techniques of measurement of body composition part i

Contents

Sports Medicine 5: 11-40 (1988) 01 12-1642188/000 1-00 11/$15.00/0 © ADIS Press Limited All rights reserved.

Techniques of Measurement of Body Composition Part II

D.A. Brodie School of Physical Education and Recreation. University of Liverpool, Liverpool

Summary ....................................................................................................................................... 12 I. Cadaveric Studies .................................................................................................................... 15

1.1 Gross Tissue Weights .............. ................. .. ...................................................................... 16 1.2 Skin fold Compressibility .................. .... .. .. ........................................................................ 16 1.3 Skin Thickness .................................................................................................................. 17 1.4 Adipose Tissue Patterning ................................................................................................ 17 1.5 Fat Fraction in Adipose Tissue .................................................................. .. ........ ........... 17 1.6 Subcutaneous Fat in Relation to Internal Fat ............................................................... 18 1.7 Surface Area ...................................................................................................................... 18

2. Fat Cell Size and N umber ...................................................................................................... 18 2.1 Site Variability .................................................................................................................. 18 2.2 Reliability .......................................................................................................................... 19 2.3 Method Variability ........................................................................................................... 19

3. Estimation from Skinfold Equations ..................................................................................... 21 3.1 Population-Specific Prediction Equations ....................................................................... 22 3.2 Generalised Prediction Equations ................................................................................... 22 3.3 Equations for Infants and Children ................................................................................ 23 3.4 Equations for Athletes ...................................................................................................... 24 3.5 Biological and Methodological Measurement Errors ..................................................... 24

4. Body Density Measurement Methods ................................................................................... 25 5. Anthropometric Assessment of Muscle and Fat Area ......................................................... 28 6. Simple Methods for Measuring Adiposity ............................................................................ 28 7. Fat-Soluble Gases .................................................................................................................... 31 8. Creatinine Excretion .................................... ........... ........... ........ .............. ........ ........................ 31

8.1 Method ................................................................................. ...................... ........................ 31 8.2 Reliability and Validity ..................... .. ............................................................................. 31 8.3 Plasma and Serum Creatinine ................... ....... .. ............................. .. ...... ........................ 32

9. 3-Methylhistidine ....................................................... ................................................... ........... 32 9.1 Method ..................................... .................... ..... ... .............................. ........ ............. ........... 32 9.2 Validity .................................... ........... ... ...... ..... ........... ........ ........... ... ........ ............. ........... 33

10. Total Body Water ........................ .. ... .. ....... .. .. ..................... ............ ....... .... .... .. ...... .. .............. 33 10.1 Methodology .......................... .. ............ .. .... .... .. .......... ........ .... .... .. ..... ....... .. .. .. ... ............... 35

II. Total Body Potassium ............................... ...... ...... ... ...... .. .... .. .. .......... .... ....... .. ............ .......... 36 12. Other Nuclear-Based Techniques .... .. ............ .. .................................... .. ............................... 37

12.1 Neutron Activation Analysis .................. .. ....... .. ......... .. ................. .. ..... .. ...... .. ............... 37

I Part II including references ~ill appear in the next Issue of the Journal

Measurement of Body Composition 12

12.2 Total Body Nitrogen ...........................................................•....................................................... .. 37 12.3 Total Body Carbon ..... .................................................................................................................... 38 12.4 Photon Absorptiometry .................................................................................................................. 38

12.4.1 SinaJe Photon Absorptiometry ................................................................................................ 38 12.4.2 Dual Photon Absorptiometry .................................................................................... .. ........... 38

12.S Nuclear Resonance Scalterinl ............................... .. ...................................................................... 39 12.6 Multiple Isotope Dilution .............................................................................................................. 39

The measurement of lean body and fat mass has developed with the increase in sports participation and the prescription of exercise. Quantification of body fat is also related to the treatment of obesity and to assess nutritional status. Different levels of evaluation have bHn proposed depending on expertise and need.

Body density estimation /rom skin/old measurements has the advantage of simplicity, low cost and reasonable validity with predictions to within J to 4% for 70% of the population. The choice of prediction equation will depend on whether a generalised equation or a population-specific equation is appropriate. In all cases tester reliability and standard error of esiimation should be established. There is strollg evidence that a quadratic equation should be applied and that measures of the lower limb should be included with circumference and diameter measures to strengthen the prediction. Methodological errors need to be reduced by careful training of experimenters. Cross validation of regression equation will strengthen their validity, particularly when fat loss is to be quantified. The popularity of skin/old assessment of body fat is enhanced by the use of nomograms to predict body fat, although some accuracy will be lost. Skinfold estimation of body fat will continue to be a useful guide to adiposity for epidemiological studies andfor popular usage.

The hydrostatic weighing procedure to estimate body density is considered by many to be the criterion method. Under carefully controlled conditions with maximum subject compliance, it is highly reliable. particularly if residual volume can be determined accurately. The conversion of body density to percentage body fat is based on a number of assumptions which need to be considered with respect to the population being studied.

Simple methods for adiposity include various weight for height indices. for example, the body mass index of weight (kg) + height} (m) which is often used to define obesity. frame size for the prediction of ideal weight and visual estimation for predicting body fat.

Differences in the literature concerning fat cell size and number have their origins in variations of methodology. The choice of site for removal of tissue will influence the size of adipocytes. those obtained /rom deep sites are generally smaller than subcutaneous fat cells. If total cell number is determined from cell size. there will also be variability of cell number. The correlation between different methods of sizing cells is high for most techniques. but as the validity of the criterion method is not based on statistical evidence, the validity of the other methods cannot be readily accepted. When comparing the findings of different investigations. the methodology and type of analysis and sample site must be considered as they can exert a profound influence on the results. The controversy concerning changes in cell number and size during obesity will only be resolved when the limitations and differences in methodologies are fully resolved.

Measuring fat /rom fat-soluble gases may be used concurrently with other in vivo measurements. e.g. body water or body potassium. to establish average proportions and biological variability offatfree mass components. With such in/ormation it would be possible to evaluate fundamental assumptions. and increase the usefulness of more widely applied techniques of body fat measurement. The procedure is prolonged and tedious for most subjects. and cannot easily be applied to large populations. Its potential is related to the direct measurement of fat. but it has not developed into a widely-used method.

U-Hour urinary creatinine excretion is a widely used biochemical marker for body muscle mass estimation. After a creatine-controlled diet, the totalU-hour urine is analysed

Measurement of Body Composition I3

for creatinine content. Howelw. no definitil'e creatinine equil'alence for human muscle has been established. An alternative approach for estimating muscle mass is to use plasma or serum creatinine values. although as yet these methods are not widely used.

3-Methylhistidine is another excretory product which is proportional to human muscle mass. Excellent correlations hal'e been found with total body potassium andfat:free body mass. The method is essentially similar to the measurement of 24-hour urinary creatinine. although it does require the use of an amino acid anal.vser and dietary control is essential.

Total body water measurement can employ a range of dilution techniques. Deuterium oxide dilution is a common method andfu(fils the methodological criteria.

Total body potassium is a commonly used criterion method for el'aluating lean body mass. It involl'es the measurement of the naturally occurring 40K using screened scintillation detectors.

Nuclear-based techniques are generall.v expensive. require a high lel'el of operator training and are ethically questionable because of the use of radioactivity. The dosage is generally considered to be within an acceptable range and the specific information would be d(flicult to obtain by other techniques. The methods include neutron actil'ation analysis. total body nitrogen. total body carbon. photon absorptiometry (single and dua/). nuclear resonance scattering. and multiple isotope dilution. These methods are more likely to be restricted to direct clinical applications and will be used relativel.v rarely on healthy populations for body composition assessment.

Ultrasound can be used to measure the depth of tissue interfaces. typically fat and muscle. Two types are commonly used. the B scan system to provide grey scale images. and the A scan del'ice which records depth from reflected echoes. The latter method is reliable. portable. and could be of particular l'alue in measurements on the obese.

In bod\' composition research computed tomography (CT) scanning. subject to the ethical considerations of radiograph,l'. is commonly used. A CT scanner produces a crosssectional image of the distribution of x-ray attenuation. It can be used to validate existing anthropometric techniques and to provide an understanding of inter-relationships between metabolic actil'ity and body composition. Disorders oIfat distribution can be detected and may be sign(ficant in I'arious clinical conditions. e.g. Cushing's disease and diabetes. In prolonged weight reduction it may be useful to apply CT to establish the relative quantities offat. muscle and bone. Similarly CT can detect the details of apparent weight increase with patients sufferingfrom renal. cardiac or liver diseases which may hal'e oedema masking muscle wastage. In sports medicine research the hypertrophy and atrophy of muscle after rehabilitation or immobilisation can be studied with precision by the CT method.

Nuclear magnetic resonance imaging requires a large bore magnet capable of accepting the human body. The images. received by a radio frequency coil. are dependent on the behal'iour of h,l'drogen nuclei in the magnetic field. The time taken for the protons to realign is detected and the image constructed similar to computed tomography. The realignment characteristics relate to speqfic tissues both in health and disease. Various mod!fications such as chemical sh(fi imaging hal'e improved the quality of the image such that it could now be usefully employed in body composition assessment. The capital cost of an NMR unit means that opportunities for use will be limited. Howel'er. the absence of radioactivity makes this approach ethicall.v advantageous.

The total body electrical conductil'ity method is a current induction approach in which the conductil'ity of the human body perturbs the electromagnetic .field such that fat and non:fat tissue can be evaluated. It appears to be both reliable and valid. Compared with total body water and total body potassium, total body electrical conductil'ity seemed to track short term changes in nitrogen balance more effectil·el.l'. Its main disadl'antage is that of capital cost, but it is safe. simple, conl'enient, rapid, non-inl'asil'e and requires low operator and recurrent expense. It undoubtedly distinguishes between fat and non:fat tissue. so mar hare considerable ben~fit in body composition measurement. particularly (f a 101l'-cost I'ersion can be produced. If this does nor occur. researchers are likely to pursue current injection techniques as a more ,financially viable alternative.

Electrical impedance or impedance plethysmography is a current injection method of


relating body conductivity to composition. Typically a 50 kHz. 800pA electrical current is passed bet~ the hand and foot and the resistance is recorded. This value is used in a ~ession equation to determine lean body mass. although the measurement is more directly related to total body water. The procedure is reliable. and has been shown to be valid in comparison with hydrostatic weighing. The quoted standard e"ors of estimale range from 1.7 to 6.J'Ib. but population specific regression equations may improve this aspect. Its speed of use, safety and portability may make this approach particularly useful for epidemiological studies.

Infrared interactance is a method of body composition based on the principles of light absorption, reflectance and near i'lfrared spectroscopy. It developed from work on animal foodstuffs but has been used successfully on humans, although only to a limited extent.

A true cross-valida/ion using a distinct populalion is necessary as is the requirement for intra- and inter-reliability stalistics on the methodology. Co'lfounding results could occur because of inaccurate positioning of the fibre optic probe. The pressure of applying the pr. could also i'1fluence the degree of energy scalter and absorption. The method uses 5 sites, so the precision of site location would. like ski'lfold readings. provide a p0-

tential source of error. However. the method has virtue in being rapid. safe and noninvasive so may, with further experimentation. prove to be a use/ul addition to body composition analytical techniques.

Interest in body composition has developed in parallel with the increased application of scientific' methods in sports medicine and exercise studies. Improvement in sports performance has resulted from the application of knowledge from many scientific and medical subdisciplines. Appropriate active mass is an essential ingredient of optimising physical activities. This not only applies to sports involving specific weight categories but to most activities where the transportation of excess fat is detrimental to performance.

However, not only the sporting population should be interested in the estimation of body composition especially with the correlative evidence indicating an association t¥tween obesity and a variety of diseases. Quantification of body fat is needed to study the nature and treatment of obesity, to assess nutritional status, and to determine the response of patients to a range of metabolic disorders (Cohn et al. 1981). Intravenous hyperalimentation techniques (Dudrick 1977) dramatically improved surgical procedures and indicated that concurrent malnutrition was responsible for many deaths in surgical patients. It is important to measure nutritional status in certain categories of patients, with immediate postoperative patients requiring 2500 to 4000 Calories per day and severely

septic, traumatised or burned patients requiring up to 10,000 Calories per day (Salmond 1980). Morgan (1982) reviewed the clinical application of body composition measures and argued strongly for a better understanding of abnormal values and assessment of the difference in disease, and for the need to measure body composition as part of the assessment of nutritional state.

