SPECIAL COMMUNICATION
Unreliable use of standard muscle hydration value in obesity

G. Mingrone1, A. Bertuzzi2, E. Capristo1, A. V. Greco1, M. Manco1, A. Pietrobelli4, S. Salinari3, and S. B. Heymsfield4

1 Istituto di Medicina Interna e Geriatria, Università Cattolica del Sacro Cuore, 00168 Rome; 2 Istituto di Analisi dei Sistemi ed Informatica del CNR 00185 Rome; 3 Dipartimento di Informatica e Sistemistica, Università di Roma "La Sapienza", 00184 Rome, Italy; and 4 Obesity Research Center, St. Luke's-Roosevelt Hospital, Columbia University College of Physicians and Surgeons, New York, New York 10025


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intramuscular water content is assumed to be constant in humans independent of their anthropometric characteristics. To verify whether this assumption is correct, intramuscular water, proteins, glycogen, and both total and intramyocytic triglycerides were measured in 51 samples of rectus abdominis muscle obtained from 16 lean and 35 overweight and obese subjects (body mass index cutoff 24.9 kg/m2). Data (referred to as wet tissue) were analyzed by means of a composition model at the cellular level of the skeletal muscle (SM). The average SM water content was 76.3 ± 3.3% in normal-weight individuals and 65.7 ± 5.8% in obese subjects (P < 0.0001). Total triglycerides were 5.5 ± 2.3% in controls and 19.0 ± 7.0% in obese subjects (P < 0.0001). The intramyocytic triglyceride fraction was also increased in obese subjects. The composition model provides an explanation for the negative correlation between total triglycerides and intramuscular water, and some of the model parameters were determined from the experimental data. In conclusion, although the hydration of fat-free SM mass may be unchanged in obese subjects, the hydration of in toto muscle mass decreases as its lipid content increases.

muscle triglycerydes; mathematical model


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE (SM) accounts for up to 40% of body weight (36) and is a major determinant of whole body energy expenditure, even at rest, because of its large mass. Quantification of muscle mass is important for cross-sectional studies and when normalizing various physiological parameters such as muscle force production, metabolic rate of oxygen uptake, blood flow, and protein turnover. Furthermore, investigations into the causes of skeletal muscle loss with advancing age have stimulated a renewed interest in the quantification of this important component of body composition (3, 5, 10, 16).

Although it is well established that myocytes represent the largest body reservoir of carbohydrates, in the form of glycogen, their role in lipid storage and metabolism is not completely understood. Skeletal muscle is thought to be the major site for removal of both nonesterified fatty acids (NEFA) and triglycerides (TG) from the circulatory stream, although the exact role and function of the intramyocytic TG pool have not been thoroughly ascertained. According to the few studies which have addressed this topic, the TG pool appears to be in dynamic and rapid equilibrium with substrate utilization and supply (7, 24). In addition, high levels of either circulating NEFA (25, 26, 29-32) or TG (14, 27) along with supranormal levels of intramyocytic TG (TGm) (28) may play a pivotal role in the pathogenesis of insulin resistance in humans.

Electron micrographs reveal that the storage of TGm in lipid droplets is less homogeneous than the storage of glycogen (1, 9, 18, 19, 40). However, attempts to quantify the TGm content in skeletal muscle biopsies have so far been technically inadequate due to contamination with adipose tissue lipids (11, 33, 47). On the other hand, the small specimens (75-100 mg) of needle biopsy muscle tissue available for testing make it difficult to perform the analysis of TGm in duplicate or in triplicate, thus explaining the large coefficients of variation (20 to 50%) observed (47). Recently, the use of 1H magnetic resonance spectroscopy has been validated to quantitatively differentiate between adipocyte and intracellular TGm stores in both animals and humans (38). However, despite the excellent results obtained, this technique is still too advanced and expensive to be used for large population studies.

