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. Author manuscript; available in PMC: 2016 Aug 1.
Published in final edited form as: Clin Nutr ESPEN. 2015 Aug 1;10(4):e140–e146. doi: 10.1016/j.clnesp.2015.05.003

Maternal post-absorptive leucine kinetics during late pregnancy in US women with HIV taking antiretroviral therapy: a cross-sectional pilot study

W Todd Cade a, Gautam K Singh b, Mark R Holland c, Dominic N Reeds d, E Turner Overton d, Nancy Cibulka e, Karen Bahow a, Rachel Presti d, Andrea Stephens f, Alison G Cahill f
PMCID: PMC4528644  NIHMSID: NIHMS696139  PMID: 26273702

Abstract

Background

Despite the success of combination antiretroviral therapy (cART) for the prevention of mother to child transmission of HIV, infants exposed to cART in utero frequently are born smaller and have mild cardiac abnormalities. The mechanisms responsible for lower birth weight and cardiac abnormalities in children exposed to cART are unclear but could be related to dysregulation of maternal amino acid metabolism during pregnancy. Previous data in HIV(−) women have shown a relationship between abnormal maternal protein metabolism during pregnancy and low infant birth weight and animal data demonstrate a relationship between altered maternal protein metabolism and increased risk for offspring cardiovascular abnormalities.

Objective

The objectives of this study were to:

  1. characterize post-absorptive maternal leucine kinetics during late pregnancy and

  2. examine the relationships between maternal leucine kinetics and offspring birth weight and cardiac function.

Design

Post-absorptive maternal leucine kinetics (evaluated by using stable isotope tracer methodology) in 16 HIV(+) women receiving cART and 14 HIV(−) US women during the 3rd trimester of pregnancy were compared. Relationships between post-absorptive maternal leucine kinetics, cardiac function (echocardiography) and birth weight were statistically examined.

Results

Maternal plasma leucine concentration (HIV(−): 82.8 ± 10.7 vs. HIV(+): 72.3 ± 13.5 μM, p=0.06) and leucine oxidation rate (HIV(−): 6.1 ± 1.6 vs. HIV(+): 4.9 ± 1.8 μmol/kgBW/min, p=0.03) were lower in HIV+ women compared to controls. Total leucine turnover rate, non-oxidative leucine disposal rate and post-absorptive maternal glucose and palmitate kinetics did not differ between groups. Left ventricular fractional shortening tended to be lower in children born to HIV(+) compared to controls (HIV(−): 42 ± 1 vs. HIV+: 36 ± 5 %, p=0.08) and associated with lower maternal plasma leucine concentration (r= 0.43, p=0.08).

Conclusions

Preliminary results indicate that post-absorptive maternal leucine metabolism during late pregnancy is mildly altered in HIV+ US women taking cART. The clinical significance of maternal leucine metabolism on adverse infant outcomes is unclear and should be further explored in more expansive studies.

Keywords: HIV, metabolism, pregnancy, amino acid, nutrition

Introduction

Annually over 1.4 million children are born to women with HIV-infection world-wide(1). Combination antiretroviral treatment (cART) is effective in preventing the vertical transmission of Human Immunodeficiency Virus (HIV) from mother to child during pregnancy and parturition (2–4). However children exposed to cART in utero, regardless of their HIV status appear to be at a greater risk for lower birth weight (5, 6) and stunted growth and wasting(7, 8), particularly in less developed countries. In addition, children exposed to cART in utero are born with minor cardiac abnormalities (9) that persist into pre-adolescence (10).

Mechanisms for lower birth weight and impaired cardiac abnormalities in offspring exposed to cART are unclear. In HIV(−) women, abnormalities in protein metabolism (i.e. intake) during pregnancy is associated in lower offspring birth weight (11, 12). In non-HIV animal models, maternal protein restriction during pregnancy results in lower offspring birth weight(13) and abnormalities in molecular regulators of cardiac growth(14). In addition, in a rodent model of intrauterine growth restriction (a condition where offspring are born smaller and frequently have cardiovascular abnormalities), maternal leucine turnover, concentration and fetal leucine delivery is blunted(15, 16).