The effects of obesity range from reduction of performance in a sporting contest to 'morbid' obesity. The Framington study showed that hypertension increased IO-fold in subjects who were more than 20% over ideal weight (Gordon &. Kannel 1976) but a causal relationship was inconclusive. Similarly angina pectoris and sudden death were more common in the obese, although no significant correlation existed between actual myocardial infarction and obesity. Periods of weight gain are associated with hypercholesterolaemia. Thus, although the effects of obesity per se on the cardiovascular system are not clear, there are mortality risks associated with both hypertension and increased lipids, both of which are positively correlated with excess fat. There is a relationship between diabetes mellitus and obesity, and operative mortality and the risks of anaesthesia are increased in the massively obese (Mann 1974). Orthopaedic

Measurement of Body Composition

complications such as degenerative changes in the weight-bearing joints of the legs and vertebral column are related to obesity as are muscular disabilities. particularly of the lower back. One of the less well understood aspects of obesity is psychosocial health. The obese may suffer a restricted social life and employment discrimination which could result in a variety of psychological handicaps.

The relationship between work capacity and body fat is of most concern to those involved in sports medicine. The composition of energy stores in humans has been reviewed by Garrow (1982a), who adopted Passmore's (1965) distinction between energy stores and reserve. Reserve is essential fat whereas fat store is an accumulation which may be in excess of current requirement. It is the fat store which is of specific interest in sport because excess fat can be detrimental to performance. Garrow (1982b) reviewed the methods of body composition and in so doing provided a 'buyers' guide' to the available techniques. The present review attempts no such direct comparison but will in certain cases allude to perceived advantages and disadvantages. Body composition profiling is well advanced, with many researchers applying the information directly to athletes (Katch & Katch 1984) or in the field of general physical education (Montoye 1974). A round-table discussion, moderated by Wilmore et al. (1986). suggested 3 levels of expertise in body composition assessment. These were techniques to be used in clinical practice. more advanced measurements to be used in sports medicine or health surveys. and methods adopted in research laboratories. No attempt has been made to categorise measurements by level of expertise because equipment standards change and financial situations vary from country to country. It is not always easy to classify the available methods into distinct groupings but this review will attempt to keep methods which operate upon similar principles together. Research progress in the field of body composition is an evolutionary process as indicated by Roche (1984a). This review has tried to extend the reader's knowledge on certain tech-

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niques whilst providing a resource base for the better known methods.

Some sections have been considered in far greater detail than others. This is because certain areas have been reviewed elsewhere. but also in an attempt to provide more emphasis to recent and less popular methods.

1. Cadaveric Studies

Direct measurements of body composition can only be made from cadaveric analysis. Since the nineteenth century human tissues have been weighed (Bischoff 1863; Schwann 1843; von Liebig 1874), but in some cases (Theile 1884) total bodyweight was not reported so the results were of limited value. Between 1945 and 1984 body composition analyses have only been completed on 8 cadavers (Forbes et al. 1953. 1956; Mitchell et al. 1945; Moore et al. 1968; Widdowson et al. 1951). A full tabulation of these studies was given in Clarys et al. (1984). Since 1984 the results of the Brussels cadaver study have appeared in the literature, based on an analysis of 25 corpses measured in 1978-80 and a further 7 corpses in 1983. This work has added significantly to the literature on direct measurements of body composition and the detailed methodology can be obtained in Martin (1984), Martin et al. (1985) and Clarys et al. (1984, 1987). Clarys et al. (1984) state 'None of the individual methods have ever been validated against direct human cadaver evidence ... there is no human body for which both whole body density and body fat are known. None of the previously mentioned dissections included any skinfold measurements or extensi ve anthropometry.'

The Brussels study provides the opportunity for a number of body composition issues to be reassessed. These include sex differences in the composition of muscle and bone. the assumption that the density of fat-free weight is constant, the validity of the formulae used to predict body surface area. the errors and assumptions in the prediction


of body fat from skinfold measurements, the subcutaneous patterning amongst individuals and the change in bone density during ageing.

1.1 Gross Tissue Weights

The ~ports of the Brussels study made the distinction between the 2-component model of fat weight and fat-free weight and the alternative anatomical model of dividing the body into tissue components such as muscle, skin plus subcutaneous adipose tissue, bone and the remainder proposed by Matiegka (1921). Body fat can only be defined as the ether-extractable constituents (Keys & Brozek 1953). Ether extraction was not used in the Brussels study but adipose tissue was dissected such that the adipose-tissue-free weight could be used for comparative purposes. Although adipose tissue is a close anatomical analogue of body fat, it will be slightly at variance because the major organs, undifferentiated soft tissues such as the major vessels, and the muscles will contain fat. The Brussels study divided the total bodyweight into skin, adipose, muscle, bone and undifferentiated tissue. Clarys et at. (1984) concluded that because of the variation in adipose-tissue-free weight composition, with bone weights varying from 16.3% to 25.7% and muscle weights varying from 41.9% to 59.4%, it would be unwise to accept fully the assumption that fat-free weight has a constant density. This clearly has implications in the prediction of fat percentages from assessments of subcutaneous adipose tissue.

The mean values of the gross tissue weights, expressed relative to adipose-tissue-free weight (skin 8.5%, muscle 50.0%, bone 20.6%) compared favourably with the nineteenth century data whereas the other twentieth century data indicated a lower muscle value, possibly due to a lower nutritional status in those SUbjects.

Another interesting finding was a bone density range twice that reported previously (Martin 1984). This questions the assumptions used in densiometric methods of predicting body fat and could explain in part the negative fat value found for lean subjects in certain categories of sport.

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Skeletal density showed a decrease with age approximating to a 2% loss of bone mass per decade in older populations. The best single predictive measure of skeletal mass for the combined sexes was established by Martin (1984) to be the wrist width (r = 0.875). The multiple regression equation to produce the lowest standard error in the prediction of skeletal mass (SM) was for males (all values in cm; giving SM in grams):

SM = (28.0 X XI) + (0.4815 X X2) + (1.377 X XJ) + 4265 (r2 = 0.98, SE = 222g)

where XI is (wrist width)2 X ankle width, X2 is (head girth X humerus width X biacromial width), and XJ is (head girth X humerus width X femur width) and for females:

SM = (0.1822 X X4) - (6.415 x Xs) + (1.l45 x X6) + 787 (r2 = 0.79, SE = 479g)

where X4 is (head girth x stature x wrist width), Xs is (femur width)2 x wrist width, and X6 is (humerus width)2 x ankle width.

1.2 Skinfold Compressibility

Before the use of skinfold as a predictive measure can be discussed, the measurement technique itself merits consideration. Many observers have noted the change in caliper readings soon after application (Booth et al. 1966; Aetcher 1962; Orpin & Scott 1984). Brans et al. (1974) proposed an exponential decline in the values in neonates over 60 seconds, and Laven (1983) also showed that in neonates compression was a source of skinfold error and that it could be used to measure changes in hydration status. There have been a number of factors associated wi~h skin-plus-fat compressibility including the skinfold site (Brozek & Kinsey 1960; Qeg & Kent 1967; Himes et al. 1979; Lee & Ng 1967), the skinfold thickness (Edwards et al. 1955; Hammond 1955), sexual dimorphism (Clegg & Kent 1967; Lee & Ng 1967) and age (Brozek &


Kinsey 1960). Becque et al. (1986) in a more detailed analysis of the changes of skin compressibility over time considered that the compression conformed to a 2-component exponential model. Sexual dimorphism was only evident at the iliac site, and regional differences were only observed in males with the iliac site being the only one to show greater compressibility than the others. Measurements based on minimal compression required the readings to be taken within 4 seconds of applying the caliper.

Compressibility has been defined as:

(Uncompressed value - compressed value)

Uncompressed value

but Martin et al. (1985) used the definition:

(Incised depth - caliper reading)

I ncised depth

and thus were able to compare skinfold measurement with the direct measurement. There was a large variation in compressibility with supraspinale and biceps giving a mean of over 60% compared with front thigh and medial calf at just over 30%. This may result in identical skinfold values having large differences in actual adipose tissue thickness.

1.3 Skin Thickness

The situation is further complicated by the effect of skin thickness because each skinfold measure will include a double layer of skin of unknown thickness. Various studies have measured the thickness of skin (Lee 1957; Shephard & Memma 1967) with age causing a decrease in thickness with males having thicker skin than females. This was confirmed in the Brussels study (Clarys et al. 1987). The effect of skin thickness is most marked with lean individuals. The subscapular site has the highest proportion of skin thickness as a percentage of the caliper reading (28.1 %) and may thus be limited in the prediction of adiposity.

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1.4 Adipose Tissue Patterning

The anatomical positions of adipose tissue are known to vary widely (Edwards 1951; Garn 1955, 1971: Mueller & Stallones 1981). Alternative methods of comparison have been made with Garn (1955) using Z score profiles, Feldman et al. (1969) using ratios, and Mueller and Reid (1979), Mueller and Wohlleb (1981) and Bailey et al. (1982) using a range of statistical approaches. Skin folds were considered by Ashwell et al. (1978, 1982, 1985) to be unreliable in obese subjects so alternative methods based on diameters and circumferences have been used to examine fat distribution. The findings from the Brussels study (Martin et al. 1985) indicated that the prediction of directly measured adipose tissue mass from subcutaneous adiposity should involve a number of skinfold sites and particularly those of the lower limb. The caliper reading of front thigh, for example, correlated at r = 0.89, whereas the triceps measurement, so often favoured for adiposity prediction, was not significantly correlated at the 5% level. These results suggest that it would be advisable to include lower limb sites in any prediction formulae, thus confirming the observations of Jackson and Pollock ( 1978).

1.5 Fat Iraction in Adipose Tissue

A necessary assumption concerning fat prediction is that the fat content is relatively constant in adipose tissue. Not only do the published values represent a broad range (Martin 1984), bUi Pawan and Clode (1960) and Thomas (1962) have shown that as adiposity increases so does the fat content in the adipose tissue. The Brussels study did not undertake chemical analysis but a decreased water content and an increased fat content was noted as adiposity increased. This supported the notion that similar skinfold measurements could contain different fat concentrations, once again making fat prediction prone to error.


1.6 Subcutaneous Fat in Relation to Internal Fat

From the aspect of pathology and metabolic disease the proportion of internal fat may be of great significance, and thus it is important to assess the relationship between the measurable subcutaneous fat and the internal fat. The third compartment of intramuscular fat must not be overlooked, but in the Brussels study this was included within the internal adipose tissue. Clarys et aI. (1987) stated that 'there will be no fixed proportion of internal to external fat', yet Martin et al. (1985) contend that as the slope of the regression lines are so similar both males and females accumulate about 200g of internal adipose tissue for every kilogram of subcutaneous adipose tissue. The correlations between internal adipose tissue mass and subcutaneous adipose tissue mass were r = 0.75 in males and r = 0.89 in females (Martin 1984). Although the slopes relating internal and external adipose tissue are similar in males and females, the intercept is very different, with males having a higher proportion of internal adipose tissue mass per kilogram of external adipose tissue. This difference may account for the distinction between the sexes in terms of mortality from fat-related conditions such as diabetes and coronary artery disease.

1.7 Surface Area

The Brussels study enabled a comparison to be made between 9 anthropometrically based prediction formulae and dissected skin surface areas. The surface area formulae were given in Martin et a1. (1984) with only 2 of the 9 producing significantly different results from the direct measurements. They considered that the Dubois and Dubois (1916) formula was as accurate as any of the available formulae and should, therefore, be retained for the prediction of body surface area.

2. Flit C~ll Sitt 11l1li N"mbtr

During the past 2 decades a great deal of interest has been shown in the cellularity of adipose tissue. Two opposing viewpoints were held concerning the

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relationship between adipocyte cellularity and obesity. Obesity was considered to be primarily related to an increase in fat cell number or due to increased adipocyte size (Bray 1970; Brook & Lloyd 1973). These differences may have arisen as a result of variations in the methodologies adopted by the different researchers. The main reasons for disagreement included the sites chosen for removal of adipose tissue samples, the methods of measuring adipocyte size and cell number within the body. The current view is that obesity is caused by both fat cell number and increased adipocyte size.