Another related problem is the intramuscular water content, which is assumed to be constant in humans independent of their anthropometric characteristics and is derived from the data of the so-called "reference man" (36). For instance, the ratio of tissue water to lean tissue mass was assumed to be constant (and = 0.81 in SM) in the analysis of data of 1H magnetic resonance spectroscopy (38). This point is a relevant one in the assessment of body composition, because it is on the aforementioned data as well as on animal studies (8, 15) that the mean whole body cellular hydration is assumed to be equal to 0.70 (43). This factor is used in the fat-free body mass hydration model recently proposed (43) as the most reliable method for the calculation of the ratio of total body water to fat-free mass and also serves as the basis of the dual-energy X-ray absorptiometry body composition model.

To clarify the hydration level of SM across subjects differing in body weight, a large sample of SM tissue, taken during abdominal surgical operations in subjects with body mass index (BMI) ranging between 17.1 and 66.7 kg/m2, was chemically analyzed. An improved technique was used to avoid contamination of muscle fibers by adipocyte lipids and employing a large amount of skeletal muscle tissue to obtain a low within-biopsy variability. Data were analyzed by means of a composition model at the SM cellular level, and estimates of model parameters were obtained.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Subjects were 51 nondiabetic patients, 26 males and 25 females, who underwent abdominal surgery for colecystectomy due to the presence of cholesterol gallstones (21 patients) or biliopancreatic diversion for morbid obesity (30 patients). The subjects were divided into a lean (control) group and a group including overweight and obese subjects, denoted in the present paper as the obese group. Subjects with BMI >24.9 kg/m2 were included in the obese group, so some of the gallstone patients were classified as obese. The average age was 52.2 ± 11.2 yr in lean subjects (n = 16) and 45.1 ± 15.6 yr in obese subjects (n = 35); weight was 59.9 ± 11.0 vs. 117.7 ± 35.2 kg and BMI was 21.8 ± 2.4 vs. 43.0 ± 11.3 kg/m2 (P < 0.001), respectively, in the two groups. None of the subjects had abnormalities in renal function or serum electrolytes.

The study was approved by the Institutional Review Board as designated by the Helsinki Declaration. Patients gave their informed consent before their enrollment in the present study.

Body composition. The day preceding the surgical procedure, body weight was measured to the nearest 0.1 kg with a beam scale and height to the nearest 0.5 cm with a stadiometer (Holatin, Crosswell, Wales, UK). Subjects were measured in the morning after an overnight fast, in a hospital gown, and after emptying of the bladder.

Muscle biopsy. During surgery, a rectus abdominis muscle biopsy (>= 6 g) was obtained after opening of its investing fascia. Each sample was free from any visible contamination from subcutaneous adipose depot store and was divided into three parts to determine chemical components. All of the analyses were performed in triplicate.

Skeletal muscle water and protein analysis. SM water (~1 g of tissue) was determined by freeze drying with continuous pumping. The difference between wet weight and dried weight was used to calculate the water content of the sample examined. The coefficient of variation for SM water determination on 3 aliquots of muscle for the same biopsy (within-biopsy variability) was 2.3 ± 0.9% (mean ± SD). The dried samples were stored at -80°C for no more than 1 mo until the analyses were performed.

To measure the protein content, the dried tissue was suspended in 200 µl of a lysis buffer and homogenized by use of a homogenizer (Ultra-Turrax TP 18-10; Janke and Kunkel, Ikawerk, Breisgau, Germany). The lysis buffer was composed of a 100-ml solution of TRIS · HCL (1 M, 2 ml, at pH 7.4), NaCl (5 M, 2 ml), MgCl2 (1 M, 0.5 ml), and Tween-20 (0.1%, 10 ml). The homogenate was centrifuged for 20 min at 6,000 rpm at 4°C. Protein content was determined from the clear supernatant by spectrophotometry at a wavelength of 565 nm by use of a colorimetric test kit (Bio-Rad protein assay; Bio-Rad Laboratories, Hercules, CA).

