not included in the fat component. The underwater weighing method of fat-free body mass makes no provision for nontriglyceride lipids and thereby assumes their amount is negligible or that they do not contribute measurably to body density.
The current study is the first in a series aimed at expanding knowledge of the lipid portion of body mass with the purpose of clarifying some of the above-mentioned ambiguities. We selected the rodent model as one that allows complete carcass analysis and an opportunity for experimentally manipulating energy and lipid balance by dietary and exercise interventions.
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METHODS |
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Study Hypothesis
Two specific questions were examined in the present study: 1) What is the proportion of total lipids as triglycerides in the adult female rat? and 2) Is this proportion constant or does it change with negative energy balance and weight loss produced by calorie restriction and increased exercise? These questions are based on the overall hypothesis that nontriglyceride lipids constitute an insignificant proportion of total body lipid. If the hypothesis is inaccurate and there is a large nontriglyceride lipid fraction, and if this component behaves with interventions in a manner distinct from triglycerides, it would suggest an expanded role for these lipids in molecular-level body composition models and in future body composition studies.The two main lipid-related questions were explored in experimental animals before and after energy deficits that varied in duration and magnitude. Other body composition components were also estimated to explore their relationship to lipid components during the experimental protocol phases with varying levels of negative energy balance.
Protocol
The experimental procedure involved two phases, an 8-wk weight gain phase followed by a 9-wk food restriction-weight loss phase (Fig. 1). During the weight gain phase, rats were fed a defined diet that provided 45% of total energy as fat. At the end of the 8-wk weight gain phase, rats were divided into four weight-matched groups. One group was euthanized to serve as the baseline control group (n = 7). During the weight loss phase, the remaining three groups of animals were food restricted by placing them on an energy intake equal to 75% of the energy intake at the end of the ad libitum period. The diet was low in fat (12% of total energy) and meals were provided twice daily. The three groups of animals were randomly assigned to the restricted diet for 2 (n = 5) or 9 (n = 8) wk and an additional group to restricted diet plus exercise (n = 7). The exercise group swam three times a week at 1 PM for up to 35 min.
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Female Sprague-Dawley rats were housed individually in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. Rats were fed twice per day, at 9 AM and 4 PM. Body weight was recorded three times per week. Animals were euthanized at the end of 2- or 9-wk protocols, and carcass weights were recorded after removal of hair and gastrointestinal contents.
Analytic Methods
Tissue preparation.
Euthanized carcasses were frozen at 10°C. At study
completion, 500 ml of distilled water were added to the frozen
carcasses, which were placed in a preweighed beaker and heated to
125°C for 60 min. The cooled carcasses were homogenized using a
large-bore polytron (PT6000; Brinkman Instruments, Westbury, NY) for
7-10 min, and a 45-ml aliquot was then stored at
10°C.
Total body lipid. The following procedure for total carcass lipid purification is based on previously established methods of lipid purification (3, 24). Approximately 1 g of carcass homogenate and 27 ml of methanol were added to an Erlenmeyer flask. The sample was stirred, 14 ml of chloroform were added at 30 min, and the mixture was set aside overnight. Samples were run in triplicate.
Clean conical Teflon-lined glass capped tubes were heated to 90°C and weighed after cooling to room temperature. The homogenate mixture was filtered into the preweighed tubes, and the flask was rinsed with 2:1 chloroform-methanol. The volume of liquid in the tubes was then compared with a preprepared capped tube of known volume (21 ml). The volume of sample was then made up with 2:1 chloroform-methanol, and 4.2 ml of 0.88% KCl solution were added as the wash (3, 14, 24, 31). The liquid mixture was homogenized for ~20 s, and samples were then centrifuged at 1,500 rpm for 10 min. The top layer was aspirated and discarded, and the tubes were placed in a 55-60°C water bath until volume decreased to ~4 ml. Tubes were then dried in an oven at 90°C for several hours until only an oily lipid layer remained. Tubes were then cooled, and lipid weight per sample was obtained by calculating the difference between the conical tube weight with and without lipid. Triplicate values of total lipid weight per gram of homogenate were averaged and multiplied by carcass weight to obtain total body lipid.Total body triglyceride. To prevent autoxidation of the lipids, a solution of 15 mg of butylated hydroxytoluene (BHT), an antioxidant, in 200 ml chloroform was prepared and used as the storage solvent (7, 14, 31). Four milliliters of BHT in chloroform solution were added to the extracted lipid, and the solution was then poured into glass vials. Nitrogen gas was infused into the vials, which were then capped (7, 14, 31).