Disruptions in amino acid metabolism are well-known in HIV(+) adults both taking and not taking cART(17–19), however, little is known regarding maternal amino acid metabolism during HIV(+) pregnancy and its potential effects on infant birth weight and cardiac function. Based on previous findings of insulin resistance(20, 21) and impaired fatty acid oxidation(22) in non-gravid HIV(+) adults, we hypothesized that maternal leucine utilization (i.e. oxidation rate) during HIV(+) pregnancy would be higher in order to meet maternal energy needs (i.e. to compensate for insulin resistance and lower fatty acid oxidation) leaving less leucine (i.e. lower plasma concentration and leucine non-oxidative disposal rate) available for fetal growth and metabolism. Subsequently, reduced maternal amino acid delivery to the fetus could contribute to lower birth weight and cardiac function in the offspring by limiting the amount of leucine or by disrupting growth signaling needed for fetal organ growth and maturation. Therefore, our primary aim was to compare post-absorptive maternal leucine kinetics in late pregnancy between women with and without HIV taking cART. Our secondary aim was to examine the relationships between maternal post-absorptive leucine kinetics and infant birth weight and cardiac function. We also measured maternal glucose and fatty acid kinetics in order to examine leucine kinetics in the context of overall maternal substrate metabolism. Identification of mechanisms for abnormalities in infant body composition and cardiac structure and function in children exposed to HIV and cART may lead to nutritional optimization strategies for HIV+ pregnancy; especially important in resource limited countries and in those who are socio-economically disadvantaged, where nutrition is often sub-optimal.

Participants

Pregnant women were recruited from January 2009–December 2010. HIV-infected women (n=16) were recruited from the AIDS Clinical Trials Unit and the Infectious Diseases Clinics at Washington University School of Medicine (WUSM), and HIV-negative women (n=14) were recruited from the WUSM/Barnes Jewish Hospital Women’s Health Clinic. Twenty HIV(+) pregnant women were screened and 16 agreed to participate and were enrolled; and 19 HIV(−) pregnant women were screened and n=14 agreed to participate and were enrolled in the study (total n=30). All HIV(+) women were infected less than 10 years and did not have Acquired Immune Deficiency Syndrome (AIDS). HIV(+) women were taking combination antiretroviral therapy (cART) during their pregnancy and all but three HIV(+) women had undetectable viral loads at the time of the metabolic study (Table 1). Twelve (n=12) of 16 HIV(+) women were taking combivir (lamivudine/zidovudine) and all were taking a protease inhibitor. Eleven (n=11) of 16 HIV(+) women received intravenous zidovudine during parturition. All enrolled HIV(+) and HIV(−) women were excluded if they had current or a history of gestational diabetes, were not sedentary (exercise ≥ 2x/week), had multiple pregnancies, had a fetal abnormality (as determined by routine standard of care ultrasonography), and if they were currently using illegal drugs or had greater than moderate alcohol consumption (by self-report). The Human Studies Committee at Washington University approved the study and all women provided informed consent before participating.

Table 1.