Skeletal muscle TG analysis. To measure total TG, a skeletal muscle sample of ~500 mg was homogenized (Ultra-Turrax TP 18-10) in a 2-ml solution of chloroform-methanol (2:1 vol/vol) acidified with 5-10 mg of trichloroacetic acid (TCA) to precipitate proteins. Lipids were extracted twice with 8 volumes, the solution being stirred at 6°C for 30 min.

To measure TGm, another specimen of ~500 mg was taken and immediately placed into a calcium-free Hanks' solution added with EDTA and bubbled with 95% O2-5% CO2. The sample was washed and then immersed in a fresh Hanks' solution added with collagenase type IV (50 mg) and calcium ions and agitated in a Dubnoff water bath maintained at 37°C until the tissue appeared soft. At this point the specimen was gently removed, and cells were brushed with a blunted spatula, filtered, suspended in PBS, and centrifuged twice at 50 g for 2 min. The supernatant was discarded, and the muscle cells were dried under a nitrogen stream. After protein precipitation with 5-10 mg of TCA, lipids were extracted twice with 8 volumes of chloroform-methanol (2:1 vol/vol) with the solutions being stirred at 60°C for 15 min. The combined extracts were dried in a GyroVap apparatus (GV1, Gio.DeVita, Rome, Italy) operating at 60°C, coupled with a vacuum pump and a gas trap (FTS System, Stone Ridge, NY). The dry weight of lipid extracts was obtained by weighing the sample tube before and after drying the extracts. The low within-biopsy variability (~5%) suggested that the TGm measurement technique was reliable and that the between-biopsy variability was not a result of measurement error.

Skeletal muscle glycogen analysis. After a 10-min incubation with 0.1 M NaOH at 80°C of the fat-free material to destroy background hexose monophosphates and glucose, glycogen was degraded to glucose by amyloglucosidase, and glucose was measured by a Beckman Glucose Analyzer II (Beckman Instruments, Fullerton, CA) as described by Bergmeyer (4). Glycogen was corrected for the highest total creatine content in the skeletal muscle specimens of a given subject.

Skeletal muscle composition model at the cellular level. Taking as a reference the model of Wang and Pearson (42) and the model of the fat-free body mass at the cellular body composition level developed by Wang et al. (43), we propose the skeletal muscle model presented in Fig. 1. This model contains three components: the muscle cell mass (MCM), the extracellular space (ECS) containing fluids and the solid matrix, and the adipose tissue mass (ATM). Thus muscle mass (MM) is equal to the sum of the three components
MM<IT>=</IT>MCM<IT>+</IT>ECS<IT>+</IT>ATM (1)
These three components consist of an aqueous and a solid compartment. Following Wang et al., and denoting by ICW the intramyocytic water, by ECW the extracellular water, and by E/I the ratio ECW/ICW, we write MCM = ICW/a and ECS = ECW/b = ICW × (E/I)/b, where a and b are the fractions of water in the myocytes and in the extracellular space, respectively. Furthermore, denoting by ATW (adipose tissue water) the water content of adipose tissue, we may write ATM = ATW/alpha , alpha  being the fraction of water in adipose tissue. The total muscle water (TMW) is thus expressed as
TMW<IT>=</IT>ICW<IT>+</IT>ECW<IT>+</IT>ATW (2)
and we note that this amount of water was actually measured as skeletal muscle water in the SM samples examined.


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Fig. 1.   Model of skeletal muscle composition at the cellular level. MM, muscle mass; MLM, muscle lean mass; MCM, muscle cell mass; ECS, extracellular space; ATM, adipose tissue mass; ICW, intramyocytic water; ECW, extracellular water; ATW, adipose tissue water. The fraction of intramuscular water is represented by the hatched area.