2.1 Site Variability

The literature is not in agreement over the preferred sites for removal of adipose tissue samples. Analysis has been made on tissue removed from I or more subcutaneous fat depots and from tissue surgically removed from deep depots. Hirsch and Knittle (1970) investigated the cellularity of obese and non-obese subjects and based their findings on subcutaneous fat tissue. They used the method developed by Hirsch and Goldrick (1964), which involved a series of stabbing motions with a syringe into the subcutaneous fat layer to remove shreds of tissue, each containing between I ()4 and lOs cells. A problem with this technique is the difficulty of obtaining a representative sample of the entire depot. When Hirsch and Knittle (1970) analysed 3 samples of subcutaneous tissue removed from the buttocks, arm and abdomen of 23 subjects, they found the average coefficient of variation of the samples was 10.44%.

Work indicating variations in cellularity between different fat depots casts doubts on the validity of using tissue samples from only one site. Salans et a1. (1973) investigated the variability of adipocyte size within the body in 78 obese and 21 non-obese subjects and found size variations between different subcutaneous depots and different deep depots, as well as between subcutaneous and deep depots. A similarity in intersite variability was shown between obese and non-obese subjects, with a total variance of 18% attributed to variation between sites within each subject. The cell size within


subjects differed by more than 100% in some subjects but only slightly in others. The reliability of these results was checked by analysing duplicate samples and this variability was found to be quite small. Clarkson et al. (1980) found gluteal cells were significantly larger (p < 0.01) than abdominal and subscapular adipocytes. confirming the variation in subcutaneous cell size.

From these studies it can be seen that extrapolation of cell size from a single fat depot to the whole body may be misleading. This is particularly important when estimates of total adipocyte number are based on cell size.

2.2 Reliability

Clarkson et al. (1980) also tested the reliability of adipocyte size assessment over time. comparing samples obtained by the same method on 2 separate days. No significant difference between the measurements for days I and 2 were found for any region.

2.3 Method Variability

The variability introduced in estimating total adipocyte number by choice of sample site is compounded by variability in the methods of determining fat cell size. Hirsch and Gallian (1968) developed a method of preparing tissue samples which has been widely used by other researchers, and is considered the criterion method. It involved incubating washed samples of known weight in a solution of osmium tetroxide in a collidine buffer for 24 to 72 hours. The contents were then filtered to remove fibrous tissue, and the number of free cells was determined by a Coulter counter. The average adipocyte size was expressed as the cell lipid content (mg/cell) using the following equation:

(Wet weight. ILg) x (ratio of lipid to wet weight)

Total number of cells in sample

This method of determining cell size is dependent on the assumption that the diameter of the fat

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cell is the same as the diameter of the fat droplet within the cell.

Hirsch and Gallian (1968) compared this method of analysing samples with 3 other methods. One of the other methods also involved fixation with osmium tetroxide, but the cells were sized in a Coulter automatic particle size distributor analyser. In the other 2 methods the samples were incubated in a solution of collagenase to obtain free cells and fixed in a mixture of trichloroacetic acid and glutaraldelhyde. After fixation the cells were filtered and processed. Cell size was determined either from a photomicrograph and Zeiss particle cell analyser, or by a Coulter counter. They used the mean of the 3 methods as an estimate of 'true' cell size and compared the individual data with it to determine the most accurate method of measurement. However, they only compared the different methods visually and did not use any statistical tests to compare similarities or differences between the methods. From their visual comparison they concluded that fixation with osmium tetroxide was the most accurate method for determining cell size. They tested the reliability by measuring cells from epididymal fat pads of 4 Sprague-Dawley rats and found the standard error for the mean cell number and size was between 5 to 20% of the mean. They recommended this process for accuracy and general applicability to adipocytes of all sizes in humans and animals.

All counting techniques have inherent reliability problems, but the Coulter counter has the additional problem of coincidence error. This occurs when 2 or more cells simultaneously enter the counting aperture and are recorded as a single larger cell. To minimise this error, Hirsch and Gallian (1968) used a correction curve for each aperture size. The validity of the osmium tetroxide technique has been criticised for its inability to measure cells smaller than O.OIILg of lipid.

For a more detailed analysis of the methods of measuring the number and size of fat cells see the review of Gurr and Kirtland (1978). The image analysing computer (Pallier et al. 1985) may be used in the determination of fat cell sizes when a large


number of samples are necessary in a specialised hospital service.

Recent work on hyperplasia and obesity has shown that the failure to detect small cells in the past has been responsible for creating the impression of constancy of cell number in adulthood. This has been challenged with the development of photo microscopic methods capable of detecting cells too small to be measured by Coulter counter. Sjostrom et al. (1971) compared the reliability of a photo microscopic method with automatic determination of osmium-fixed cells. For the microscopic measurements they used frozen cut tissue, which they found easier to prepare than normal methods, and reduced errors due to shrinkage and thinness of slices. One hundred cell diameters were measured with a Zeiss photomicroscope and the results compared with cells measured by the other method. The microscope technique had a standard error of 2.6% which decreased with increasing diameters, and the result correlated closely with the osmium tetroxide method.

The reliability of photomicrographs was also researched by Lavau et al. (1977). In duplicate experiments the photomicrographs had a correlation of r = 0.98, with one-quarter of cells showing more than 15% deviation. Samples analysed by osmium tetroxide had a correlation ofr = 0.99 for duplicate counts, but slightly larger variability, which may have been related to sampling artifacts due to the small samples. Comparison of the cell size distribution showed excellent agreement. Lavau et al. (1977) felt the less favourable comparison by Hirsch and Gallian (1968) was influenced by their methodology. In their study, the latter had a 2-hour period between preparation of the coUagenased fixed cells and taking the photographs. On the photomicrographs they observed a great deal of breakage of large cells due to cell lysis. Lavau et al. (1977) thought this was responsible for the difference in their results. They avoided this problem by using a quicker method of measurement. Other advantages of this method were its cheapness (osmium tetroxide is expensive, although it can be recycled) and provision of a permanent visual record of the sample. This allowed flexibility of time between

20

collecting the sample and taking the measurements. By its close comparison with the osmium tetroxide method, this method is considered to be suitable for analysing tissue from humans.

These advantages of the photomicrographic method were the reasons Gurr et al. (1982) chose to use it in their study of the relationship between adipose cellularity and the rnass-distribution of body fat in humans. In contrast to the claim of Sjostrom and Bjomtorp (1974) they found this method was unable to measure cell diameters smaller than 15"m. Because of this limitation they made the distinction between cell number and measurable cell number, using the latter term throughout their study.

Whilst these are the 2 main methods employed In determining cell size, other methods have been used. Lemmonnier (1972) measured adipocyte size and number by a histological procedure. The cells were stained for fat cell membranes and the volume of the cell was calculated from its diameter. The results of this method were confirmed by the osmium tetroxide method, but it is not generally used as it is a very time-consuming method.

The DNA content of a tissue sample has also been used to estimate adipocyte number, but it provides a very inexact estimation due to the contribution of DNA from many other cell sources within the sample of tissue.

Interest has been shown in the minimum number of cells that have to be measured to give reliable results. DiGirolamo et al. (1971) extensively investigated the reliability and validity of their methods for determining adipose cellularity. They concluded when using photomicrographic measurements the reliability of the determination of mean cell measurements increased as the number of cells increased. A minimum of 300 cells was recommended to keep the coefficient of variation for estimated cell volume below 5%. Using sequential estimation analysis, Oarkson et al. (1980) found it was possible to estimate reliably adipocyte diameter from less than 100 cells.

The reliability and validity of cell size measurements influences the estimation of total adipocyte number. Many researchers estimate total cell

Measurement of Body ComposJllOn

number by dividing the total body fat by fat cell size. The site of tissue removal and method of determining cell size may result in an over- or underestimation of total cell number. Total body fat has been determined from skin fold measurements. total body water. immersion and 40K methods. Any unreliability in these methods will be another source of error. There is debate in the literature concerning the effect of using mean cell sizes obtained from I or more fat depots. Bjorntorp (1983) states that as deep fat is included in the total body fat measurement. deep fat cells should be included in the mean cell size measurements. Failure to include these cells would result in an underestimation of total adipocyte number. Salans et al. (1973) found the mean cell size from all 3 subcutaneous depots correlated higher (r = 0.97 for non-obese; r = 0.93 for obese) than did the size at any individual subcutaneous site. Clarkson et al. (1980) found that if only changes in cell number were being compared. cells from just the gluteal tissue gave valid results.

Estimates of total cell number of organisms have been at the centre of investigations into obesity. The original concept of adipose cell number increasing during early development and remaining constant during maturity was based on longitudinal studies of rats by Hirsch and Han (1969) and supported by Stiles et al. (1975) and Oscai et al. (1974). This was further corroborated in humans by Johnston et al. (1984) during an examination of mean fat cell diameters from 1 to 48 months of age. In this study the fatness on the trunk may be explained by changes in fat cell size with mean fat cell diameter greatest in the 9- to II-month group. When Hirsch and Knittle (1970) performed crosssectional studies on people they found similar results. These were reinforced by observing cellular changes in 19 adults who individually lost approximately 50kg bodyweight. The fat cell number in these subjects remained elevated. but cell size was greatly reduced. From these studies they concluded that obesity of early onset was associated with adipocyte hyperplasia. The findings of Sjostrom and Bjorntorp (1974) supported this view. They found adipocyte cellularity was dependent on age. with

21

cell hypertrophy associated with the onset of obesity after 40 years of age.

This distinction between hyperplasia and hypertrophic obesity has been confirmed and rejected in the literature. Jung et al. (1978) highlighted the lack of suitable controls. inclusion of severely obese subjects and failure to measure small deep cells. In their view, if hypercellularity exists. it does so due to the inadequacy of assessment techniques. They found a slight increase in the number of fat cells in the obese, but that no relationship existed between cell number and the age at onset of obesity. From the differences in the mean size of subcutaneous and omental cells, they considered that the techniques for counting resulted in an underestimate of cell number. The constancy of cell number has been challenged by several studies. Lemmonnier (1972). Faust et al. (1978) and Betrand et a1. (1984) all found new cells developed in adult rats fed on a high fat diet. However. care has to be exercised in extrapolating the results of studies on rats to people with. in some cases. different results being obtained with different species of rat. In one of the few cross-sectional c;tudies performed on mainly non-obese human subjects. Chumlea et a1. (1981) found total body fat and percentage of body fat increased with age and these increases were associated with increased adipocyte number, especially in men.

These studies suggest the idea of a critical period for determination of cell number needs reexamination. Faust et a1. (1978) found the cell number increased when adipocytes in rats reached a specific size. This has been confirmed in other investigations. but it was not known whether the increase was due to cell division or differentiation. General opinion suggests precursor cells are present and these become committed and gradually filled with fat when other adipocytes are full. The small size of these cells makes them difficult to measure. hence the original concept of constancy of cell number was due to limitations of methodology compounded by poor empirical design.

3. Estimation from Skinfold Equations

The interest in body composition assessment. particularly the fat component. has prompted in-


vestigators to find a simple, inexpensive method of prediction. The use of skin folds and skinfolds combined with other anthropometric measures, seems in part, to provide the solution. The technique of regression analysis has meant that numerous workers have been able to predict body density, and hence body fat, from skinfolds. These have been validated against criterion methods such as hydrostatic weighing or whole body potassium. Many of the formulae that have been produced are specific to a particular sample of the population, dependent on age, sex and even specialist activities (Davies 1981).

Lohman's review (1981) pays particular attention to measurement errors, to the importance of crossvalidation studies and the use of curvilinear equations. He recommends that body density should be the criterion-dependent variable, a standard measurement procedure be adopted, and large and random samples be used in future studies.

3.1 Population-Specific Prediction Equations

Most prediction equations are population specific. Equations have been produced for college age men (Brozek & Keys 1951; Durnin & Rahaman 1967; Wilmore & Behnke 1969), college women (Katch & McArdle 1973; Weltman & Katch 1975; Wilmore & Behnke 1970; Young et al. 1962), nonCaucasians (Nagamine & Suzuki 1964; Satwanti et al. 1977), soldiers (Haisman 1970; Pascale et al. 1956), male youths (Katch & Michael 1969), previously somatotyped groups (Bulbulian 1984), boys and girls separately (Brook 1971; Parizkova 1961), children of specific ethnic groups (Harsha et al. 1978), obese girls (Seltzer et al. 1965) and premenarcheal and postmenarcheal girls (Young et al. 1968). Socioeconomic status has even been examined with respect to skinfold sites (Mueller 1986), although no body fat regression equations were established.