The procedure for triglyceride quantification was adapted from the colorimetric serum triglyceride assay kit of Diagnostic Chemicals (Oxford, CT). The kit contained a triglyceride standard of 2 mM triolein (177.08 mg/dl). Two additional standards were prepared by using corn oil purified by first mixing with Zeolite (Sigma Chemical, St. Louis, MO). The high standard contained 3.31 mM (293.3 mg/dl) purified corn oil, and the low standard contained 0.99 mM (87.73 mg/dl) purified corn oil. Samples for the triglyceride assay were prepared in dilutions so that the estimated triglyceride concentration (~80% of total lipids) would be close to that of the standard solution.Other body composition components. Total body water was determined by drying duplicate 1-g samples of homogenate overnight at 90°C to stable weight. Total body nitrogen was estimated by adjusting homogenates to pH 2 with hydrochloric acid and then measuring nitrogen with an adaptation of the Kjeldahl method (23). Protein was calculated by assuming a nitrogen-to-protein ratio of 0.16 (19). Total body ash was measured by placing ~3 g of well-stirred carcass homogenate in crucibles. The crucibles were then heated to 90-95°C overnight in a drying oven, placed into a muffle furnace for ~4 h at 600°C, and cooled, and the ash weight of the sample was then determined. Water, protein, and ash contents per gram of homogenate were multiplied by carcass weight to obtain total body water, protein, and ash, respectively.
Statistical Methods
The study hypothesis was tested by examining the triglyceride-to-total body lipid ratio with progressive levels of energy deficit. Specifically, the hypothesis states that no statistically significant change in the triglyceride-to-total lipid ratio would be observed with energy restriction. Three depletion levels were examined (2 wk, 9 wk, and 9 wk + exercise), and, using the Bonferroni test for multiple comparisons (17), we accepted POther statistical tests used in the study were of a more exploratory and descriptive nature. Student t-tests were used to compare the most extreme energy-deficit group (i.e., 9-wk low-energy diet plus exercise) with the control group. These statistical analyses are provided in Tables 1 and 2 but are not discussed in the text.
Results are expressed in the text and Tables 1 and 2 as group means ± SD and in Fig. 2 as group means ± SE of the estimate. All statistical calculations were performed using the SAS statistical software (Carey, NC) package for personal computers.
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RESULTS |
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Body Weight Kinetics
Weight curves for the three chow-restricted groups are presented in Fig. 2. The 2- and 9-wk weight losses for the low-energy diet groups were 17 ± 5.4 and 34 ± 9 g, respectively. Expressed as a percentage of prediet weight, the two low-energy diet groups lost 5.5 ± 0.3 and 10.9 ± 0.3% of initial weight, respectively. More weight was lost by the 9-wk low-energy diet plus exercise group (43 ± 10.3 g; 14.1 ± 0.1%; P < 0.001 vs. control) than the comparable 9-wk nonexercise group.
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Lipid Changes
The results of total lipid and triglyceride analyses for the four groups of rats are presented in Table 1. In the control group, total lipid and triglyceride constituted 17.1 and 14.3% of carcass weight, respectively. Of the total amount of lipid present, 83% was triglyceride. Nontriglyceride lipid, the difference between total lipids and triglyceride, was 7.6 ± 4.2 g or 2.8% of body weight.
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Both total body lipid and triglyceride decreased with lengthening energy restriction (9 wk > 2 wk) and energy deficit (9-wk exercise > 9-wk nonexercise). At 9 wk of energy restriction in the nonexercise group, weight loss, total lipids, and triglycerides were reduced by 10.9, 50.4, and 57.7%, respectively, compared with the control group. In the greater-energy-deficit 9-wk exercise group, the corresponding reductions were 14.1, 67.1, and 72.0%, respectively.