We can now consider the fraction of intramuscular water, TMW/MM, as the (observed) hydration level of the in toto skeletal muscle mass. This ratio differs from the hydration of fat-free mass defined by Wang et al. (43), because it takes into account adipose tissue and the water associated with this tissue. Following the approach of Wang et al., from Eqs. 1 and 2 we have
<FR><NU>TMW</NU><DE>MM</DE></FR><IT>=</IT><FR><NU>ICW<IT>+</IT>ECW<IT>+</IT>ATW</NU><DE>MCM<IT>+</IT>ECS<IT>+</IT>ATM</DE></FR><IT>=</IT><FR><NU>ICW<IT>+</IT>ICW<IT>×</IT>(E<IT>/</IT>I)<IT>+&agr;</IT>ATM</NU><DE>ICW<IT>/</IT>a<IT>+</IT>ICW<IT>×</IT>(E<IT>/</IT>I)<IT>/</IT>b<IT>+</IT>ATM</DE></FR> (3)
To rewrite Eq. 3 in a more suitable form, we introduce as a new parameter the ratio of adipose tissue mass to muscle lean mass (MLM), ATM/MLM, with MLM = MCM + ECS (MLM includes the TGm). This parameter can be considered an "adiposity index" of skeletal muscle. Thus we have
<FR><NU>TMW</NU><DE>MM</DE></FR><IT>=</IT><FR><NU>ICW<IT>+</IT>ICW<IT>×</IT>(E<IT>/</IT>I)<IT>+&agr;</IT>(ATM<IT>/</IT>MLM)<IT>×</IT>MLM</NU><DE>ICW<IT>/</IT>a<IT>+</IT>ICW<IT>×</IT>(E<IT>/</IT>I)<IT>/</IT>b<IT>+</IT>(ATM<IT>/</IT>MLM)<IT>×</IT>MLM</DE></FR>
Because MLM = ICW/a + ICW × (E/I)/b, we obtain
<FR><NU>TMW</NU><DE>MM</DE></FR><IT>=</IT><FR><NU><IT>1+</IT>E<IT>/</IT>I<IT>+&agr;</IT>(ATM<IT>/</IT>MLM)<IT>×</IT>(<IT>1/</IT>a<IT>+</IT>(E<IT>/</IT>I)<IT>/</IT>b)</NU><DE>(<IT>1/</IT>a<IT>+</IT>(E<IT>/</IT>I)<IT>/</IT>b)(<IT>1+</IT>ATM<IT>/</IT>MLM)</DE></FR> (4)
For ATM/MLM = 0 the quantity TMW/MM in Eq. 4 represents the hydration of MLM, which is thus given in the present model by the expression (1 + E/I)/[1/a + (E/I)/b].

Other important quantities that have been found to exhibit differences in SM samples from nonobese and obese patients are the fractions of TGm and of total TG in skeletal muscle mass, TGm/MM and TT/MM, respectively, where TGm denotes the content of intramyocytic triglycerides and TT denotes the total content of TG in the muscle mass. We will indicate by TGat the content of TG in SM adipose tissue and by beta  the fraction (referred to myocyte mass) of TG in the myocytes, and we will assume that adipocytes are essentially composed of water, proteins (5% according to Ref. 36), and lipids (75% according to Ref. 42). Neglecting the lipids in the ECS and following the same procedure as above, we may write
<FR><NU>TT</NU><DE>MM</DE></FR><IT>=</IT><FR><NU>TG<SUB>m</SUB></NU><DE>MM</DE></FR><IT>+</IT><FR><NU>TG<SUB>at</SUB></NU><DE>MM</DE></FR>

=<FR><NU>&bgr;MCM</NU><DE>MCM<IT>+</IT>ECS<IT>+</IT>ATM</DE></FR><IT>+</IT><FR><NU><IT>0.75 </IT>ATM</NU><DE>MCM<IT>+</IT>ECS<IT>+</IT>ATM</DE></FR> (5)