A range of statistical procedures have been used to optimise the regression equation. Johnston (1982) considered that stepwise regression produced the best linear combination of variables. This approach, although producing the best model for a

22

given sample, may predict for another sample with less precision. Mukherjee and Roche (1984) suggested that the maximum r2 improvement method is superior to conventional stepwise selection techniques. They estimated the final regression coefficients and associated standard errors by the 'jackknife' method which divided the observations into a number of subgroups and obtained the same number of partial estimates by omitting I subgroup at a time. Mueller and Stallones (1981) utilised principal components analysis to predict overall fatness from a number of skinfold sites. They considered that leg fat was an important indication of differences in anatomical fat distribution. Factor analysis was also the method used by Jackson and Pollock (1976) to identify body composition factors from anthropometric measurements including skinfolds. The multiple correlations between factor scores ranged from r = 0.88 to r = 0.94 (both sexes) and the use of multivariate scaling models was considered to provide a valid measure of body composition for use in the field.

The dilemma of population specificity has been examined differently by those workers who have attempted to apply theoretical principles in the derivation of equations. Included in these would be Katch et al. (1974), Katch and Weltman (1975), Weltman and Katch (1975, 1978). Their approach was to predict body segment volumes from geometric approximation and thus calculate total body volume by summation. Prediction equations for total body volume were considered to be less population specific if based on these theoretical principles.

3.2 Generalised Prediction Equations

Population specificity of prediction equations has been confirmed by crossvalidation studies (Jackson & Pollock 1977), and it is clear that age (Durnin & Womersley 1974), physical fitness (Flint et al. 1977) and lack of homogeneity (Pollock et al. 1977) will necessitate their use. However, general applicability of prediction equations has been attempted to increase scientific economy (see Jackson & Pollock 1982). Although the commonly cited


article by Durnin and Womersley (1974) took full account of the ageing process, many populationspecific regression equations failed to accommodate age adequately. Prediction bias could be caused by the continued use of linear equations when a curvilinear relationship between skinfold and percentage fat has been established in a number of studies (Allen et al. 1956: Chen 1953: Chien et at. 1975a: Jackson & Pollock 1978). Mayhew et at. (1985) examined a range of prediction formulae to predict body composition in female athletes. They found that the most satisfactory formulae were either population specific athletic formulae (Mayhew ct al. 1983) or the Jackson et al. (1980) generalised formula. They confirmed that the relationship between the sum of skinfolds and body density was curvilinear and was best described by a polynomial equation.

Generalised equations based on curvilinear relationships have been crossvalidated in males (Jackson & Pollock 1978), females (Jackson et at. 1980), male athletes (Sinning et al. 1985), and in female athletes (Sinning & Wilson 1984). Care needs to be taken in using generalised equations for predicting percentage body fat in women over 40 (Jackson et al. 1980), but there appears some merit in the provision of these population non-specific equations. However. the biological factors imposing on body composition should never be underestimated, with fat patterning, the proportion of subcutaneous fat and the compressibility of skin fold all providing potential specificity. Norgan and Ferro-Luzzi (1985) compared 5 generalised equations and found them all to be statistically different. Their conclusion was consequently to question the validity of the generalised equation. Lohman (1981) in his review paper, applied an alternative strategy, combining the data of Boileau et al. (1971), Lohman et al. (1978). Sinning (1974) and Sloan (1967) into a single regression equation utilising the sum of triceps, abdomen and subscapular skinfolds only.

3.3 Equations for Infants and Children

Infants and children present a special population for whom specific equations have been produced. Oakley et al. (1977) produced standards of

23

triceps and subscapular thicknesses for nearly 1300 Caucasian newborn infants. They showed that females had greater skinfold thickness than males and that in both sexes the values declined after 40 weeks of gestation. They concluded that skinfold thickness could be a useful pointer to nutritional status in neonates. Dauncey et al. (1977) were able to compare total body fat with published data from cadaveric analyses and to produce a formula to predict total body fat from skinfold measures at 2 sites. Growth rates were examined serially up to 40 weeks after birth, and it was noted that the boys increased musculoskeletal tissue more than girls, who showed a relatively greater increase of fat.

Older children have been studied by AAHPERD (1980), Boileau et al. (1981), Corbin and Zuti (1982), Johnston (1985), Lohman et al. (1975), Nelson and Nelson (1986) and Parizkova (1961). Triceps and subscapular skinfolds were considered to be adequate predictors (r = 0.71) by Corbin and Zuti (1982) but did not produce a sufficiently high correlation in the girls. Johnston (1985) attempted to predict obesity from triceps skinfold and relative weight alone. The conclusion was that only when both indicate obesity could the diagnosis be considered confidently.

A larger number of anthropometric indices were examined as independent variables by Lohman et al. (1975), but the step-down multiple regression procedure established that bodyweight and the 2 skinfold measures of upper arm and back (as described by Allen et al. 1956) accounted for 91.3% of the variation. This study applied a crossvalidation procedure satisfactorily and also established no racial differences in the prediction of lean body mass. Nelson and Nelson (1986) found that White children had larger skinfold thicknesses at most sites, but there was only a significant difference (p < 0.01) at the total of the limb sites. For all classifications of age, sex and race they found that the combination of triceps and subscapula was the best predictor. Boileau et al. ( 1981 ) returned to the subject of biological variability considered in the section above. They concluded that in the case of 8-to II-year-olds, although body density can be estimated from 2 skinfolds as well as from a range


of anthropometric measures, the poor crossvalidation from one sample to another still required further examination of methodological and biological variability in children.

3.4 Equations for Athletes

A number of regression equations have been reported for specific sporting groups, e.g. distance runners (Pollock et al. 1977), volleyball, synchronised swimming, field hockey (Hall 1977), college wrestlers (Sinning 1974), baseball, track and field, football, tennis (Forsyth & Sinning 1973), gymnasts (Sinning 1978), swimmers (Hall 1977; Meleski et al. 1982), male athletes (Forsyth & Sinning 1973; Pollock et al. 1977; Sinning 1974) and female athletes (Mayhew et al. 1985; Meleski et al. 1982; Sinning 1978). Thorland et al. (1984a,b) have more recently reported a crossvalidation study in which the original regression equations were based on the events of track and field, gymnastics, diving, and wrestling. The equations were validated against a sample of male wrestlers and female track and field athletes. The equations thus derived either \,nvolved the sum of 3 or 7 skinfolds and both were considered sufficiently accurate for the field testing to estimate body density. It was interesting to note that in the Thorland (1984a) paper the quadratic equation proposed by Jackson and Pollock (1978) was the most suitable one, thus supporting the contention expressed earlier that the standard error of the mean is reduced by using a quadratic as opposed to a linear equation.

3.5 Biological and Methodological Measurement Errors

The estimated biological variation in reference man is considered to be up to 3.8% when determining body density. This will be compounded by a further error when estimating percentage fat from body density of about 3.9% in the general population and 2.6% in a restricted population. Total body fat, for a given skinfold thickness, is greater in older people. Also for a given skin fold, women have more fat than men due to the high level of

24

sex-specific essential fat. As total fatness increases so does a greater percentage of the fat stored internally. Body size in relation to fatness is inevitably going to cause errors when using skinfold predictions. Katch et al. (1979) have attempted to resolve this by proposing that skinfolds and surface area formulations are incorporated. This would ensure that both body size and fatness measures are included in body composition estimation.

Reproducibility of test scores can be high as a result of careful site location, high quality of training and repeat measuring until values become consistent. This was considered fully by Katch and Katch ( 1980) and their recommendations for measurements and statistical considerations are recommended for the aspiring anthropometrist. Site locations based on surface measurement, and marking based on photographic information (e.g. Behnke & Wilmore 1984; Grant 1979; Jackson & Pollock 1985; McNair et al. 1984; Pollock et al. 1980) are essential, with particular attention being paid to the angle of the skinfold measurement. A novel technique for determining anatomical landmarks has been proposed by Drerup and Hierholzer (1985). They used surface coordinates to establish local surface shape and measured these by optical methods such as moire topography or rasterstereography. This method, although providing a sensitive index, would be more appropriate for clinical pathologies such as idiopathic scoliosis than for skinfold site assessment. Test-retest reliability should be established as a matter of course for each tester. Intratester correlations from r = 0.96 to r = 0.98 for skinfold measurements (Tanner & Weiner 1949) and r = 0.92 to 0.96 for abdomen, chest and arm skinfolds have been reported (Keys & Brozek 1953). Jackson et al. (1978) reported significant differences (F ratios resulting in p < 0.01) for chest, subscapula, suprailiac and thigh skinfold between 3 testers. However, the sum of 3 or 7 skinfolds was not significantly different. Lohman et al. (1984) found that the triceps and subscapular sites showed the least variation between 4 testers, whereas the suprailiac, abdomen and thigh sites showed the most variation. Edwards et al. (1955) found that the pressure exerted by the caliper had a significant


effect both on the skinfold measurements and the consistency with which the measure was repeated. Keys (1956) recommended a caliper pressure of 10 g/mm2 as caliper pressure over 15 g/mm 2 may cause subject discomfort. Results have also shown a significant difference when the range of pressures changed within the range of the jaw openings and conclusions have stated that the calipers should not vary in pressure by more than 2.0 g/mm 2 over a range of 2 to 4mm. With the introduction of cheaper, plastic calipers, Leger et al. (1982) looked at the validity of measurements obtained and compared them to the more expensive steel Harpenden caliper. The results indicated consistency amongst calipers of the same company; however, the Ross calipers systematically overestimated the true aperture by 16.1 % over the investigated aperture range as compared to 4.1 % for the Harpenden calipers. Leger and his coworkers also compared 2 plastic calipers - the Ross and the McGaw (the latter validated by results published by Burgert & Anderson 1979). They found that both plastic calipers displayed almost identical skinfold thickness and fat percentage results. Womersley et al. (1973) and Sloan and Shapiro (1972) did not find any systematic difference between measurements at any site made by the Harpenden and Lange steel calipers. In contrast to this, Lohman et al. (1984) found that the Lange consistently overestimated the results of the Harpenden caliper at least 1 to 2mm per site. Lohman et al. (1984) also compared 4 different calipers and the standard error of measurement (SEM) was generally ± 1 mm or less, with the Lange and Adipometer calipers showing a larger SEM than the Holtain and Harpenden calipers. A recently developed electronic body fat calculator (Syndex) will disregard all values that are not within Imm of each other during multiple measurements (personal communication). This reduces the variability in repeat measurements and with the automatic calculation of percentage fat reduces the possibility of transcription errors.

As Pollock and Jackson (1984) state: .... perhaps it is not as necessary to be as concerned as previously thought about using only certain standardised and calibrated calipers for skin fold meas-

25

urements.' They go on to recommend greater validation of new calipers and that future research which uses these instruments should take into account which caliper the tester has most experience with and with which they feel most comfortable.

These methodological factors became increasingly more important when changes due to experimental intervention (e.g. diet) are assessed. A study by Zwiren et al. (1973) compared 5 equations during a reduction experiment and found that only those of Allen et al. (1956) and Sloan et al. (1962) produced values that were not significantly different from the criterion method of hydrostatic weighing. This paper is, however, some 14 years old and the many alternative regression equations established since that time may reveal some more suitable equations for examining changes in body fat. The most commonly used skinfold site for nutritional assessment has been the triceps, but in the examination of weight loss as part of nutritional assessment, Bray et al. (1978) and Bradfield et al. (1979) considered that more measurements would be appropriate. The sum of triceps and subscapular skinfolds were found to be the best correlate with weight change (r = 0.75 for males, r = 0.74 for females). Grant et al. (1981) appreciated the influence that oedema or subcutaneous emphysema could have on skinfold readings as could the altered pliability of skin caused by the administration of intravenous flutd.

4. Body Density Measurement Methods

Fat has a lower density than lean tissue, therefore relatively fat subjects will have a lower overall density. Thus, a measure of body volume in relation to total body mass will measure body density and an estimate of the proportion of fat to lean tissue can be established. The most commonly used method of measuring body volume is the hydrostatic weighing technique which incorporates Archimedes' principle.

