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1 From the Africa Centre for Health and Population Studies, Somekele, South Africa (PCP, NCR, and MLB); the Program in International Nutrition, University of California, Davis, Davis, CA (PCP, KHB, and MVL); the Department of Paediatrics, Nelson R Mandela School of Medicine, University of KwaZulu Natal, Durban, South Africa (NCR); the Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Boston, MA (MLB); the Nuffield Department of Clinical Medicine, University of Oxford, Oxford, United Kingdom (MLB); and the Western Human Nutrition Research Center, United States Department of Agriculture, Davis, CA (MVL)
2 Supported by grants no. 063957 and 067181 from the Wellcome Trust (to the Africa Centre for Health and Population Studies); a Fullbright Scholarship (to PP); Mid-Career grant no. 1 K24 AI/HDO1671-01 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and grant 62925 from the Wellcome Trust (both to MB); and grant 050524 from the Wellcome Trust (to NR). The Western Human Nutrition Research Center, United States Department of Agriculture, Davis, CA, provided the isotope, bioimpedance equipment, and technical assistance for deuterium measures of total body water. 3 Reprints not available. Address correspondence to PC Papathakis, Department of Pediatrics, University of California, Davis, Medical Center, 2516 Stockton Blvd, Sacramento, CA 95817. E-mail: pcpapathakis{at}ucdavis.edu.
ABSTRACT
Background: The effect of breastfeeding on the nutrition of HIV-infected (HIV+) mothers is unknown. Simple, valid methods are needed for body-composition assessment of HIV+ women.
Objective: We compared the ability of bioimpedance spectroscopy (BIS) and anthropometry with that of isotope dilution (2H2O) to measure fat-free mass (FFM) and fat mass (FM) in HIV+ and HIV-uninfected (HIV–) breastfeeding South African mothers.
Design: Total body water (TBW) content of 68 lactating mothers (20 HIV+, 48 HIV–) was measured 10 wk after delivery by using BIS and 2H2O to measure FFM and FM. Anthropometric measurements included body mass index (BMI; in kg/m2), midupper arm circumference (MUAC), and 4 skinfold thicknesses.
Results: TBW, FFM, and FM measurements determined by BIS were correlated with 2H2O measurements in HIV+ (r = 0.664, 0.621, and 0.872, respectively; P < 0.01) and HIV– (r = 0.876, 0.868, and 0.932, respectively; P < 0.001) mothers. TBW measured by BIS was greater than that measured by the 2H2O method in both HIV+ (1.8 L) and HIV– (1.5 L) women; FM or FFM did not differ significantly by method. BMI, MUAC, and all skinfold-thickness measurements correlated strongly (r > 0.62, P < 0.001) with FM measured by 2H2O in both groups. BMI and MUAC correlated (r > 0.64, P < 0.001) with FFM in HIV– mothers but not in HIV+ mothers.
Conclusions: In HIV+ and HIV– breastfeeding mothers, BIS provides an estimate of body composition comparable to that obtained with the 2H2O method. BMI and MUAC are useful in predicting FM in both groups but are not valid measures of FFM in HIV+ mothers.
Key Words: HIV • breastfeeding • body composition • South Africa • fat mass • fat-free mass • anthropometry • isotope dilution • women • AIDS • lactation • bioimpedance spectroscopy
INTRODUCTION
Despite concerns about the risk of transmitting HIV to infants via breastfeeding (1) and the unknown effect of breastfeeding on the health and nutrition of HIV-infected [HIV-positive (HIV+)] mothers (2), the use of breast milk replacements is often considered unacceptable, unaffordable, or unsafe (3-6). Because it is likely that breastfeeding will remain the norm among HIV+ mothers in most of sub-Saharan Africa, it is important to determine whether breastfeeding may be detrimental to their nutrition and health. To assess nutritional status in these women, valid, inexpensive, and easily utilized field methods of measuring body composition are needed.