=<FR><NU>&bgr;/a</NU><DE>[<IT>1/</IT>a<IT>+</IT>(E<IT>/</IT>I)<IT>/</IT>b](<IT>1+</IT>ATM<IT>/</IT>MLM)</DE></FR><IT>+</IT><FR><NU><IT>0.75 </IT>(ATM<IT>/</IT>MLM)</NU><DE><IT>1+</IT>ATM<IT>/</IT>MLM</DE></FR>
Another quantity that can be derived from the model is the fraction of proteins (P) in the SM mass. Denoting the fractions of proteins in myocytes and extracellular space as gamma  and gamma ', respectively, and because the fraction of proteins in adipocytes has been assumed equal to 0.05, the fraction P/MM can be expressed as
<FR><NU>P</NU><DE>MM</DE></FR> (6)

=<FR><NU>&ggr;/a<IT>+&ggr;′</IT>(E<IT>/</IT>I)<IT>/</IT>b<IT>+0.05</IT>(ATM<IT>/</IT>MLM)<IT>×</IT>[<IT>1/</IT>a<IT>+</IT>(E<IT>/</IT>I)<IT>/</IT>b]</NU><DE>[<IT>1/</IT>a<IT>+</IT>(E<IT>/</IT>I)<IT>/</IT>b](<IT>1+</IT>ATM<IT>/</IT>MLM)</DE></FR>
Similarly, assuming that the glycogen in the SM sample is almost exclusively present in myocytes with a fraction delta  (referred to myocyte mass), an equation for the glycogen fraction in the SM sample, G/MM = (delta /a)/[1/a + (E/I)/b](1 + ATM/MLM), can be easily obtained.

The preceding equations show that the quantities that have been measured in the SM samples: TMW/MM, TGm/MM (given by the first term on the right hand side of Eq. 5), TT/MM, P/MM, and G/MM can be expressed in terms of the following factors: a, b, E/I, alpha , ATM/MLM, beta , gamma , gamma ', and delta . Although these parameters will exhibit variability from sample to sample, it is not possible to estimate all their individual values on the basis of the present measurements. However, some of these parameters can be assumed to be constant in the population of samples. Parameters a and b are likely to have a reduced variability, because they appear to be maintained stable by regulatory mechanisms (43). On the basis of data reported in Wang et al. (43) for the SM cells, we assumed a congruent  0.72. Regarding parameter b, we note that the organic extracellular solids like the fibers are included in the extracellular space in the present model, whereas they were included in a compartment of extracellular solids in Wang et al.; thus we took for b a smaller value than in Wang et al., b congruent  0.97. Adipose tissue hydration was found by Wang and Pierson (42) to be relatively invariant, so the value of alpha  was set to 0.14, according to Wang and Pierson. Moreover, because the term representing the extracellular proteins is scarcely influent in Eq. 6, at least in the absence of atrophy or fibrosis, when the content of extramyocytic proteins may become large, we gave to gamma ' the constant value of 0.02 (2, 36).

As far as the other parameters are concerned, we note that a relative increase of extracellular with respect to intracellular fluid has been found in obesity (42), so E/I was assumed to have a linear increase with the adiposity index ATM/MLM. In particular, we took E/I = 0.4 + 0.5(ATM/MLM), and thus larger than the experimental value of 0.2 found for rats and humans (6, 13). With the previous assumptions, the parameters ATM/MLM (and thus also E/I that is a given function of ATM/MLM), beta , gamma , and delta  were reliably estimated for each sample from the experimental data of TMW/MM, TGm/MM, TT/MM, P/MM, and G/MM.

Statistical analysis. Each single value represents the average of triplicate determinations. The results are presented as means ± SD unless otherwise specified. The variability of the determinations from three samples of the same biopsy was expressed as the coefficient of variation (CV = 100 × SD/mean).

The Fisher's exact test was used to compare sex proportions between the two groups. Between-group comparisons were performed using a Student's t-test for the difference of means, with significance at P < 0.05. The linear correlation between anthropometric data and total lipids or intramyocytic triglycerides was assessed by the correlation coefficient. In the linear regression, the sum of absolute deviations was minimized to reduce the effect of outliers. The estimates of the regression parameters are given as estimate ± SE (34, 37).