The technique has been described in detail by Luft et al. ( 1963), Katch et al. ( 196 7), Katch (1969), Wilmore (l969a,b) and Sinning (1974). The essential requirements for the tank include sufficient size


and shape for total human submersion, an accurate method of measuring weight, a method of measuring water temperature so that water density can be corrected, and a seat, chair or cradle which has been weighted to prevent flotation. The typical fluctuations of the weighing system during immersion have to be considered. Shephard et al. (1985) report final readings agreeing to ± l00g, Mayhew et at. (1981) quote final readings agreeing within lOOg and Faria et at. (1981) state -three similar readings to the nearest 1 5g'. Shephard et at. (1985) used the mean of the 3 final readings, whereas Mayhew et at. (1981) used the maximal reading. A common procedure is to use the midpoint of the fluctuation, employing the highest value obtained. SUbject unfamiliarity with the procedure usually requires up to 10 readings to be taken ·before they become consistent.

The subject should be several hours postprandial, clean, wearing garments from which all air is expelled, and should have urinated and defecated immediately before measurement [see Noble (1986) for recommendations to reduce errors in underwater weighing).

Various modifications to the simple technique have been successfully incorporated including a snorkel to permit ease of breathing and the simultaneous measurement of residual volume.

Alternative tank shapes and sizes have been used. Katch et at. (1967) found that the prone p0-

sition caused less movement when breathing and this posture was also employed by Warner et al. (1986) when using a small oblong tank. A comparison of a sit-in tank and a Hubbard (prone p0-

sition) tank by Williams et at. (1984) revealed the prone measurements to be reliable (r = 0.997) and compared significantly (r = 0.977) with the seated position.

Inexpensive systems to measure weight hydrostatically include a simple T -bar apparatus suspended in a swimming pool (Clark & Mayhew 1980) and the use of a wooden shell placed within a pool to reduce water movement (Katch et al. 1967).

Although many researchers use autopsy scales, a force cell has the advantage that the analogue

26

output can be used to make more precise measurements from the fluctuating readings. Computer software can establish the midpoint of the fluctuations and use that as the basis for subsequent calculations. Body density can thus be calculated according to the formula of Brozek et at. (1963) which relates body weight in air (W A), body weight in water (WW), density of water (OW), residual volume (RV) and the volume of gas in the intestines (VOl) in the following manner:

Body density = WA

(WA - WW) - (RV + VGI)

OW

The volume of gas in the intestine is often taken as a standard lOOml (Buskirk 1963), although this value may be too high for children, and become relevant if increased by ingestion of carbonated drinks (Durnin & Satwanti 1982).

The measurement of residual volume can take place whilst in the tank or separately, and there is considerable controversy as to which is the most appropriate procedure. Using gas dilution methods and with the body submerged, residual volume has been shown to increase (Brandom et at. 1981; Girandola et al. I 977a), to decrease (Agostini et al. 1966; Bondi et al. 1976; Jarrett 1965; Ostrove & Vaccaro 1982; Robertson et al. 1978; Sawka et al. 1978), and to remain the same (Arborelius et at. 1972; Craig & Ware 1967; Dahlback & Lungren 1972; Etheridge & Thomas 1978; Prefaut et at. 1976; Sloan & Bredell 1973). Lohman (1981) indicated that measurement of residual volume produces similar errors whether measured in the tank or elsewhere, yet Oirandola et at. (1977a) have shown significant differences. Marks and Katch (1986) examined biological and technological variability in the measurement of residual lung volume. They found that the within-subject variance was accounted for by biological variance (72%), technological variance (19%) and trend effect variance (9%)

respectively. The oxygen dilution method across subjects produced a reliability coefficient of r = 0.95.

The options available include nitrogen washout (Wilmore 1969b), helium dilution (Com roe et at.


1962). and oxygen dilution (Wilmore et al. 1980) and the method of choice usually depends on the facilities available.

For those unable to gain access to such equipment. Mayhew et al. (1980) concluded that the 2 most acceptable techniques for estimating residual volume was 24% of vital capacity (Wilmore 1969a) and the anthropometric method of Polgar and Promadht (1971). Total lung capacity was compared with residual volume by Timson and Coffman (1984) and provided the measurement was made in water. no significant differences were detected. However, the prediction of body density from total lung capacity measured in air resulted in an overestimation.

It has been common practice to convert the value for body density into an estimate of percentage of body fat. but this procedure is based on a number of assumptions enumerated by Wilmore (1983). The density of fat and lean is assumed to be known and to be 0.90 and 1.10 g/cm 3 respectively (Schemmel 1980). Leonard et al. (1983) used mean densities from the studies of Pace and Rathburn (1945), Siri (1956a). Brozek et al. (1963) and Chien et al. (1975b). This produced an average value of 0.9168 g/cm3 for adipose tissue and 1.0997 g/cm 3 for lean tissue. This would thus provide a further prediction of fat from the more commonly used equations of Behnke (1945), Keys and Brozek (1953). Siri (1956b), and Brozek et al. (1963). Of these. the Brozek et al. (1963) equation:

4.570 % Body fat = (---- - 4.142) x 100

D and that of Siri (1956b):

4.950 % Body fat = (---- - 4.500) x 100

D

are the most commonly used. The assumptions concerning constancy of den

sity have also been criticised. Athletes are considered by Macdougal et al. (1983) to have denser bone and muscle which could lead to overestimation of body fat (Wilmore 1983). Osteoporosis in the elderly will cause a decrease in bone density and an overestimation of body fat (Weredin & Kyle 1960)

27

with Durnin and Womersley (1974) also showing that the mineral content of bone increased in the young and decreased in the elderly. The bone minerai content in preadolescents has been shown by Lohman et al. (1984) to be 5.4%. leading to a value of 1.084 g/cm 3 for estimated fat-free body density. It is thus proposed to revise the prediction equation for preadolescents so that body fat = (5.30/D - 4.89) x 100. Eight body densities in excess of 1.1 g/cm 3 were reported by Adams et al. (1982) in an assessment of 22 professional football players. According to the Siri (1956a) formula this would result in negative fat percentages, supporting the contention of Jackson and Pollock (1985) that a curvilinear relationship exists between body density and subcutaneous fat 'which will increase errors at the extreme ends of the distribution'. Until more precise cadaveric information is provided, the solution may be to transform values to percentage fat only when necessary. and to retain body density measures for scientific comparisons.

Other experimental errors refer to the state of the subjects prior to testing. It has been shown that food consumption can change the prediction of body fat by about I % (Durnin & Satwanti 1982) with carbonated drinks increasing the error by a further 0.5%. Durnin and Satwanti (1982) also showed that the extent of the underwater expiration had a similar effect on body fat determination. The influence of hyperhydration and dehydration also influenced body fat determination (Girandola et al. 1977b), but this error was considered only to become influential when relative measures were being made such as during training or dieting. However, with high fluid loss during intensive training, and high fluid retention during premenstTUation. it is important to assess these aspects whilst using the hydrostatic technique.

The measurement of body density from water displacement has been used in some laboratories as an independent measure and a combined system was described by Macdougal et al. (1983). In this case water displaced from the tank caused a proportional rise in a manometer attached to the tank. Air or helium displacement has also been used to measure body density (Gnaedinger et al. 1963). This


has the advantage of the subject not being required to exhale underwater and thus increases the likelihood of compliance. A more recent alternative method has been proposed by Gundlach et al. (1980). This involves measuring body volume by plethysmQgnlphy and requires the subject to be inside a polyurethane foam-filled chamber while volume and pressure changes are recorded. It is considered to be as accurate as hydrostatic weighing (r = 0.99, p < 0.001), is quicker, is less intrusive, all ages of subjects can be tested and the measurement of residual volume is not required.

5. A"thropoIMtric Allftlme"t 0/ MIIICU a"d Fat Ami

In nutritional analysis such as protein-calorie malnutrition (Gray & Gray 1980) or in cancer (Neumann et al. 1982) specific body fat areas (e.g. the arm) are often measured in addition to total body fat. Similarly muscle cross-sectional areas and volumes can be a useful indication of nutritional status in addition to strength.

Mid-upper arm circumference has been used in nutritional assessment to measure the degree of protein-calorie malnutrition. The technique has been described by the Committee on Nutritional Anthropometry (1956), by Jelliffe (1966) and Fomon (1978), and reference data for children was provided by Wolanski (1969). Upper arm circumference provides an undifferentiated measure of muscle, fat and bone, so various attempts have been made to estimate cross-sectional fat and muscle area. Gurney and Jelliffe (1973) used triceps skinfold (TSF) and mid-upper arm circumference and produced a nomogram to assist in the calculation. Mid-arm muscle circumference is equal to:

Mid-arm circumference - [TSF x r]

and muscle area is equal to:

Muscle circumference2/4..-

Muscle volume can be estimated approximately from 20% upper arm length x muscle area (Jelliffe & Jelliffe 1982).

These equations were compared with com put-

28

erised axial tomography ~y Heymsfield et al. ( 1982). Bone free arm muscle area in men was estimated from [(muscle arm circumference - r X TSf)2j 4 r] -10 and for women [(muscle arm circumference - r X TSf)] -6.5. They also used corrected arm muscle area (AMA) to predict total body muscle using the formula (ht) [0.0264 + (0.0029 x corrected AMA»).

The use of mid-arm muscle circumference to estimate the muscle compartment of the body and thus to assess nutritional effects was used by Bertrand et al. (1984). This measure was criticised by Lerner et al. (1985) because they did not accept the assumptions that the mid-arm circumference and the mid-arm muscle compartment are of a circular configuration. They also contested that the subcutaneous fat ring surrounding the arm is concentric. This was accepted by Bertrand et al. (1985) although they maintained that the clinical findings were sustained even though a relatively crude measure was employed.

Arm fat area can also be estimated from: (arm circumference2/4r) - arm muscle area (Trowbridge et al. 1982) or from a formula involving skinfold thickness, i.e. (skinfold x circumference/ 2) + (r X skinfold2/4) [Gray and Gray (1980)]. Himes et al. (1980), using the same formula for the arm fat area, found that cross-sectional fat areas do not estimate body density any better than skinfolds. However. fat weight was found to be better estimated by fat areas than skinfold thicknesses.

6. Simple Methodl/or Mea"ri", Adipolity

Weight for height indices have been used in a variety offorms to indicate adiposity. This has often been in an attempt to predict body fat from a simple measure. Satwanti et al. (1980) was typical of these when various weight for height indices (weight/height; weight!height2; weighto.33/height; height/weighto.33) were correlated with densiometrically determined body fat. The correlation coefficients ranged from 0.57 to 0.82 depending on the index and the age, but averaged r = 0.73 for weight/height, r = 0.76 for weightjheight2 and r =

Mea5urement of Body Composition

0.76 for weighto. J3/height. These compared favourably with Brockett et al. (1956) whose correlations with body density were generally lower for men. and with Keys et al. (1972) who showed higher correlations for students, but lower ones for executives. Womersley and Durnin (1977) with 324 women showed correlations of r = 0.81 for weight/ height, r = 0.82 for weigh1/height2 and r = 0.84 for weight0 33/height.