The stable isotope deuterium oxide (2H2O) is a reference technique for measuring total body water (TBW). After the ingestion and equilibration of a known dose of 2H2O in the body's water compartments, 2H2O concentration serves as a marker for TBW from which fat-free mass (FFM) and fat mass (FM) are derived. This technique is a safe and well-established standard method for assessing body water compartments even during pregnancy (7), but it is cumbersome to use in the field. Thus, validation of a simpler method is desirable.
Bioelectrical impedance analysis (BIA), using a single-frequency measurement of resistance and reactance, has been shown to provide reliable estimates of FFM and FM in adults (8-10). It is an easy, reliable, and portable technique for measurement of body composition that is applicable for fieldwork and is less expensive than the 2H2O method. However, chronic illness and infection can cause shifts in body water compartments (11, 12) thereby introducing error in the measurement of body cell mass and making the use of single-frequency BIA in infected populations potentially problematic. To overcome this concern, multiple-frequency bioimpedance, known as bioimpedance spectroscopy (BIS), is an alternative to BIA. TBW and extracellular water (ECW) measurements made by using BIS (BIS) may be more accurate in populations in which there are potential alterations in fluid distribution, as are seen with HIV infection and during lactation (13, 14).
In healthy HIV-uninfected [HIV-negative (HIV–)] pregnant and lactating women in the United States, measurements of TBWBIS correlated well with measurements of TBW made by using isotope dilution (2H2O) (15). Multifrequency BIA has also been used to monitor changes in body water compartments during pregnancy (16). It is recommended that the validity of impedance measures be confirmed as applicable for specific populations (10), and thus it is necessary to validate this method in HIV+ breastfeeding African mothers.
The primary objective of this study was to determine the validity of BIS and anthropometric measurements to measure body composition in HIV+ and HIV– women as compared with the values obtained with the reference stable isotope dilution method. The second objective was to describe the body composition of HIV+ lactating women in rural South Africa.
SUBJECTS AND METHODS
Subjects
This study was conducted in government health clinics in the field area of the Africa Centre for Health and Population Studies in a rural, northern part of KwaZulu Natal Province, South Africa. Mothers were identified for the study when they came to clinic for their infants' immunizations. Inclusion criteria were delivery 4 to 20 wk previously, maternal age >15 y, current breastfeeding of the infant, and arrival at clinic by 0900 to allow sufficient time for the full range of measurements. Exclusion criteria were current acute illness that would influence hydration status (eg, diarrhea or fever) and admission to the hospital in the previous 2 wk.
Mothers who were eligible and who agreed to participate provided written informed consent and agreed to HIV counseling and testing. HIV testing was anonymous, and mothers returned in 2 wk to obtain their results from the HIV counselor. Thus, at the time of body-composition measurement, HIV status was not known by subjects or study staff. Mothers were enrolled from November 2001 to April 2002, and all measurements were made on the day of enrollment.
The Ethics Committee of the Nelson R Mandela School of Medicine at the University of KwaZulu Natal, Durban, South Africa, and the Human Subjects Committee of the University of California, Davis, Davis, CA, approved the study.