Maternal Demographic and Metabolic Variables

Variable HIV(−)(n=14) HIV(+)(n=16) p-value
Age (yrs) 26 ± 5(23,29) 26 ± 4(24,28) 0.89
Race (n, White/Black) 4/10 2/14 0.13
Height (cm) 164.7 ± 4.7(162.2,167.2) 165.5 ± 8.6(161.3,169.7) 0.68
Weight at study (kg) 86.0 ± 24.3(73.3,98.7) 85.3 ± 22.1(74.5,96.1) 0.70
GWG (kg) 17.2 ± 6.9(13.6,20.8) 17.7 ± 21.2(7.3,28.1) 0.20
Smoking during pregnancy (n) 4 7 0.39
HIV duration (yrs) N/A 5.3 ± 3.4(3.6,8.7)
3rd trimester CD4 (cells/dL) N/A 509 ± 205(409,609)
3rd trimester viral load (copies/mL)
 <400 (%) N/A 88
 400–1000 (%) N/A 12
Hematocrit 31.0 ± 1.9(30,32) 28.7 ± 3.3(27.1,30.3) 0.05
HbA1C (%) 5.1 ± 0.8(4.7,5.5) 5.4 ± 1.4(4.7,6.1) 0.65
Fasting Glucose (mg/dL) 77.4 ± 5.8(74.4,80.4) 76.9 ± 7.1(73.4,80.4) 0.76
Fasting Insulin (μU/mL) 12.9 ± 12.3(6.5,19.3) 9.1 ± 5.4(6.5,11.8) 0.79
TG (mg/dL) 150.2 ± 36.2(131.2,169.2) 160.3 ± 61.1(130.4,190.2) 1.00
HDL (mg/dL) 65.5 ± 15.6(57.3,73.7) 60.0 ± 12.1(54.1,72.1) 0.36
LDL (mg/dL) 109.1 ± 22.9(97.1,121.1) 86.8 ± 36.6(68.9,104.7) 0.24
Chol (mg/dL) 204.7 ± 38.9(184.3,225.1) 178.9 ± 38.2(160.2,197.6) 0.10
FFA (μmol/L) 467.9 ± 103.7(413.6,522.2) 555.0 ± 122.5(495.0,615.0) 0.06
βHB (μmol/L) 318.1 ± 149.7(239.7,239.7) 317.2 ± 166.4(235.7,398.7) 0.92
Growth Hormone (ng/ml) 0.48 ± 0.69(0.12,0.84) 0.39 ± 0.34(0.22,0.56) 0.53
Cortisol (ug/dL) 23.4 ± 6.9(19.8,27.0) 21.7 ± 4.7(19.4,24.0) 0.53
IGF-1 (ng/mL) 375.8 ± 211.2(164.6,486.4) 249.8 ± 101.6(200.0,299.6) 0.11
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Values are mean ± SD (95% CI). GWG: gestational weight gain, TG: triglyceride, HDL: high density lipoprotein, LDL: low density lipoprotein, Chol: total cholesterol, FFA: free fatty acid, βHB: beta hydoxy butarate, IGF-1: insulin-like growth factor 1.

Methods

Fasting Blood Chemistry and Physical Examination

Using a cross sectional design, all women were studied in the 3rd trimester of pregnancy, between gestational weeks 30–36. Upon enrollment, all women provided comprehensive medical history and underwent a physical examination. Fasting blood chemistry were obtained and included complete blood cell count, endocrine profiles (insulin, triglycerides (TG), total-, LDL-, and HDL-cholesterol), serum lipid, lipoprotein, and plasma HIV RNA quantitation (Roche Amplicor HIV-1 Monitor®; Roche Diagnostics Corporation, Indianapolis, IN). Plasma analyses were performed as previously described (20).

Substrate Kinetics Study

The evening prior (1800h) to the metabolic study, participating women consumed a standardized meal, based on body weight (BW)(12 kcal/kg BW) with 55% of calories from carbohydrate, 30% from fat and 15% from protein. In order to ensure adequate muscle and hepatic glycogen stores the women ingested a high carbohydrate liquid beverage (80 g carbohydrates, 12.2 g fat, 17.6 g protein) (Ensure™; Ross Laboratories, Columbus, OH) following the meal (1900h). Participating women then remained fasted (excluding water) until completion of the study the following day. The following morning at 0700h, participants reported to the Clinical Research Unit at the Washington University Institute of Clinical and Translational Sciences. An intravenous catheter for isotope infusion was inserted into a forearm vein and a contralateral intravenous catheter was inserted into hand vein (heated to 55°C) for arterialized venous sampling. At 0800h (time 0 min), a constant infusion of [2,2-2H]palmitate (0.04 μmol/kgBW/min) bound to 25% serum albumin (Centeon, LLC, Kankakee, IL), a primed, constant infusion of 1-13C leucine (0.06 μmol/kgBW/min) with a 1-13C bicarbonate prime (1.2 μmol/kgBW, in 0.9% saline), and a primed, constant infusion of [6,6-2H] glucose (0.25 μmol/kgBW/min) dissolved in 0.9% saline were started and maintained for 180 min as previously described (22, 23). Leucine, an essential amino acid, was selected for use as a tracer in the current study because leucine is oxidized in skeletal muscle(24) and has a regulatory effect on other branched chain amino acids(25) and muscle protein synthesis(26). Thus, we hypothesized that leucine might have a regulatory role in fetal muscle and heart development. All tracers were obtained from Cambridge Isotope Laboratories (Andover, MA).