There is no shortage of studies comparing weight for height indices with estimates of body fat (Baecke et al. 1982: Billewicz et al. 1962; Forbes & Amirhakimi 1970; Lohman et al. 1975: Roche et al. 1981; Rolland-Cachera et al. 1982; Seltzer et al. 1965; Shephard et al. 1969; Ward et al. 1975). Both Lee et al. (1981) and Norgan and Ferro-Luzzi (1982) expressed concern over the use of these indices as measures of adiposity because of the correlation with height. Norgan and Ferro-Luzzi (1982) found that weight/height2 had the lowest correlation with height (r = 0.07), the highest correlation with percentage fat (r = 0.75) and the lowest standard error of estimate. They also found that the addition of age improved the accuracy of estimation of percentage fat. An anthropometric index of obesity needs to be independent of stature, yet highly correlated with weight (Lee et al. 1982). Roche (1984) using data from the Fels longitudinal study, showed that this theoretical principle may be invalid for boys and doubtful for girls aged less than 14 years of age. Benn (1971) considered that weight divided by height raised to a specific exponent or power function would be a more suitable index of obesity. The so-called 'Benn Index' involves the calculation of age. sex and race specific regression coefficients of weight on height. Frisancho and Flegel (1982) found that weight/height2 produced as high a correlation with sum of skinfolds as did the Benn index. Their conclusion based on 16,459 adult Black and White subjects was that adequate information about body size and fatness could be provided from a simple height-weight ratio and skinfold thicknesses. Garn and Pesick (1982) gave further support to the limitations of the Benn index. once again based on large scale surveys totalling some 54.468 individuals. The Benn index was shown to corre-

29

late with simpler methods. e.g. bodyweight (r = 0.85 to r = 0.98). and it was only fractionally superior to simple height-weight indices in relation to skinfolds. Thus in nutritional terms, particularly for small scale surveys (i.e. less than 2,000 individuals) weight/height2 would appear to be an adequate index of adiposity. It is accepted that these correlational studies are limited in their usefulness to assess accuracy and validity. However. the more applicable measure of standard error of the estimate was not quoted in either case. Another large scale survey (10,021 adults) showed regional differences within the United Kingdom in body mass index (weight/height2) in addition to gender differences (Rosenbaum & Skinner 1985). Eight per cent of the females were above a body mass index of 30. whereas in males the value was only 6%. Wales had the highest proportion (44%) of males with body mass index above 25.0. with the lowest proportion (36%) in the southeast outside London.

Although a body mass index of 25 may appear arbitrary. this figure was used by DiGirolamo (1986) when considering the definition of obesity. He considered that for normal individuals an upper body mass index limit was 25 for men and 27 for women. Moderately obese subjects would have a body mass index between 25 and 30 for men and 27 and 30 for women. with massively obese subjects having a body mass index between 30 and 40. Anything over 40 was considered to be morbid obesity. Garrow (1983) also accepted the body mass index as providing an index of obesity and a value of over 30.0 for females and 25.0 for males is a commonly-used threshold.

Regional adipose tissue distribution has been studied in relation to lipid and carbohydrate metabolism (Krotkiewski et al. 1983). Obesity complications seemed to be associated with the ratio of waist: hip circumference in women, with a high waist: hip circumference ratio being indicative of the male risk profile. A study by Kissebah et al. (1982) showed that age-adjusted values of body mass index and skinfold thicknesses for women were significantly related only to the incidence of myocardial infarction. However. the waist: hip ratio was significantly correlated with myocardial in-


farction. angina pectoris and stroke. In a further study by Larson et at. (1984) the waist: hip ratio was significantly related to the incidence of stroke and ischaemic disease. whereas body mass index and skinfold thicknesses showed non-significance. One stral)ge statistic was noted (Jarrett 1986) in these studies, namely that the lowest risk of ischaemic heart disease occurred in men with high body mass index and low waist: hip ratio. The highest risk was in men with a low body mass index and a high waist: hip ratio. There is contradiction between the Gothenburg study as. quoted by Jarrett and the statements of DiGirolama (1986), although theoretically a highly mesomorphic person could fulfil both criteria satisfactorily.

An alternative approach is to use relative weight, which is the weight expressed as a percentage of the standard weight for age, height and sex. Moynihan et at. (1986) defined obesity as a relative weight greater than 120. This may work reasonably well for growing children, but any values compared with national norms questions the desirability of the national norms. If a population is fatter than is desirable then any relative value may appear acceptable, even though the population itself is not. This brings the debate back to the limitations of any measure involving height and weight which does not take fully into account the composition of the bodily tissues.

One of the complications of the large-scale height for weight surveys such as those produced by the Metropolitan Life Insurance Company (1959, 1983) was the use of body frame categories. The rationale for including frame size is to discriminate between those who are heavy because of either a large fat mass or a large fat-free mass. Roche (I984b) considered that frame size measures should account for a considerable proportion of the variance in weight not accounted for by stature. It should be independent of body fatness and therefore correlated with fat-free mass. Himes and Bouchard (1985) compared 6 measures of frame size with fat and fat-free mass and found similar correlations for each one. They also found that wrist and ankle diameters were the most suitable indications of frame size compared with other diameters includ-

30

ing the elbow. Katch and Freedson (1982) used the sum of the biacromial and bitrochanter diameters (I:A T) in preference to chest and bi-iliac diameters because of reduced measurement errors. They then established the regression line of I:A T against height and divided the subjects into small, medium and large frame sizes by drawing perpendicular lines to the regression line at ± I standard deviation from the mean stature. Having completed this frame size model, gender differences were observed between frame sizes. Differences in bodyweight between frame sizes in males were largely due to differences in lean body mass, whereas in females no increase in lean body mass was observed. Self-assessment of frame size is necessary when using total bodyweight to predict desirable weight as often required with scales available in public places. Katch et at. (1982) examined the differences between self-assessment, the quantitative method outlined above and the use of an expert rater. The differences indicated that neither the self-appraisal nor the expert rater produced values sufficiently close to the criterion method to justify a subjective method as being satisfactory. Inaccurate frame size estimation would cause consequential errors in predicting ideal weight from tables employing them. Frisancho (1984) incorporated frame size into new weight and body composition standards from the US National Health and Nutritional Examination Surveys on 21,752 subjects. Percentiles for weight, skinfolds, and bone-free upper arm muscle area were established by height, frame size and gerider. These percentiles when age-corrected were considered to be of value in differentiating the obese and undernourished.

An alternative, simple method of body fat assessment is to use visual estimation. Sterner and Burke (1986) found that some raters can estimate body fat from photography about as accurately as from skinfold measurements. The degree to which this could be extended to non-experts and without photography is not yet known, but it does at least suggest that a well-trained individual could question any apparent discrepancy between measured and observed body fat.


7. Fat-Soluble Gases

The degree of absorption by the body of inert gases which are more soluble in fat than in water has been used to determine total body fat. The basis of this method is that the magnitude of uptake of these gases by an individual is primarily a function of body fat content. The inert gas absorption procedure is thus essentially independent of variations within the fat-free mass. Body fat (defined as the weight of all ether-extractable material in the body) and fat-free mass can by this method be measured without the need for major assumptions as to the composition of the fat-free mass, and without concern as to differing conditions of age, sex and health (Lesser et al. 1971).

Behnke et al. (1935) attempted such an independent procedure by applying principles from studies of dissolved nitrogen in dogs to measurements of nitrogen in humans. They explained that much of the washout of dissolved body nitrogen comes out of solution in body fat, because nitrogen is about 5 times as soluble in body fat as in water. This suggested that a gas which was more soluble in fat might be applicable using the same principle. The widely used and relatively safe anaesthetic gas, cyclopropane, is about 33 times as soluble in fat as in the fat-free mass, so that in normal subjects in equilibrium, approximately 90% of absorbed cyclopropane is in total body fat and about 10% is in the fat-free mass (Lesser et al. 1960). Total body fat determination by cyclopropane absorption has been measured by Lesser et al. (1960). and cyclopropane and/or krypton by Lesser and Deutsch ( 1967).

Although measurement of the volume of cyclopropane absorbed from a closed respiratory system had proved successful for the determination of total body fat in living rats (Lesser et al. 1952). a difficulty was encountered in its application to human subjects. A direct calculation of the total body fat requires knowledge of the amount of cyclopropane taken up when equilibrium between the environment and the body tissues has occurred. In human subjects. absorption of the gas was found to be much slower than in rats. equilibrium not being

31

reached even at the end of 8 hours (Lesser et al. 1960).

8. Creatinine Excretion

24-Hour urinary creatinine excretion is the most widely used biochemical marker for estimation of body muscle mass. The basis of the method is that creatinine is the only metabolite of creatine which is largely located in muscle (98% of the body creatine pool; Heymsfield et al. 1983). Creatine is synthesised by the liver from 4 amino acids. Whether in its free state or bound to ATP, creatine spontaneously dehydrates at a relatively constant rate to form creatinine which is excreted unaltered in the urine. Thus, an assay of creatinine excretion in a 24-hour urine collection should theoretically reflect the level of total body creatine and therefore total body muscle mass (Grant et al. 1981).

8.1 Method

The method originates from animal studies which showed that urinary creatinine excretion was directly proportional to total body creatine in a number of species (Myers & Fine 1913; Palladin & Wallenburger 1915). A number of studies over the next 70 years (quoted by Lykken et al. 1980) have shown that synthesis of endogenous creatine is influenced by the amount of exogenous creatine in the diet. Therefore to keep the body creatine pool in a steady-state, the subject's diet must be creatine free in order to eliminate exogenous creatine, or the diet must be of a constant composition. This normally requires subjects to consume an appropriate diet (usually meat- and fish-free) for at least 3 days before the timed 24-hour collections are made. Accurate collections are often difficult with as little as a 15-minute error in voiding time leading to a I % error in estimation. Additional errors from spillage or inadvertent discard are sometimes unavoidable. The urine is then analysed for creatinine content by standard assay procedures.

8.2 Reliability and Validity

Studies by Moran et al. (1980) indicated a mean coefficient of variation of 36% in creatinine excretion over 5 days in serial 24-hour urine collections


on standard nursing wards. The variance is reduced in special metabolic units but is still within the range 1.4 to 27% (Bistrian et al. 1975; Forbes &. Bruining 1976; Moran et al. (980). Heymsfie1d et al. (1983) quoted a normal daily variability of 4 to 8% with reliable subjects and also quoted variability values for other factors which could influence daily urinary creatinine excretion such as strenuous exercise, emotional stress and menstruation. Urinary creatinine excretion in humans has been shown to be a good predictor of lean body mass using 4OJ( dilution, 4OJ( total body counting or total body water as the criterion measure (Boileau et al. 1972; Muldowney et al. 1957; Turner &. Cohn 1975). A correlation of r - 0.988 (p < 0.00 1) was obtained by Forbes and Bruining (1976) between lean body mass determined by 4OJ( counting (mean coefficient of variation, 1.8 to 4.1 %) and urinary creatinine excretion (mean coefficient of variation, 6.9%). Lykken et al. (1980) developed a mathematical model which related changes in creatine synthesis to changes in both creatine and protein in the diet They verified the validity of the model by comparing published changes in creatinine excretion rates with changes predicted by the model. This model may be valuable in future experimental designs concerning athletes (Buskirk &. Mendez 1984).

No definite creatinine equivalence (kg muscle! g creatinine) has been established in humans. It is approximately 17 to 20 kalg creatinine. The units in which the creatinine equivalance is expressed also differed amongst authors. Some referred to fatfree wet muscle, others to whole wet muscle and yet others to intracellular or non-collagenous protein. Until a valid, non-invasive method of measuring muscle mass in vivo becomes available it will remain difficult to establish valid estimates of creatinine equivalence with its associated variability (Heymsfield et al. 1983).

8.3 Plasma and Serum Creatinine

It was sugested by Schutte et al. (1981) that total plasma or circulating creatinine (plasma creatinine concentration multiplied by the plasma

32

volume) may provide a reasonable estimation of muscle mass. Total plasma creatinine correlated stronaly with urinary creatinine excretion (r = 0.82) and with weight, total body water and anthropometrically determined fat-free mass. The authors concluded that body composition can be predicted as accurately with a single measurement of total plasma creatinine as with an average of 3 urinary creatinine excretion measurements.

Serum creatinine concentrations have been used to predict endogenous creatinine clearance in adults and children with both stable and unstable renal function (Hallynck et al. 1981). Its main value is in estimating renal dysfunction and change in state, and has yet to be adapted for assessment of fat-free mass.

9. 3-Metltylltutidiu

3-Methylhistidine is an amino acid that is present almost exclusively in myofibrillar protein. During catabolism the released 3-methylhistidine is neither recycled for protein synthesis nor metabolised further, but is excreted in the urine. Muscle protein synthesis and degradation should be balanced processes during steady-state periods, and thus daily urinary 3-methylhistidine excretion should be proportional to muscle mass in adult humans (Mendez et al. 1984).

9.1 Method

To eliminate exogenous 3-methylhistidine from the body at least 3 days of a meat-free diet must pass before urinary collections commence. At least 3 consecutive collections of timed 24-hour collections should be obtained to gather information on the daily variability of endogenous 3-methylhistidine excretion (Buskirk &. Mendez 1984).

The method is essentially similar to urinary creatinine excretion with the same potential errors in sample collection. 3-Methylhistidine does not represent all muscle protein breakdown as it does not change with the breakdown of sarcoplasmic protein which represents about 35% of muscle protein.