The values of ATM/MLM, E/I, beta , gamma , and delta  were estimated for each sample from the measurements of TMW/MM, TGm/MM, TT/MM, P/MM, and G/MM by least squares solution of model equations, whereas the other parameters of the model were set to the values specified above. The solution was found with the constraint that the sum a + beta  + gamma  + delta  of water, triglycerides, proteins, and glycogen in the myocyte mass, a sum that represents all the main cellular components excluding nucleic acids and miscellaneous metabolites, has to be close to 97%. The standard errors were evaluated from the inverse Hessian matrix of the least squares index at the minimum (34).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Table 1 reports the data of tissue composition in the two groups: mean values ± SD refer to 100 mg of wet muscle tissue. The fractions of TT and TGm were significantly smaller in the control group compared with the obese group. The fraction of intramuscular water was significantly lower in the obese group. A positive correlation was found between BMI and both the fractions of TGm (r = 0.52, P < 0.0001) and of TT (r = 0.87, P < 0.0001). A negative correlation was found between the fraction of intramuscular water TMW/MM and the fraction of total triglycerides TT/MM (r = - 0.67, P < 0.0001). The experimental data of TT/MM vs. TMW/MM are reported in Fig. 2, where it is noted that lean subjects are represented in the right region of the plane (high fraction of intramuscular water and low lipid content). The dotted line in the figure is the regression line (slope -1.044 ± 0.032, intercept 0.874 ± 0.025).

                              
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Table 1.   Chemical composition of the skeletal muscle specimens



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Fig. 2.   Experimental data of the fraction of total intramuscular water (TMW) vs. the fraction of total triglycerides (TT) in the skeletal muscle samples (controls, ; obese subjects, ). Theoretical lines predicted by the model of skeletal muscle composition at the cellular level (Eqs. 4 and 5), with the parameter values indicated in the text [solid lines: top, ratio of ECW to ICW (E/I) = 0.68 and fractions of TG in the myocytes (beta ) = 9.7; bottom, E/I = 0.40 and beta  = 1.5); dotted line, regression line.

The individual estimates of ATM/MLM, E/I, beta , gamma , and delta  were found with standard errors that were generally much smaller than the 10% of the estimate. Table 2 gives the mean values and standard deviations of the estimates over the control and the obese group. As expected, the mean value of beta  (fraction of TGm referred to myocyte mass) is larger than the mean value of the TGm referring to the wet mass of the sample reported in Table 1 and similarly for gamma  and delta  compared with the fractions of proteins and glycogen in Table 1. The predicted values of the ratio of H2O to proteins are also reported in Table 2. All of the parameters of the model except gamma  are significantly different in the two groups. The estimates of the adiposity index ATM/MLM were found to range from 0.01 to 0.09 in controls and from 0.06 to 0.56 in the obese group. The fraction of SM lean mass that can be computed from the adiposity index as MLM/MM = 1/(1 + ATM/MLM) ranges thus from 0.91 to 0.99 in controls and from 0.64 to 0.94 in obese subjects.

                              
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Table 2.   Model parameters determined from data of chemical composition of skeletal muscle specimens

The negative correlation observed between intramuscular water content and total TG content can be explained on the basis of the skeletal muscle composition model at the cellular level of Fig. 1. The solid lines in Fig. 2 were plotted by computing abscissae and ordinates by Eqs. 4 and 5, respectively, with the model parameters a, b, and alpha  fixed at the values given in MATERIALS AND METHODS and with E/I and beta  set to the extreme values found in the SM samples (see the legend of Fig. 2). The adiposity index ATM/MLM was changed from 0 to 0.9 to cover the entire range of the measured intramuscular water fractions. These lines, which are easily shown to be straight lines, are thus parameterized by the adiposity index, and the terminal point at the right corresponds to ATM/MLM = 0. If a subject were to change from lean to obese while maintaining the SM parameters a, b, alpha , beta , and E/I constant, its representative point on the plane of Fig. 2 should move on a straight line from right to left. The two lines of Fig. 2 show the effect of the variability of three parameters of the model, the adiposity index ATM/MLM, E/I, and the fraction of intramyocytic triglycerides beta .