Baseline blood samples were collected before starting the study and additional blood samples were collected every 10 min during the last half hour of the study (t=160, 170, 180 min) to determine leucine, glucose and palmitate tracer-to-tracee ratios (TTRs) and plasma substrate and hormone concentrations(20, 22, 23). Vial preservative EDTA was used for substrate kinetics and trayslol for hormone concentrations. Following collection, blood samples were immediately placed in ice and within 30 min, plasma was separated by centrifugation (3000 rpm, 10 min, 4°C, 1006 g) and stored at −80°C until analysis. 13CO2 breath enrichment was obtained by collecting expired breath in evacuated tubes at the same time intervals as the blood samples(22). Indirect calorimetry was used to measure 15 min whole body oxygen consumption (VO2) and CO2 production rates (VCO2) at t=135 min (ParvoMedics, Sandy, UT) for the calculation of resting energy expenditure and leucine oxidation rate.

2H-glucose, 2H-palmitate, and 13C-leucine plasma tracer-to-tracee ratios were quantified using capillary gas chromatography–mass spectrometry (GC-MS; Agilent 6890 N gas chromatograph and Agilent 5973 N mass selective detector; Agilent, Palo Alto, CA, USA) as previously described(19, 27). For enrichment quantification of 2H-glucose, cold acetone was used to precipitate plasma proteins, lipids were extracted into hexane, and the aqueous phase was dried (Labconco, Kansas City, MO, USA). Glucose was derivatized to form the heptafluorobutyric derivative of glucose, and GC electron ionization (EI)-MS and selective ion monitoring was used to determine 2H-glucose enrichment. Plasma leucine was isolated using cation exchange chromatography. Plasma leucine was derivatized to the heptafluorobutyric propyl ester derivative. GC-MS in negative-chemical ionization mode was used to determine 13C- leucine enrichment. Plasma leucine concentration was determined using an internal standard ([U-13C6]-leucine). For 2H-palmitate enrichment, cold acetone was used to precipitate plasma proteins, lipids were extracted into hexane, and iodomethane and dichloromethane were used to form fatty acid methyl esters. GC-EI-MS with selective ion monitoring was used to determine plasma 2H-palmitate enrichment. The GC-MS instrument response was calibrated using gravimetric standards of known isotope enrichment. Breath 13CO2 enrichment was measured using gas isotope ratio mass spectrometry (IRMS; Finnigan Delta + XL, Bremen, Germany)(28).

Calculations

Plasma leucine, palmitate, and glucose rates of appearance were calculated by dividing each tracer infusion rate by the average tracer-to-tracee (TTR) ratio obtained during the last 30 min of the study (time 160, 170, 180 min). Leucine oxidation rate was determined by multiplying the average breath 13CO2 TTR obtained during the last 30 min of the study (time 160, 170, 180 min) by the CO2 production rate from indirect calorimetry (13CO2 TTR x VCO2) divided by the plasma leucine TTR(22). Kinetic rates were expressed per kilogram body weight where appropriate.

Infant Cardiac Function and Birth Weight

Infant anthropometrics including birth weight were obtained from the woman’s medical record. Cardiac function was measured at 1 month post-partum by 2D, Doppler and tissue Doppler echocardiography at the Heart Station at St. Louis Children’s Hospital. Specifically, transthoracic echocardiographic images were acquired using a GE Vivid 7™ imaging system (General Electric Medical Systems, Milwaukee, WI). All images were obtained at frame rates ranging from 60 to 130 fps and consisted of three consecutive heart cycles. The images were obtained by an experienced sonographer, digitally archived for subsequent analyses, and analyzed by one investigator.