Measurements
Isotope dilution method
Ideally, the isotope dilution method is conducted in a controlled environment, but that was not possible in this rural setting. Most of the women attending the clinic walked, from varied distances, to the clinic while carrying their infant. To ensure adequate and consistent hydration status, all women were encouraged to drink water ad libitum during the 30 min before the 2H2O administration. The women then provided a baseline urine specimen, after which they drank 20 g 2H2O mixed in 100 mL water. The women were asked to void 2 h after consuming the 2H2O dose; this urine was discarded, because the isotope is not equilibrated in the 2-h sample. Three hours after the 2H2O dose, a postequilibrium urine specimen was collected for measurement of the 2H2O concentration. A second urine collection 4 h after the dose was not possible in this setting, but, in other studies that we have conducted in pregnant and postpartum women (15) and in AIDS patients receiving anabolic therapy (17), we found that 3-h postdose samples were similar to 4-h postdose samples. During the 3-h equilibration period, each subject drank 500 mL bottled water but did not consume any other foods or fluids. Urine specimens were stored in screw-top containers with additional wrapping to prevent evaporation and leakage and maintained at –70 °C until they were processed at the Western Human Nutrition Research Center in Davis, CA. Urine was processed by vacuum sublimation to obtain a pure water sample. The 2H2O concentration was measured by fixed-filter infrared spectrometry. The concentration of 2H2O in urine was calculated with the following equation:
RESULTS
Sixty-eight mothers were enrolled in the study, 20 of whom were HIV+. Four of the 48 HIV– mothers were excluded from analysis because of implausible BIS measurement (negative values) due to improper electrode placement (ie, the electrode placements were reversed). Two HIV+ mothers were excluded because of incomplete isotope dilution procedures (one could not drink the full solution, and the other could not urinate at the 3-h time point), and one HIV+ mother was removed as an outlier (2H2O method indicated 3.4 kg FM, which is incompatible with human physiology).
Study participants ranged in age from 15 to 40 y (median: 24 y), had 1–8 previous pregnancies (median 2), and had delivered between 4 and 19 wk previously (median: 10.1 wk). Less than half of the women had access to piped water or electricity in their homes. HIV+ mothers had more formal education than did HIV– mothers (8.5 ± 3.4 and 6.1 ± 3.0 y, respectively; P = 0.009), but otherwise the 2 groups were not significantly different (Table 1).
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TABLE 1. Characteristics of study subjects by HIV status
The median viral load in the HIV+ mothers was 25 000 RNA copies/mL (log 4.4), and values varied from nondetectable to 170 000 copies/mL (log 5.23). The median CD4+ cell count was 631/µL (range: 179–1229), and just one subject had <200 cells/µL. Because the South African programs to prevent mother-to-child HIV transmission had not yet begun, none of the study participants had received antiretroviral drugs.
Body composition
Mean body-composition measurements made with the 2H2O method did not differ significantly by HIV status (Table 2). Of the body-composition measurements made with the BIS method, only FFMBIS and ICWBIS differed significantly by HIV status (Table 2). When FFMBIS and ICWBIS were calculated as a percentage of body weight, however, there was no difference by HIV status. As was found by using the 2H2O method, HIV+ mothers had greater TBWBIS, a difference that was of marginal significance (P = 0.056). ECWBIS compartments for both HIV+ (weight: 21.3%; TBW: 45.5%) and HIV– (weight: 21.8%; TBW: 47.0%) mothers are within normal limits (27) and did not differ significantly by HIV status.
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TABLE 2. Body composition by HIV status1
None of the anthropometric measurements differed significantly between groups (Table 3). Although the HIV+ mothers weighed slightly (3.8 kg) more than did the HIV– mothers, the difference was not significant. Four mothers (5.9% of HIV+ and 6.8% of HIV– mothers) had BMIs < 19.5, and 5 mothers (11.8% of HIV+ and 6.8% of HIV– mothers) had BMIs > 30.5 (P = 0.82).
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TABLE 3. Anthropometric measures by HIV status1
Comparison of methods
The BIS and 2H2O methods
TBW, FFM, and FM measured with the BIS method were significantly correlated with those measured with the 2H2O method in both HIV+ (r = 0.664, 0.621, and 0.872, respectively; P < 0.01 for each) and HIV– (r = 0.876, 0.868, and 0.932, respectively; P < 0.001 for each) mothers. The 2H2O and BIS methods did not differ in their ability to measure FFM and FM in HIV+ and HIV– mothers (Figure 1), and there was no significant difference between the 2 groups in slope (P > 0.60) or intercept (P > 0.70). In contrast, TBW was significantly different by method (P < 0.001), but there was no significant group effect.