The model in Fig. 1 shows that the hydration level of skeletal muscle can be reasonably defined in three ways. The ratio TMW/MM is the observed hydration of the in toto muscle mass; this quantity was seen to change in the range from 0.5 to 0.8 in the SM samples examined (see Fig. 2). We can also consider the hydration referred to the muscle lean mass, TMW/MLM, and the hydration defined as (ICW + ECW)/MLM. This latter quantity is given by Eq. 4 for ATM/MLM = 0, as seen above, and depends on the obese or nonobese status of the subject only through the parameters a, b, and E/I. In contrast, the quantity TMW/MM decreases with the adiposity index, because it takes into account the water contained in the adipose tissue that has a small water content. For instance, with E/I = 0.6, the ratio TMW/MM decreases continuously from a value of 0.797 for ATM/MLM = 0 to a value of 0.492 for ATM/MLM = 0.9.

To assess the predictive capacity of the model, we computed the theoretical values of the ratio of total triglycerides to proteins (TT/P) and of the ratio of total muscle water to proteins (TMW/P), as predicted by Eqs. 4-6, with the parameters estimated on the individual samples as described above. A Bland-Altman plot of the experimental and theoretical values of TT/P is shown in Fig. 3. It is seen that the experimental ratios exhibit a remarkable variability from sample to sample especially in the obese group, mainly because of the presence in this group of five samples in which a very reduced protein content (from 5.5 to 3.6%) was measured and thus a high value of the ratio was obtained. The correlation coefficient for experimental and predicted values of TT/P is equal to 0.998. For the TMW/P ratio, whose experimental values are in the range of 2-18, the predicted mean and SD are reported in Table 2 for the two groups, and the correlation coefficient is equal to 0.995. 


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Fig. 3.   Bland-Altman plot of measured values and of theoretical predictions of TT/protein (P) in the SM samples (controls, ; obese subjects, open circle ). The solid line, mean difference; broken lines, mean difference ± 2 SD.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Only recently has the importance of maintaining the SM mass in dynamic disease conditions stimulated new approaches to the in vivo assessment of this variable (12, 16, 17, 45). A fundamental limitation to the availability of a useful technique for the measurement of SM mass is represented by the relative lack of direct information on anatomic tissue composition. In fact, data on SM mass by cadaver dissection have been reported in only 25 subjects (23, 39, 41, 46). Total body water, fat, protein, minerals, and carbohydrates were measured in two cadavers, one underweight (BMI 19.35 kg/m2) and the other with cachexia (BMI 10.92 kg/m2) (21). Indeed, the study in Ref. 21 was addressed to demonstrate the usefulness of the in vivo neutron activation analysis in severely wasted patients. However, the extrapolation of data obtained by cadaver dissection to the assessment of body composition, and of SM mass in particular, in living individuals with a wide range of body weights appears at best inaccurate.

A few studies have been performed on the SM composition in humans but without addressing the question of whether the lipid content of the SM mass may influence the assessment of body composition. Landin et al. (22) found in muscle biopsies that only obese men, but not women, had a higher fat content than did lean men. Using computed tomography of the thigh, Kelley et al. (20) reported similar findings in obese men, and these data were later confirmed in women (35). However, these investigations failed to demonstrate whether fat is accumulated in the connective tissue between muscle fibers or is stored within muscle cells. By use of data obtained in the "reference man" studies (36), the water content of human skeletal muscle as a percentage of its total weight has been generalized for all humans to be a constant value of 81%. However, the assumption that the water content of skeletal muscle is constant throughout the population may turn out to be incorrect in light of different amounts of fat mass among different subjects (44).