Indexes quantifying systolic and diastolic left ventricular (LV) function were determined from analyses of M-mode images to determine LV fractional shortening (FS = [LV end diastolic diameter − LV end systolic diameter]/LV end diastolic diameter) and 2D images were utilized to measure LV ejection fraction using the modified Simpson method(29). The Tei Index(30) and isovolumetric contraction and relaxation times describing cardiac function were determined from analyses of mitral Doppler flow and tissue Doppler (mitral annulus) patterns, respectively(31).

Statistical Analyses

Maternal and infant group differences in demographics, substrate kinetics and cardiac function were determined by Independent Samples Mann-Whitney U test (due to n<30 and non-normal distribution on some variables) and chi-square analyses where appropriate. Relationships between maternal leucine kinetics and infant birth weight and cardiac function were determined by Pearson Product correlation analyses. Results are expressed as means ± SD. Statistical significance was determined at p<0.05 level.

Results

Maternal demographics and plasma metabolites

Age, height, weight, and gestational weight gain (GWG) were similar between the 2 groups of women (Table 1). Fasting maternal plasma HbA1C, glucose, insulin, triglycerides, growth hormone, cortisol, beta-hydroxybutyrate and cholesterol levels were not different between groups. Maternal plasma hematocrit was significantly lower and free fatty acid (p=0.06) concentration was significantly higher in HIV(+) than HIV(−) (Table 1). Maternal plasma concentration of insulin-like growth factor-1 tended to be lower in HIV(+) than HIV(−) (Table 1).

Infant demographics and cardiac function

All infants were born HIV(−). Gestational birth week, birth length, head circumference, chest circumference, and delivery mode were not different between groups (Table 2). There was no difference in birth weight between groups (Table 2). A greater percentage of males (p=0.07) and those admitted to special care nursery were born to HIV(+).

Table 2.

Infant Demographics and Echocardiographic Variables

Variable Born to HIV(−) (n=14) Born to HIV(+)(n=16) p-value
Demographics
 Gestational age (wks) 38 ± 2 38 ± 2 0.55
 Sex (Male/Female, %) 36/64 67/33 0.07
 Delivery mode (Spontaneous/C-section, %) 86/14 69/31 0.27
 Birth weight (g) 2977 ± 561 3187 ± 626 0.22
 Birth length (cm) 50.3 ± 3.1 49.6 ± 2.8 0.58
 Head circumference (cm) 33.6 ± 1.9 34.2 ± 1.7 0.25
 Chest circumference (cm) 31.6 ± 2.4 31.8 ± 2.5 0.82
 APGAR 1-min 7.8 ± 0.7 6.3 ± 2.8 0.27
 APGAR 5-min 8.8 ± 0.4 7.9 ± 1.8 0.53
 Disposition (Nursery/Special Care, %) 83/17 62/38 0.04
Echocardiography
 HR (bpm) 153 ± 18 170 ± 15 0.02
 MM LVPWD (cm) 0.37 ± 0.06 0.40 ± 0.04 0.20
 MM IVSD (cm) 0.42 ± 0.09 0.45 ± 0.05 0.40
 MM LVEDD (cm) 1.95 ± 0.26 1.98 ± 0.16 0.75
 MM LVESD (cm) 1.14 ± 0.18 1.28 ± 0.15 0.15
 FS (%) 42 ± 1 36 ± 5 0.08
 2D LVEDV (ml) 7.6 ± 1.7 7.4 ± 2.1 1.00
 2D LVESV (ml) 2.7 ± 0.8 2.4 ± 0.4 0.62
 EF (%) 62 ± 6 65 ± 6 0.28
 Doppler E′ 0.9 ± 0.2 1.0 ± 0.2 0.10
 Doppler A′ 1.0 ± 0.2 1.0 ± 0.2 0.24
 Doppler AoV (m/s) 1.0 ± 0.2 1.0 ± 0.2 0.71
 Doppler AoV ET (ms) 179.0 ± 15.4 186.0 ± 9.0 0.24
 Tai index 0.49 ± 0.04 0.46 ± 0.08 0.92
 LV Vs (cm/s) 5.8 ± 1.5 6.3 ± 0.9 0.18
 LV Ve (cm/s) 8.6 ± 3.0 10.4 ± 3.1 0.16
 LV Va (cm/s) 10.3 ± 2.9 11.5 ± 2.8 0.04
 IVCT (ms) 38.0 ± 6.4 39.8 ± 5.5 0.48
 IVRT (ms) 41.6 ± 8.2 40.4 ± 6.3 0.70
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Values are mean ± SD. HR: heart rate, MM LVPWD: M-mode left ventricular posterior wall diameter, MM IVSD: M-mode intraventricular septum diameter, MM LVEDD: M-mode left ventricular end diastolic diameter, MM LVESD: M-mode left ventricular end systolic diameter, FS: fractional shortening, 2D LVEDV: two dimensional left ventricular end diastolic volume, 2D LVESV: two dimensional left ventricular end systolic volume, EF: ejection fraction, Doppler E: early filling velocity measured by Doppler echocardiography during diastole, A′: filling velocity measured by Doppler echocardiography during diastole, AoV: aortic velocity, AoV ET: aortic velocity ejection time, LV Vs: left ventricular wall velocity measured by tissue Doppler imaging during systole, LV Ve: left ventricular wall velocity measured by tissue Doppler imaging during early diastole, LV Va: left ventricular wall velocity measured by tissue Doppler imaging during late diastole, IVCT: intraventricular contraction time, IVRT: intraventricular relaxation time.