FIGURE 1.. Regression between deuterium (2H2O) and bioimpedance spectroscopy (BIS) to measure fat-free mass (FFM) and fat mass (FM) by HIV status. •, HIV-positive; , HIV-negative.
Compared with the 2H2O method, the BIS method overestimated TBW by a mean of 1.8 L (P < 0.05) in HIV+ mothers and 1.5 L in HIV– mothers (P < 0.001). Although the difference was not significant, the BIS method tended to overestimate FFM by 1.4 kg in HIV+ and by 0.9 kg in HIV– mothers and to underestimate FM by 1.4 kg in HIV+ and by 1.2 kg in HIV– mothers (P > 0.53) (Table 4). These differences are shown in Bland-Altman plots (Figure 2). Also evident in these plots is the distribution of values both above and below the mean, which indicates a lack of systematic bias in methods for the 2 groups.
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TABLE 4. Correlations between body-composition measurements obtained by using the 2H2O method, the bioimpedance spectroscopy (BIS) method, and equations derived from anthropometric measurements1
FIGURE 2.. Bland-Altman comparisons of total body water, fat mass, and fat-free mass by HIV status. •, HIV-positive; , HIV-negative. Limits of agreement: 2 ± SD.
With the use of analysis of covariance to adjust for either weight or BMI, there was no significant effect of HIV status on FFM2H2O or FFMBIS results (data not shown). After adjustment for CD4+ or viral load in the HIV+ mothers only, there was no effect on FFM2H2O or FFMBIS results (data not shown).
Anthropometric measurements and the 2H2O method
All individual skinfold-thickness measurements and their sum, BMI, MUAC, and midupper arm muscle circumference were significantly (r > 0.62, P < 0.001) correlated with FM2H2O in both HIV+ and HIV– mothers; BMI and MUAC had the highest correlation with FM2H2O. These same indicators were significantly (r > 0.64, P < 0.001) correlated with FFM2H2O only in HIV– mothers (Table 5). Compared with the 2H2O method, the Durnin-Womersley equations significantly overestimated FFM (P < 0.05) and correspondingly underestimated FM significantly (P < 0.05) in both groups of mothers (Table 4).
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TABLE 5. Correlations between body-composition results determined by the 2H2O method and individual anthropometric measurements and by HIV status1
As indicated in the description of statistical analyses, the initial equations used to predict FFM and FM from anthropometric equations were based on a subsample of 60% of the population and were cross-validated in the remaining 40%. The correlations between the results obtained with the 2H2O method and those obtained with the newly developed equations for this population were strong and significant for FFM (r = 0.915) and FM (r = 0.859), which suggested that these equations were appropriate for use in this population. Therefore, we pooled the dataset to develop separate versions of a more robust equation for use in HIV+ and HIV– mothers (Table 6). The results from the new equations were well correlated with those obtained with the 2H2O method, but the bias between methods (or the difference between the results from the anthropometric equation and from the reference standard) was much smaller than when the Durnin-Womersley equations were used.
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TABLE 6. Anthropometric measurement equations as predictors of fat-free mass (FFM) and fat mass (FM)1
DISCUSSION
We have shown that BIS provides a reliable estimate of body composition in HIV+ and HIV– breastfeeding mothers. Results obtained with the BIS method correlated well with those obtained with the 2H2O method. There was no significant difference between methods in the measurement of FFM or FM. Although the TBW estimate obtained with BIS was larger than that obtained with the 2H2O method in both HIV+ and HIV– mothers, the difference was fairly small (5–6%) and was considered acceptable. It is difficult to ascertain which of the methods is the correct estimate in this field setting.