In the present paper, the composition of samples of skeletal muscle from subjects with a wide range of BMI was analyzed. The fractions of intramuscular water, proteins, glycogen, and both total and intramyocytic triglycerides, which represent the typical lipids stored for energy supply, were measured. Our data showed that obese subjects have a significantly larger amount of SM total triglycerides, whose fraction was found to be threefold in the obese subjects. Also the content of intramyocytic triglycerides increased with the BMI, albeit to a lesser extent. The increase of SM triglycerides in obese subjects was thus mainly due to the accumulation of adipocytes between muscle fibers. As a consequence, the presence in obese subjects of larger amounts of lipids resulted in a lower fraction of intramuscular water, as confirmed by the negative correlation found. In our study, the measured intramuscular water percentage for normal-weight individuals was 76.3 ± 3.3% and thus comparable to the reference man value and to the value of 0.8034 obtained from Ref. 43, whereas the water percentage in obese subjects was much lower (65.7 ± 5.8%), with a decrease of ~15% compared with the reference value.

Our data were interpreted on the basis of a model of SM composition at the cellular level, which was developed starting from previously described models of total body mass (42, 43). The present model considers, in addition to the lean mass, the adipose tissue mass (adipocytes between muscle fibers) and is thus suitable to represent a sample of in toto muscle mass. The model describes the SM composition in terms of factors whose variations can account for the interindividual variability of SM composition. In particular, one of these factors, denoted as adiposity index (ratio between adipose tissue mass and lean mass in the skeletal muscle), takes specifically into account the amount of triglycerides that is present in the adipocytes between muscle fibers. This factor is likely to show a wide intersubject variability (values from 0.01 to 0.56 were estimated on the present samples). A certain degree of variability, both between control and obese groups and within the two groups, has to be expected also for the other parameters of the model, as seen in Table 2.

The model shows that the hydration level of SM (interpreted as the ratio of total intramuscular water to muscle mass) decreases when the adiposity index increases, whereas all other parameters of the model, and, hence, the hydration of SM lean mass, are unchanged. In other words, SM hydration may still be normal in obese subjects after correction for intramuscular lipids. However, in the transition from the lean to overweight and, eventually, to obese status, all of the model parameters are likely to change to some degree. A complete study of the physiological (or associated to obesity) variability of the parameters of the model, in conjunction with the plausible effects of the experimental errors, could account for the scatter observed in the data. It has to be stressed that the measurement of the extracellular water in the samples would provide a more accurate estimate of E/I, whose between-sample variability was only partially considered in the present study.

The model allowed us to reasonably reproduce the variability of the data, as shown in Fig. 3. We note that a subgroup of samples from obese subjects had a greatly increased value of TT/P, so a larger deviation from model prediction was found. This increased TT/P is possibly associated with a specific loss of muscle proteins. All of these subjects, in fact, in addition to morbid obesity, also presented us with associated complications, namely degenerative arthritis of the hip and knee, which severely restricted their walking. For these cases, some specific modification of the model, to take into account the possible effects of the loss of proteins, should be considered.

Because skeletal muscle accounts for a major portion of body cell mass, the finding that the water fraction of skeletal muscle is highly variable with BMI implies that the estimation of the fat-free mass should be revised. The assumption of a constant water fraction in skeletal muscle should also be reconsidered. It is clear, therefore, that knowledge of a more accurate estimate of the SM water content might allow a more appropriate calculation of the intramyocytic triglyceride amount in individuals with different anthropometric characteristics, with particular reference to lean and obese subjects. In conclusion, the classical 81% as a standard reference value for the water content in skeletal muscle is not appropriate when used across lean and obese subjects. However, the error in lean subjects is smaller compared with that found in obese individuals, in whom the high lipid content of SM causes a decrease of the percentage of water.


    ACKNOWLEDGEMENTS

We are grateful to A. Caprodossi for technical support.


    FOOTNOTES

Address for reprint requests and other correspondence: G. Mingrone, Istituto di Medicina Interna e Geriatria, Università Cattolica del Sacro Cuore, Largo A. Gemelli 8, 00168 Rome, Italy (E-mail: gmingrone{at}rm.unicatt.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 March 2000; accepted in final form 20 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 280(2):E365-E371
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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