With progressive energy deficit, the relative reduction in triglyceride exceeded the lowering of total lipids, and there was a decline in the triglyceride-to-total lipid ratio (Table 1) from control (0.83 ± 0.08) to 2 wk of energy restriction (0.82 ± 0.04; P = 0.80), 9 wk of energy restriction (0.70 ± 0.15; P = 0.05), and energy restriction with exercise (0.67 ± 0.09; P = 0.003; significant with Bonferroni correction) (17). Absolute nontriglyceride lipids also decreased with energy restriction and were 2.7% of carcass weight in the 9-wk nonexercise group and 2.2% (P = NS) in the exercise group compared with 2.8% in the control group. Finally, a trend analysis (17) across the four experimental conditions yielded a significant linear decrease (P = 0.005) in the triglyceride-to-total lipid ratio.
Nontriglyceride lipids are part of the triglyceride-free body mass (TGFM), which is defined as the difference between body weight and triglyceride. Nontriglyceride lipids were 3.3% of TGFM (Table 2) in the control group and increased with energy restriction at 2 wk (3.6%, P = NS) and decreased to 2.9% (P = NS) at 9 wk of energy restriction without exercise. With exercise added to energy restriction, the fraction of TGFM as nontriglyceride lipids declined further to 2.3% (P = 0.01).
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Other Component Changes
The results of other component analyses are presented in Tables 1 and 2. The last column in Table 1 represents residual mass, which is the difference between body weight and all measured components. This unmeasured component represents mainly glycogen, although small amounts of other unmeasured substances are also included.Lipid-free mass (i.e., body weight total lipids) and TGFM both
decreased at 9 wk of energy restriction relative to the control group,
without and with exercise. Compared with losses of lipid, the relative
lowering of lipid-free mass and TGFM with energy restriction was
substantially less. For example, compared with the control group,
lipid-free mass decreased by 2.5% (P = 0.19) and 4.7% (P = 0.02) at 9 wk
of energy restriction without and with exercise, respectively.
Hydration, defined as the ratio (in percent) of total body water (TBW) to lipid-free mass and TGFM, differed between denominators (i.e., lipid-free mass and TGFM) and also changed progressively with energy restriction. Mean hydration was 72.7% relative to lipid-free mass and 70.4% relative to TGFM in the control group, a significant (P = 0.02) difference that persisted throughout energy restriction. For both forms of hydration expression, relative water content increased as animals lost weight with energy depletion. For example, relative to lipid-free mass, hydration increased from a mean of 72.7% in the control group to 75.4% with energy restriction plus exercise (P < 0.0001).
As with hydration, other components also changed relative to TGFM with energy restriction (Table 2). The general pattern was for water and mineral ash to increase and for protein, nontriglyceride lipids, and residual mass to decrease relative to TGFM when control and energy restriction groups were compared. The relative contributions of the various components to total TGFM were substantially different with extreme weight loss from the control animals.
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DISCUSSION |
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To our knowledge, this is the first study to systematically explore both the total lipid and triglyceride components of body weight in either rodents or humans under various dietary and exercise conditions. Our results clearly show that triglycerides are not all of total lipids and that triglycerides do not maintain a constant proportionality to total lipids with energy restriction produced by food deprivation and exercise.
Specifically, our findings indicate that, in animals fed a defined "weight gain" chow, triglycerides represented 83% of total lipids, whereas the remaining 17% presumably represented phospholipids, sphingolipids, and other nontriglyceride lipid classes. Although absolute amounts of both triglycerides and nontriglyceride lipids decreased with energy restriction, there was a lowering of the triglyceride-to-total lipid ratio in the animals from control (0.83 ± 0.08) to 9 wk of energy restriction without (0.70 ± 0.15) and with exercise (0.67 ± 0.09). Hence, with severe weight loss brought about by tissue energy-depleting methods, approximately one-third of total body lipid was in the form of nontriglyceride chemical moieties. Although the precise mechanisms accounting for the differential rate of lipid loss are unknown, we presume that triglycerides are mobilized with energy deficit at a relatively greater rate than the essential nontriglyceride lipid compartment. Regardless of the underlying mechanisms accounting for the differing rates of lipid loss with energy restriction, our findings indicate that the proportion of total lipids as triglycerides cannot be assumed constant.