The assay requires an amino acid analyser which is less commonly available.

9.2 Validity

In an experiment on 16 male subjects. Lukaski and Mendez (1980) found that 3-methylhistidine was highly correlated with fat-free mass (r = 0.89, p < 0.00 1). Data obtained in a later experiment by Lukaski et al. (1981 b) were combined with the previous results and subjected to covariance analysis. This showed that the regression lines were similar and that a significant relationship existed between 3-methylhistidine output (mean coefficient of variation, 4.5%) and fat-free mass (r = 0.86, p < 0.(01). A highly significant relationship was observed between 3-methylhistidine and muscle mass (r = 0.91, p < 0.00 I). Excellent correlations were also obtained between 3 methyl histidine and other body composition markers (Lukaski et al. 1981 b), with total body potassium (r = 0.83), fat-free body mass by densitometry (r = 0.81) and urinary creatinine (r = 0.87). Mendez et al. (1983), quoted by Buskirk and Mendez (1984), compared 16 wrestlers after a 5-week period of intensive training with the 14 men studied by Lukaski et al. (1981 b). The results indicated that both 3-methylhistidine and urinary creatinine excretion gave a better estimate of muscle mass than hydrodensitometry and supports the use of 3-methylhistidine and urinary creatinine excretion measurements during athletic training.

10. Total Body Water

About 60% of the average male bodyweight is water. with a smaller proportion (about 50%) in women, and the neonate having a value as high as 80 to 90%. Most of the body water (62.5%) is in the intracellular compartments, the medium for cellular metabolism. with the remaining extracellular water providing a supply route to and from the cells for gases, food and waste products.

The earliest methods of measuring body composition involved weighing the organs at post-mortem. By the middle of the nineteenth century chemical analysis was developed and early data on

33

the amounts of water, protein and fat were being collected. As early as 1863, Bischoff combined chemical analysis with dissection. He weighed all the organs and visible fat of 6 humans and determined the body water in the muscles and viscera of one of the men and one of the infants.

The use of dilution techniques to determine total body water is a relatively recent innovation which has been developing since the 1930s. The basis for all the dilution methods, regardless of what substance is used as the medium, is an intake 'of a known amount of a substance and the determination of its concentration in the plasma or other body fluids after allowing time for equilibrium to be established within the body' (McCance & Widdowson 1951). The medium or substance used needs to conform to certain characteristics in order to produce data.

Ideally the medium should be: (a) easy to use and preferably non-toxic; (b) inert rather than radioisotopic; (c) metabolised fairly slowly to allow time for data to be collected; (d) evenly and rapidly distributed throughout the body and capable of achieving equilibrium as quickly as possible; (e) accurate and convenient in the estimation and calculation of body composition values; (f) cheap and readily available.

Some substances do not diffuse in all the body water but are confined to certain spaces. They can still be used provided corrections are made to the calculations of the data. For example, inulin, which is water-soluble but does not penetrate the cells, can be used to calculate the extracellular fluid volume and then applied by difference to the total body water. Similarly, sodium and potassium used as the medium are injected, and after the time allowed for equilibrium has passed, the concentration in plasma is measured as the chlorides are largely confined to the extracellular fluids.

Other techniques for calculating total body water that evolved at about the same time as the dilution principle include nitrogen solubility and specific gravity. These, although totally different, were both developed by Behnke et al. in the early to mid I 940s. Using the principle that nitrogen is more soluble in fat than in water, Behnke et al. (1942)


. used a known amount of nitrogen gas dispersed in the body fluids and fat under a known pressure to estimate the percentage of body fat and water. The studies they produced on specific gravity rely on major assumptions, e.g. that a standard mammalian body is 10% fat (Behnke 1942, 1945).

Table I provides a summary of the advantages and disadvantages of a selection of dilution methods. Lewis et a1. (1986) consider that the tritium (3"20) dilution method is the most utilised indi-

reel method of determining total body water. Tritium is a soft /t-emitter and because a scintillation counter can be employed with minimal sample preparation, it has been easier to measure in some respects than deuterium. The precision of this measurement was estimated by Hill et al. (1979) to be 1.0kg, i.e. 2.5% of a typical body water content of 40L Yasumura et at. (1983) considered that the precision of measuring total body water by 3H20 to be less than I %. However, Sheng and

T ..... I. Advantages, disadvantages and methodological r.f .... ence for the dilution methods of estimating total body water

Medium Advantages Disadvantages Reference

ThIocyanate Convenient; .. sy to handle; e .. y to Penetrates erythrocytes (needs Lavietes et al. (1938)

administer; non-radloactlve correction); only measures

extracellular fluid; questionable reliability

Ur .. Easy to handle; although Invasive Excreted quickly by kidneys; McCance & Widdowson may be teken by mouth; can be equilibrium dlfflcuH to achieve; (1951)

measured In urine (no IV sample unequal distribution needed); non-radioactive

Inulin Easy to handle; relatively cheap; Often falls to achieve equilibrium; Benedict et al. (1950)

non-radloactive extracellular fluid only

Antipyrene UnHormly distributed; excretion BInds with protein affecting Soberman et al. (1949)

negligible; metabolised relatively equilibrium

slowly; non-toxic; .. sy analysis

Tritium Oral administration; metabolised Complicated equipment and Cohn et al. (1983. 1985)

slowly; good equilibrium; equal measuring techniques; radioactive;

distribution hydrogen Isotope exchanges with

hydrogen In organic body parts

Ethanol Non-radloactive; br .. th analysis Intoxicant to subject in large doses Loeppkyet al. (1977)

may be used to calculate blood or needed for accurate calculation; sma. urine ethanol level do .. s metaboll .. quickly; equilibrium

dlfflcuH

SodIum and Both used In early studies on body Confined to extracellular fluid; diffuse Forbes & Perley (1949);

potassium compoaItion due to .... and with body chlorides; radioactive Cor .. et al. (1950)

accuracy of measurement

Deuterium oxide Equilibrium achieved quickly; non- Complicated equipment and Haschke (1983); Lukaski & radioactive; not metabolised quickly measuring techniques; hydrogen Johnson (1985)

or bound with protein; equal Isotope exchanges with hydrogen in distribution; relatively cheap; oral organic body compounds; needs amaH

administration; results may be correction calculated from urine or saliva; may

be uMd In tracer doses for

repeated measurements; no Invasive

(IV) procedures needed


Huggins (1979) concluded that. compared with total body water measurements determined by dessication. 3H 20 overestimates total body water by 4 to 15%.

Important considerations for experimenters in choosing the substance with which they are going to work include availability, ethics. cost, accuracy. validity and limitations. Deuterium oxide fulfils these as well or better than any other available medium and has fewer limitations. The basic method of determining total body water using D20 as the medium is simple. ethical. non-invasive (insofar as no venepuncture techniques are required), accurate and easily validated (by comparing levels of D20 in samples of saliva. urine and blood). The only limitations to the method being the relatively costly equipment needed to analyse the sample for D20, i.e. an infrared spectrometer. For these reasons and in relation to those summarised in table I. deuterium oxide is the method of choice and will be used to illustrate the dilution method of estimating total body water.

10.1 Methodology

Haschke (1983) describes a method whereby the subjects were given Ig D20/kg bodyweight to drink over a period of I minute. They then drank two 50ml rinses of tap water added to the beaker. The subjects were weighed immediately before ingestion of the D20, and 3 and 4 hours after. At the same time as being weighed. 2ml samples of saliva were obtained. No food or other fluids were allowed to the subjects during the duration of the trial. which was for 4 hours. I ml of saliva was vacuum sublimated to near total dryness and the sublimates condensed in traps were then immersed in liquid nitrogen and frozen at -20°e. The D20 concentration was determined using a Perkin-Elmer infrared spectrometer. The apparent total body water in grams was calculated using the following formula:

TBW = D - [(Mo - Md x Dd

35

where D = amount of D20 (grams) ingested. Mo = bodyweight in grams before ingestion of D20, M J = average weight measured 3 and 4 hours after ingestion. Do = D20 concentration before ingestion of D20. and DJ = average D20 concentration 3 and 4 hours after ingestion. The formula assumes that the weight difference (Mo - M J) represents loss of water and that water lost has D20 concentration D J.

Haschke validated his results in 6 subjects by simultaneous determination of D20 concentration in urine and saliva. Samples were collected at varying times up to I week after the trial. In 18 paired urine and saliva samples the concentration in the saliva was 100.34% (average) with an SD of 3.25% from that of the urine sample. He described the relationship by the equation:

D (saliva) = 0.00154 + 0.991 x D (urine) = (r = 0.99. p < 0.001)

In his concluding comments Haschke (1983) states that this method of calculating total body water is 'simple and suitable for field studies ... non-invasive. and the equilibrium time of 3 to 4 hours should make such studies quite acceptable to prospective subjects: essential where subject compliance is extremely important.

Lukaski and Johnson (1985) measured total body water using a tracer dose of D20 (lOg per subject regardless of their weight). The advantages of this method were that equilibrium within the body fluids was achieved within 2 hours rather than 4. the total amount of D20 needed for the trial was smaller so the cost was reduced and repeated trials could be carried out over a relatively short period of time as there was no significant build-up of background levels of D20 within the body.

The subjects fasted overnight for a minimum of 10 hours before the trial took place. Before administration of the D20 a 10mi blood sample and a 5 to 7ml saliva sample was taken from each subject. The 109 of D20 was given orally with fruit juice or deionised water. The subjects were then asked to 'remain in a quiet state during the 4-hour period during which blood and saliva samples were taken at 30, 60, 90. 120, 180 and 240 minutes fol-


lowing the dose'. They found that the absorbance of 0 20 was at IS·C, so this was chosen as the optimum temperature for 020 analysis. They also found that the treatment of urine by centrifugation of charcoal caused the sample to become too cloudy for infrared analysis. Distillation of the urine caused 020 recovery to be too low and only vacuum sublimation provided samples of sufficient quality to be used for experiments. The D20 concentrations for the fint hour after ingestion were higher in saliva than plasma, but then levelled off to remain relatively constant for the remaining collection time.

In their conclusions the authon state that their choice of 020 as the medium and infrared absorbance as the analytical method has only I main disadvantage. This is the lowering of D20 concentrations by the exchange with labile hydrogen ions which result in minimally overestimated values for total body water. The many advantages include ease of administration, non-toxicity (particularly in the smaller tracer doses), good distribution, quick equilibrium, short experimental time and good subject compliance.

11. Total Body Potali"m

Total body potassium is an indication of cellular or lean tissue mass because more than 90% of the potassium is within non-fat cells. Its measurement depends on the naturally occurring isotope 40K in non-fat tissue which is a constant fraction (0.012%) of body potassium. The measurement technique depends on counting the 1.46 MeV"Y radiation using a whole-body radiation counter (Burch & Spien 19S3). These counten vary in design but usually involve a number of scintillation detecton placed such that their geometry covers the patient under test. Two types are described by Burkinshaw and Spiers (1967), one consisting of 3 large plastic scintillation detecton grouped around a chair and the other made up of four 6-inch ( l5cm) diameter x 4-inch (I Oem) thick scintillation crystals of thallium-activated sodium iodide, 2 above and 2 below a horizontal couch. The detecton would move along the couch during the measure-

36

ment period, typically taking 30 minutes. The counten have different properties with some having high sensitivity and moderate energy resolution and othen the opposite characteristics. To reduce background radiation from various sources the whole-body counter will be housed in the basement of multi storey buildings (thus reducing cosmic radiation) and will be screened with thick lead or steel walls, the steel usually obtained from pre-1945 sources such as scuttled German battleships. The Brookhaven whole-body counter (Cohn et al. 1982) has S4 detecton and the inherent geometry is considered to produce a relatively invariant response with respect to both the size of the individual and the internal location of the radionucleide. Using an anthropometric phantom, the accuracy and precision of the Brookhaven system is considered to be ± 3.3% (Cohn et al. 1969; Cohn & Drombrowski 1970), and the standard error of estimates for the Leeds counter is stated as approximately 4% of the body content (Burkinshaw 1978; Burkinshaw et al. 1981 ).