Echocardiography parameters were only obtained in n=23 infants because only 14 HIV(+) and 9 HIV(−) mother/infant pairs returned for the 1-month post-partum echocardiogram. Reasons for lack of follow up included change of contact information and not returning phone calls after repeated attempts. Heart rate was significantly higher in infants born to HIV(+) than HIV(−) (Table 2). Fractional shortening tended (p=0.08) to be lower and late filling during late diastole measured by tissue Doppler was higher in infants born to HIV(+) compared to HIV(−). Other cardiac parameters were not different between groups of infants (Table 2).

Maternal substrate kinetics

All participants were studied during the mid 3rd trimester of pregnancy (mean: 32.4 ± 2.4 weeks gestation). Total energy expenditure expressed per kg body weight tended (p=0.09) to be lower in HIV(+) compared to HIV(−) (Table 3). Maternal post-absorptive glucose and palmitate turnover rates expressed in absolute or per kg body weight were not different between groups. Maternal fasting plasma leucine concentration was significantly lower (p=0.06) in HIV(+) compared to HIV(−). Leucine oxidation rate expressed per kg body weight was significantly lower in HIV(+) vs. HIV(−) (Table 3). Non-oxidative leucine disposal rate was not different between groups (Table 3). Maternal substrate kinetics are reported in Table 3.

Table 3.

Post-Absorptive Substrate Metabolism

Variable HIV(−) (n=14) HIV(+)(n=16) p-value
Basal
 Energy expenditure (kcal/day) 1959 ± 594 1727 ± 374 0.61
 Energy expenditure (kcal/kgBW/day) 22.9 ± 2.9 20.7 ± 3.9 0.14
 Glucose turnover rate (μmol/min) 5408 ± 1313 5198 ± 1310 0.67
 Glucose turnover rate (μmol/kgBW/min) 64.2 ± 8.5 62.0 ± 10.7 0.53
 Palmitate turnover rate (μmol/min) 183.9 ± 86.1 177.9 ± 87.4 0.98
 Palmitate turnover rate (μmol/kgBW/min) 2.1 ± 0.7 2.1 ± 0.9 0.68
 Fasting plasma leucine (μM) 82.8 ± 10.7 72.3 ± 13.5 0.06
 Leucine turnover rate (μmol/hr) 6760 ± 1233 6541 ± 1415 0.58
 Leucine turnover rate (μmol/kgBW/hr) 81.5 ± 16.3 78.8 ± 15.1 0.76
 Leucine oxidation rate (μmol/hr) 510.3 ± 152.4 419.2 ± 179.3 0.07
 Leucine oxidation rate (μmol/kgBW/hr) 6.1 ± 1.6 4.9 ± 1.8 0.03
 Non-oxidative leucine disposal rate (μmol/hr) 6250 ± 1140 6122 ± 1252 0.86
 Non-oxidative leucine disposal rate: (μmol/kgBW/hr) 75.4 ± 15.4 73.9 ± 14.0 0.92
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Kcal: kilocalorie, BW: body weight, g: gram, μM: micromolar, hr: hour, min: minute

Correlation analyses

Lower cardiac fractional shortening tended to correlate with lower fasting plasma leucine concentration (r= 0.43, p=0.08) and smaller birth weight (r=0.43, p<0.07). There were no significant associations between maternal leucine kinetic rates, infant birth weight or cardiac function.