Comparison of methods
The BIS and 2H2O methods
Correlations between the BIS and 2H2O methods were significant in both groups but stronger in the HIV– mothers. The difference between HIV+ and HIV– mothers in this study may be due to differences in the women's body composition, or they may be an artifact of the larger sample size and broader range of values in the HIV– group. Grinspoon et al (28) found that the correlation of FFM results obtained with dual-energy X-ray absorptiometry and BIA was greater (but not significantly so) in HIV– women than in HIV+ women. One explanation for this difference maybe that the quality of FFM in HIV+ persons is likely to be different from that in the HIV– persons who are usually studied for the development of body-composition reference values (ie, by the 2H2O method) (29). Although the HIV+ mothers in this study were asymptomatic and most were not immunocompromised, it is possible that some of these women may have had changes in body water compartments, and, thus, more time was needed for the 2H2O to equilibrate into TBW in their bodies.
The difference between the results from 2H2O and BIS methods to measure TBW in all mothers may be partially due to the less-than-full equilibration of the 2H2O at the 3-h time point for the postdose urine sample from lactating women. In previous studies of pregnant and postpartum women in our laboratory and of AIDS patients, we found that 3-h postdose samples were similar to 4-h postdose samples and that they provided reasonable estimates of TBW in a 4-compartment model. Therefore, we believed the 3-h postdose time point for a urine sample was appropriate. Each woman was encouraged to breastfeed before the TBWBIS measurement, but the larger effect may have been on the TBW2H2O estimate. It was not possible to repeatedly weigh the infant to adjust the TBW2H2O estimate for milk production on the basis of infant weight gain. The possible lack of full equilibration of 2H2O would result in TBW results that were lower than the BIS results, as was found in this study. In retrospect, because of lactation, a longer time may be needed for equilibration of isotopic tracers. Wong et al (30) suggested that an equilibration time of 6 h is needed in lactating women. Such a long equilibration time will also result in a greater nonaqueous H+ exchange, greater insensible losses through respiration and urine production, and the need to keep the lactating subject hydrated. Therefore, trade-offs between benefits and detriments must be made when designing study procedures in a field setting.
The reasonable intermethod difference (26) for each group increased our confidence that BIS accurately measured FFM and FM in both groups of mothers. The bias and precision between methods in our study were comparable to those found in other studies that compared methods of assessing body composition (29, 31).
Maintenance of FFM and FM is considered important for health and for the attenuation of HIV disease progression (11, 32). Accurate measurements of FFM and FM are therefore important in the clinical assessment of HIV+ persons. The 2H2O method is not possible for routine use in poor areas of Africa for methodologic and technical reasons. In contrast, BIS is simple to perform with adequate operator training, can be operated on battery power in almost any setting, is quick and without patient discomfort, and requires only one person to take the measurement. The major initial cost is the BIS instrument; the recurrent cost of the gel electrode pads and alcohol wipes is minimal compared with the costs of other laboratory-based methods.
Anthropometric measurements and the 2H2O method
Circumference and skinfold-thickness measurements are inexpensive, noninvasive techniques for measuring subcutaneous fat depots and estimating total body fat in this population. However, accurate measurements are dependent on appropriately trained observers whose measurements are standardized against each other, as was done in this study. In addition, given the relation between lipodystrophy and antiretroviral therapy, the anthropometric measurements have the advantage of measuring changes in the location of body fat stores. Our study found that all skinfold-thickness measurements, BMI, and MUAC were well correlated with FM2H2O in both HIV+ and HIV– mothers. It is interesting that none of the anthropometric measures was correlated with FFM2H2O in HIV+ mothers, whereas they were so correlated in HIV– mothers but less well than with FM. This is a finding similar to that of Grinspoon et al (28), who noted that BMI was not significantly correlated with FFM determined by dual-energy X-ray absorptiometry in 33 HIV-infected women and that skinfold-thickness measurements correlated least well to FFM.