Linkage with Earlier Studies
The definition of lipid or fat components in the field of body composition research has hitherto been vague and inconsistent. These inconsistencies are spread across the widely used methods of estimating body fatness, including the total body water, underwater weighing, total body potassium, in vivo neutron activation, and imaging methods (26).Specifically, Pace and Rathbun (22) first introduced the total body water method of estimating fat and "fat-free body weight" or "lean body mass" in 1945, when they demonstrated a mean TBW-to-lean body mass ratio (TBW/lean body mass) of 0.724 in 50 guinea pigs that was similar to the TBW/lean body mass of 0.732 found in a compilation of five other animal species ranging in size from the rat to the monkey (22). Fat in this classic study was extracted from the guinea pigs with petroleum ether, and the residual mass thus includes an unspecified amount of phospholipids, sphingolipids, and other polar lipid classes (25). In a thorough composite review of the literature on three male human cadavers, Brozek et al. (4) estimated mean TBW-to-fat-free body mass ratio as 0.737. Fat in the three evaluated cadavers was extracted from whole body homogenates by use of ether extraction methods similar to those employed by Pace and Rathbun. These classic studies therefore provided primarily triglyceride estimates as the fat component. In the present study we observed in control animals a TBW-to-lipid-free mass of 0.727 and TBW/TGFM of 0.704. Hence, our comparable estimate, the ratio of TBW to TGFM, is lower than ~0.72-0.74 as observed in earlier studies. Nevertheless, our findings emphasize that hydration estimates are critically sensitive to the employed extraction method and quantified lipid components.
Variation in lipid terminology can also be found in relation to the underwater weighing method. Specific gravity as a measure of fatness was first suggested by Bull in 1896 (5) and 1897 (6). Tester (32) in 1940 reported specific gravity as a means of quantifying the ether (i.e., oil) extract of Pacific herring. Behnke and co-workers (1, 2) extended this work to humans in 1942 and suggested a two-component model consisting of fat and lean body mass. Lean body mass was defined by Behnke as including an "undetermined and probably constant percentage (2-3%) of essential lipids in bone marrow, the central nervous system, and other organs" (1). This definition is consistent with the study of Fidanza et al. (10), in which the density of fat was measured on the ether extract of surgically harvested human adipose tissue samples. Very little nontriglyceride lipid would be present in ether extracts of adipose tissue. Fidanza included this human data along with comparable animal data to arrive at a fat density of 0.900 g/ml at 37°C (10). The analysis by Brozek et al. (4) of the three human cadavers, however, provided a density estimate of lipid-free mass (i.e., sum of water, proteins, and minerals) of 1.100 g/ml. They used Fidanza's fat density to develop their two-component model, and therefore no consideration was given to nontriglyceride lipids in the underwater weighing method (4). Later workers such as Siri (28a) used the original density estimates developed by Fidanza et al. in developing their own body composition models. Varying terminology is thus applied to the underwater weighing method, and the models now in use make no clear provision for nontriglyceride lipids.
Forbes et al. (11) first reported the total body potassium method of evaluating body composition in 1961. This method was based on the assumed potassium constancy of lean body mass. According to Forbes et al., lean body mass is a "term taken to mean body weight minus chemically determined neutral fat" (11). The tissues from four cadavers cited by Forbes and Lewis (12) were extracted using ether, and these authors' reference to neutral fat is indeed consistent with tissue extraction of mainly triglycerides. Lean body mass as originally defined by Forbes is thus largely TGFM. The total body potassium approach thus appears to quantify total body triglycerides and TGFM.
There are a number of in vivo neutron activation analysis models in current use (18, 20), but the most widely cited model is the one proposed by Cohn et al. (9), in which total body fat is calculated as the difference between body weight and the sum of total body water, proteins, and minerals. With the assumption that glycogen is a minor component in the fasting subject, Cohn's model estimates total body lipids and lipid-free mass (9).