There is some controveny over the value of the ratio of body potassium to fat-free mass (Boddy et al. 1973; Burkinshaw & Cotes 1973) with 68.1 mmol/kg having been used by Forbes et at. (1961) based on limited cadaver analysis, and a value of 64.2 mmoljkg suggested for women (Forbes 1974). Morgan and Burkinshaw (1983) suggest that there is not a gender distinction and that total body p0-

tassium increases with fat-free mass regardless of sex with values as low as 49 mmoljkg for a fat-free mass of 30kg to 62 mmoljkg for a fat-free mass of 80kg. A series of assumptions are made in order to determine these values (Forbes 1984) but the essential issue of whether fat-free tissue in men differs from women is still under debate.

A number of factors influence whole-body p0-

tassium counting and these include the amount of background depression by the subjects' mass (Anderson 1968; Coffman 1968; Lohman et at. 1968; Miller et al. 1968), the potassium concentration in various tissues and body segments (Lohman et at. 1968; Martin et al. 1968) and pre-existing radioactive contamination (Anderson 1968; Lohman et at. 1968). It has additionally been shown by Lon-


deree and Forkner (1978) and Thomas et al. (1979) that prolonged exercise produces an increase in the 4llK count which may be caused by changes in plasma potassium concentration and increased cutaneous blood flow for the dissipation of heat. It is thus recommended that 4 hours after exercise should elapse before 4°K counting.

12. Other Nuclear-Based Techniques

Emphasis in this discussion has been given to those methods either in common usage or to newly developing techniques or those which may have particular value in sports medicine if resources can be made available. Computer tomography and nuclear magnetic resonance would be included in the latter category. Others require the support services of hospital departments of medical physics (e.g. total body potassium) but are sufficiently useful to merit a separate section. A further group also require nuclear support services beyond the scope of most sports medicine laboratories and have greater clinical application. It is these methods which are reviewed in this section.

12.1 Neutron Activation Analysis

Cohn and Parr (1985) in their review of nuclearbased techniques provide details of in vivo neutron activation analysis. They review clinical applications. neutron sources. detection systems. cost. radiation dosage and quality assurance.

The normal procedure is to irradiate the patient with fast neutrons and after placement in a wholebody radiation counter. measure the radioactivity induced in the body by the neutrons (Almond et al. 1984). The resulting spectrum of emitted j'-rays can be analysed to determine a variety of total body elements including chlorine. potassium. phosphorus and calcium. The measurement of total body calcium and total body chlorine is described by Cohn et al. (1972) using 2J8Pu_Be as the source of radiation.

Further information on neutron activation analysis. particularly the derivation of the equations to calculate the muscle and non-muscle compo-

37

nents of fat-free weight. can be examined in Buskirk and Mendez (1984). Burkinshaw et al. (1978) and Lukaski et al. (1981 a).

12.2 Total Body Nitrogen

Body nitrogen is in proportion to the amount of tissue protein in the body. and may thus be a more useful indication of cellular mass than total body potassium or fat-free mass from hydrodensitometry (Burkinshaw et al. 1981). The technique has been described by Harvey et al. (1973). Mernagh et al. (l971). Oxby et al. (1978). Vartsky et al. (1979) and Williams et al. (1978).

Burkinshaw et al. (1981) describe the neutron activation method for total body nitrogen in which subjects were irradiated bilaterally to a nominal dose equivalent of 50 mrem (0.5 mSv) with 14 MeV neutrons and then counted in a whole-body radiation counter as described in the section under whole body potassium. The j'-ray spectrum was then analysed to establish the radionuclide level induced in the body by the neutrons. The total body nitrogen was estimated from the 13N activity produced by the reaction 14N(n. 2n)\3N after correction for oxygen. The details of the comparison with irradiated phantoms are given by Burkinshaw et al. (1981). Lukaski et al. (1981) and Vartna et al. (1979) describe the prompt j'-ray analysis. 14N (n:y)15N. The prompt emission (10-20 sec) of a cascade of -y-rays follows the capture of a thermal neutron by 14N. 10.83 MeV j'-rays are emitted which reflect 14N because it is the only body element having a neutron capture j'-ray at this energy. The origin of the fast neutrons is an 85Ci source of 238pu_Be delivering 26 mrem. although Cohn et al. (1982) report a dosage of 50 mrem with a 90Ci source. The precision of the total body nitrogen procedure is estimated by Vartsky et al. (1979) to be ± 3%. although Shephard et al. (1985) consider the experimental error to be nearer 6%. The detectors were sodium iodide in all cases (Burkinshaw et al. 1981: Cohn et al. 1982: Lukaski et al. 1981 a: Shephard et al. 1985).

Muscularity. body hydration. protein and mineraI content of the skeleton. and ageing effects may


all influence the relationship of total body nitrogen to lean tissue mass (Shephard et al. 1985), although aaeing as an independent variable requires further confirmation.

Measurement of both body potassium and body nitrogen provides the opportunity to use body nitrogen as a reference for total body potassium when examining potassium depletion (Burkinshaw et al. 1981). The ratio of N : K in the non-muscle compartment of lean tissue is considered by Cohn et al. (1982) to be twice that of the corresponding ratio in the muscle compartment. There is still, however, some debate as to whether an N: K ratio can be ascribed to non-muscle tissue. Garrow (1978, 1982b) maintained that in human skeletal muscle the N : K ratio was about 8.45, but that there was considerable variation in non-muscle tissue. It also permits measurements of changes during wastage (James et al. 1984), particularly cancer (Cohn et al. 1981, 1982) and in studies involving dieting (Cohn et a1. 1982).

A further measure to be combined with total body nitrogen is total body chlorine. Knight et al. (1986) described the total body nitrogen and chlorine measurements in 2 human cadavers by neutron activation and chemical analysis. The close agreement between the two methods supported the use of in vivo neutron activation analysis as an accurate measurement of total body nitrogen and chlorine, although systematic and random errors in this technique should not be underestimated.

12.3 Total Body carbon

carbon content of the body is a measure of its energy store and thus as the body's principal enel'lY store is fat, total body carbon can estimate fat content (Kinney &. Moore 1956). The method involves the detection of 'the 4.43 MeV 'Y-rays emitted when carbon nuclei are excited by inelastic interactions with fast neutrons' (Kyere et al. 1982). A supine patient receives collimated beams of 14 MeV neutrons from a sealed tube neutron generator. The thallium-activated sodium iodide scintillation counter is shielded by boric acid powder to reduce extraneous radiation. Repeated measure-

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ments on a sugar-solution filled phantom obtained a coefficient of variation of ± 2.9%. The method may be of particular value for use in clinical research when skinfold estimates of body fat are less appropriate. An equation has been established to estimate total body fat from total body carbon but the authors concede that the long irradiation periods currently required may limit the technique for clinical work. However, the use of a number of semiconductor detectors arrayed around the subject could improve precision and reduce the irradiation time.

12.4 Photon Absorptiometry

The determination of body composition in specific parts of the body can be achieved by the attenuation of x-rays or 'Y-rays. This process is photon absorptiometry. It can either involve a single energy transmission when 2 clearly defined tissues are involved, or a dual energy system when variable thicknesses such as the trunk are involved.

12.4.1 Single Photon Absorptiometry There is commercially available equipment to

measure bone mineral. Cameron and Sorenson (1963) describe a method for the ulna and radius and Atkinson et a1. (1978) used 241 Am as the radiation source when scanning the femur to establish bone and soft tissue composition.

1251 is a common source of radioactivity, but Atkinson et a1. (1978) used 241 Am in their scanning of the femur in growing boys. The measurement is quick, but precision error is generally below 4%, provided the patient movement can be minimised by placing the limb in a plastic trough and the scanning position can be reproduced exactly. The Cameron and Sorenson (1963) method was also used by Lohman et al. (I984b) in measurements of the bone mineralisation of the radius and ulna. The reliability of the method was assessed over a test-retest period of 7 days and was found to be ± 0.06 g/cm for adults and 0.04 g/cm for children.

12.4.2 Dual Photon Absorptiometry Dual photon absorptiometry is a technique re

cently applied to body composition assessment (Mazess et al. 1984) and was originally applied to


the measurement of total body bone mineral or to the lumbar spine bone mineral content (Dunn et a1. 1980; Gotfredsen et a1. 1984a.b; Peppler & Mazess 1981).

The best differentiation between bone and soft tissue is to use 2 energy levels. with a lower energy in the range 40 to 60 keY and a higher energy of about 100 keY. 153Gd is considered to be an excellent source of radiation (Madsen 1977; Kf0lner & Pors Nielsen 1980; Price et a1. 1977; Wilson & Madsen 1977), but Roos and Skoldborn (1974) were successful by combining 241 Am and 137CS. A typicallumbar scan takes 20 to 30 minutes, delivering a skin dose of about 0.1 mGy which is twice the value from a single photon measurement.

A specific method was described by Gotfredsen et a1. (1986) when using dual photon absorptiometry to measure lean body mass and total body fat. The radiation source was I Ci 153Gd with principal photo peaks at 44 and 100 keY. The lean/fat ratio of the soft tissues was averaged on the basis of multiple rectilinear scans. The 2 photo peaks at each of approximately 4000 pixels provided information which, in comparison with known ratios for pure lean and pure fat. gave an average lean: fat ratio. The lean fraction was determined from an equation which uses the ratio for soft tissue, the lowest value obtained in soft tissue and the highest value obtained in soft tissue. The total mass of soft tissue was calculated from the equation given in Gotfredsen et a1. (1984) and the accuracy error for the measurement of lean body mass from this method was considered to be about 2.5% (Gotfredsen et a1. 1986). These data are preliminary but justify the technique as a precise and accurate method of measuring lean body mass and total body fat. It is suitable for in vivo measurements, and with a low radiation dosage of less than 5 mrad (Gotfredsen et a1. 1984; Peppler & Mazess 1981) can be used in repeated trials when monitoring changes in body composition.

It correlated well with anthropometrically determined total body fat (r = 0.80, p < 0.00 I). However, the use of skinfolds (Durnin & Womersley 1974) relates most closely to subcutaneous fat, and the dual photon absorptiometry method measures

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the sum of the fatty elements of all soft tissues. It is thus suggested that comparison with a densiometric method of estimating fat may improve the correlation further. thus providing more support for this approach to body composition assessment.

12.5 Nuclear Resonance Scattering

There are certain elements (e.g. Fe, Cu, Mn) which are difficult to measure by in vivo neutron activation analysis in the human body. The method of choice for such measurements is nuclear resonance scattering. This involves irradiating with 'Y

rays to excite the target nuclei. As they de-excite, characteristic -y-rays are emitted and these are measured with a suitable detector. Cohn and Parr (1985) describe the method in more detail, particularly for the measurement of iron in the liver and heart.

12.6 Multiple Isotope Dilution

Shizgal (1985) administered a simultaneous intravenous injection of 8Ci of 22Na and 500 I-'Ci of 3H20, preceded and followed by plasma sampling. Extracellular water volume was determined by plotting the logarithm of the plasma concentration of 22Na against time. The volume was determined by the reverse extrapolation of the straight line obtained by least squares fitting. Total body water was determined from the equilibrated plasma tritium concentration at both 4 and 24 hours. The same author in an earlier article (Shizgal 1981) described the measurement of body composition using human serum albumin labelled with 1251, red cells tagged with SICr. 22Na and 3H20. The total radiation dosage is 237 millirems. This procedure permits the assessment of red cell mass. extracellular water volume. total body water and plasma volume. Szeluga et al. (1984) described the assessment of total body water and extracellular fluid using 3H20 and NH482Br respectively by extrapolation. They consider that the value of these studies was particularly with patients requiring an accurate


measure of nutritional status. Watson and Sammon (1980) also estimated intracellular and extracellular water volumes in cachectic patients by using the multiple isotope dilution technique. They utilised sler-labelled erythrocytes and 12S1-labelled

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human serum albumin for blood volumes. JH~O for total body water volume and 77Br-sodium bromide for extracellular water volume.

Pan II of this anicle including references will appear on pages 74 to 98 of the next issue.

1988 Olympic Scientific Congress

'Human Movement Science Toward 2000'

Date: 11-15 September, 1988 Venue: Seoul Korea

For further information, please contact: Dr Keung b-Director rgaru mg ommittee, 1988 Olympic Scientific Congres , Korea ports Science Institute, GPO Box 1106 Seoul Korea.

techniques of measurement of body composition part i

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