In our study population, the Durnin-Womersley equation for estimating body composition tended to overestimate FFM and underestimate FM in both HIV+ and HIV– mothers; this finding is similar to that from a study of HIV+ men in Australia (33). Unfortunately, the race or ethnicity of the subjects used to develop the Durnin-Womersley equation is not reported. If most of the subjects were white, that may partially explain this difference between methods. Wagner and Heyward (34) reviewed studies of ethnic patterns of body composition and found that blacks have less subcutaneous fat in the limbs than in the trunk, and that they tend to carry more fat on the back and lateral portions of the trunk than do whites. Kotler et al (35) found that FM was significantly higher in HIV– African American women than in HIV– white American women, but the it did not differ significantly from that in HIV– African women from Zaire. This is consistent with our finding in this study that black mothers in KwaZulu-Natal have more FM than do mainly white breastfeeding mothers from the northern hemisphere (36-38). These ethnic or racial differences could produce systematic errors in the use of references of body composition that do not take ethnicity and race into consideration. Further studies are needed to investigate the influence of ethnicity and race on body composition, to validate anthropometric equations for predicting FFM and FM in African women, and to ascertain the best methods for accurately measuring FFM in HIV+ women.
Effect of HIV on body composition
Our study found no evidence of wasting in either HIV+ or HIV– breastfeeding mothers living in this region of South Africa. Indeed, the mean percentage body fat (37%) is higher than that in lactating women in other countries (28, 39). Compared with 46 HIV+ women (breastfeeding status unknown) in Zaire who were measured with BIA, the HIV+ mothers in the current study weighed 20 kg more and had 2 kg more FFM and 18 kg more FM (35). This difference may be partially explained by the general good health of the mothers we enrolled. None of the mothers were acutely ill, and only one had advanced immunosuppression (CD4 cell count: <200/µL). In contrast, most mother-to-child transmission prevention trials report that 12–15% of enrolled HIV+ women have CD4+ cell counts < 200/µL. Thus, we are unable to assess the effect of more advanced disease on the validity of the BIS method.
We are not aware of any studies describing the nutritional status of either HIV+ or HIV– breastfeeding mothers in South Africa. It is therefore difficult to ascertain whether the mothers measured in this study are representative. However, the study mothers were socioeconomically representative of the location and had demographic surveillance information consistent with that obtained from the Africa Centre Demographic Information System.
In summary, we have shown that BIS provides an estimate of body composition that is 105% to 106% of the estimate obtained with the 2H2O method, as measured under our study conditions. This is a reasonable approximation of the values obtained with the reference method, and, given our study conditions, we are not sure which method provides the truest estimation. Regardless of the true value, the relation does not vary for HIV+ and HIV– mothers. BIS is a simple field technique that is useful for assessing body composition and for comparing groups of HIV+ and HIV– women in this location. Breastfeeding mothers living in our study area appear to be adequately nourished in terms of body composition and anthropometry. Studies of women with advanced HIV disease are needed to more fully test the limits of the BIS method for accurate assessment of body composition.
ACKNOWLEDGMENTS
We thank the Maternal Nutrition Study staff for subject recruitment; Carina Herbst and Ruth Bland for study management support; the Africa Centre Vertical Transmission Study HIV counselors, clinic assistants, and staff for study organization and data collection; the mothers who generously gave of their time and participated in this study; and the nursing staff at Madwaleni and KwaMsane clinics. The skillful laboratory guidance of Manual Tengonciang of the Western Human Nutrition Research Center is gratefully acknowledged. This paper is written on behalf of the Child Health Group of the Africa Centre for Health and Population studies: HM Coovadia, RM Bland, and A Coutsoudis of that group reviewed and approved the study design and the submitted manuscript.
PCP was responsible for the study concept and design, field supervision, analysis and interpretation of results, and drafting of the manuscript. KHB, NCR, MDVL, and MLB contributed to the study design, interpretation of results, and manuscript revisions. MVL was also responsible for the supervision of 2H2O analysis. The manuscript was reviewed and approved by all authors. None of the authors had a personal or financial conflict of interest.
REFERENCES