Imaging methods such as computed tomography and magnetic resonance imaging (MRI) provide whole body adipose tissue estimates (13, 15, 27). Although technically the difference between body weight and total body adipose tissue is adipose tissue-free body mass, Sjostrom (29) prefers to term this compartment lean body mass. Adipose tissue-free body mass should, under most circumstances, be roughly equivalent to triglyceride-free body mass (the latter includes water from adipose tissue). The main problem with use of the term lean body mass is its application, noted earlier, as a measure of both lipid-free and fat-free body mass.
Terminology used in the body composition field is thus both inconsistent and in some cases inaccurate with respect to lipid and its subcomponents. The initial results reported in this study strongly support inclusion of a nontriglyceride lipid component in applied models. This component was not inconsequential (2.8% of control body weight; about one-third of total lipids after 9 wk of food restriction and exercise) and varied independently from other components with negative energy balance.
The present study results, combined with the confusing terminology applied to lipids found in previous reports, prompted us to explore the definition of fat. The large majority of our sources defined fat as neutral lipids or triglycerides. Although there is no consensus on this point, applying the term fat or nonessential lipids to the ether extract of biological tissues has the advantage of making most of the earlier studies and models consistent. Accordingly, we suggest the following molecular-level model (Fig. 3)
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Nonlipid Energy-Restriction Effects
The present study results suggest other relative component changes with energy restriction, and exercise. At 2 wk of energy restriction, the weight of the animals was reduced by 5.5%, which reflected mainly lipid loss. Lipid-free mass, total body water, and mineral ash either remained unchanged or increased compared with levels observed in the control animals.With prolonged energy restriction and exercise, there were absolute reductions in most lean components with the exception of mineral ash, which continued to increase throughout the 9-wk protocol. Moreover, there was a relative increase in hydration (e.g., TBW/lipid-free mass = 0.727 in control vs. 0.754 in 9-wk exercise group, P = 0.3) and lowering of protein (e.g., protein/TGFM = 0.220 in control vs. 0.208 in 9-wk exercise group, P = 0.16). Although the mechanisms leading to these extreme relative changes in protein energy-malnourished animals are undoubtedly complex, they do suggest a lack of "constancy" in assumed component ratios such as TBW/lipid-free mass. These findings indicate that animal models may be useful as a means of investigating body composition relationships, because the required studies are extremely complex and difficult to interpret in humans, who often lose weight involuntarily with diseases that may alter water balance.
Future Directions
This study was based on a relatively small sample of 27 animals. The exploratory nature of the present study dictated the selected sample size, and future large-scale studies are needed to test the statistical significance of some observed body composition trends.Our results strongly support the need for improved molecular-level body composition models. At some future time, investigators should develop a consensus on terminology of the various lipid components. As a minimum, research reports should carefully document the nature of evaluated lipids.
An important question arising from our results is how the essential lipid component might be estimated in vivo. Our findings indicate that essential lipids are not a constant fraction of total lipids and are thus not easily estimated from total lipid mass. Moreover, we explored in our initial analyses (not presented in this report) other potential relations, such as the essential lipid-to-protein, -ash, -water, and -body weight ratios, and these also appeared unstable with energy depletion. These findings suggest that it would be difficult to infer essential lipid mass from these other components that can be measured in vivo. Hence, estimating lipid fractions in vivo poses an important challenge for future investigators.
Conclusion
This study firmly established the existence of at least two separate lipid body composition components that respond differently with energy restriction and exercise. As a minimum, body composition models should be cautiously labeled for specific lipid fractions, thereby allowing investigators to fully understand the nature of measured components. A need exists in future studies to develop estimation methods for specific lipid body composition components. ![]() |
ACKNOWLEDGEMENTS |
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We express our appreciation to Drs. Myles S. Faith, Moonseong Heo, and John Thornton for their helpful comments during the preparation of this manuscript.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant PO1-NIDDK-42618.
Address for reprint requests: A. Pietrobelli, Obesity Research Center, 1090 Amsterdam Ave., New York, NY 10025.
Received 3 September 1997; accepted in final form 4 February 1998.
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