Discussion

Overall

To our knowledge, this is the first study to quantify maternal substrate kinetics in HIV(+) pregnant women taking cART, and relate these to infant birth metrics, and cardiac functional parameters. The main finding was that maternal post-absorptive leucine kinetics were mildly altered in HIV+ pregnant women and there were trends towards relationships between maternal leucine concentration, infant birth weight and cardiac function. Currently, optimal nutritional requirements for HIV+ women during pregnancy are not clear(32) but these data suggest that they are similar in virologically well-controlled HIV(+) and HIV(−) pregnancy. These data do appear to highlight the importance of well-controlled HIV infection and pre-natal care during HIV(+) pregnancy as all of the HIV(+) women except three had undetectable plasma viremia and attended a similar amount of pre-natal visits as their uninfected peers (~9–10 pre-natal visits for all women). However, the current study did not examine maternal substrate metabolism under the absorptive or insulin-stimulated (i.e. hyperinsulinemic clamp) condition and therefore cannot make any conclusion under these conditions; thus additional research should be performed. Accordingly, the NIH/WHO Working Group executive summary recently recommended further research be performed on the effect of cART on nutritional status during HIV+ pregnancy and its impact on maternal and infant health outcomes(33).

Leucine Metabolism

We found small differences in post-absorptive leucine metabolism in virologically suppressed HIV+ women during the 3rd trimester of pregnancy. Women with HIV had no demonstrable increase in leucine turnover rate but had lower leucine oxidation rate which was contrary to our hypothesis. The effect of HIV and cART on leucine oxidation is unclear as it is elevated in virologically suppressed HIV+ men but is no different between those with AIDS wasting and non-infected controls(19). Higher plasma free fatty acids levels might have inhibited leucine oxidation in HIV(+) women(34). It is possible that lower leucine oxidation rate in HIV+ women during late pregnancy was driven, at least partially, by the lower plasma leucine concentration in the HIV(+) group. The mechanism behind lower plasma leucine concentration in HIV(+) pregnant women is unclear. Fat and carbohydrate intestinal absorption is impaired in individuals with HIV taking cART(35, 36) however the effect of HIV/cART on protein absorption in not known. Lower maternal plasma leucine concentration might lead to lower leucine delivery to the fetus, however, we did not measure cord blood leucine concentration and therefore, this is speculative. Overall, differences in leucine metabolism between well-controlled HIV(+) and HIV(−) pregnancy are small and the clinical meaning is unclear however; these findings might be more clinically important in populations where food insecurity is high and maternal nutrition (i.e. protein) is suboptimal.

Glucose and Fatty Acid Metabolism

In the current study, post-absorptive glucose and fatty acid metabolism kinetics were not different between HIV(+) and HIV(−) women during late pregnancy. This finding was surprising in light of the evidence supportive of dysregulated glucose(37, 38) and fatty acid(20, 22, 39) metabolism in non-gravid HIV(+) adults taking cART, including when women were examined independently(40). HIV(+) women had higher plasma free fatty acid level; however this was not associated with any infant outcome.

Birth Weight and Cardiac Function

Birth weight was not different in infants born to women with and without HIV in the current study. Studies examining the effect of HIV/cART on infant birth weight have been conflicting(5, 6, 41, 42). In studies demonstrating lower birth weight in infants born to HIV(+) women, low maternal CD4 count and high viral load were variables associated with this adverse outcome(43–45); associations not seen in our cohort. Some studies(7, 10) have shown a relationship between cART exposure and lower birth weight(46, 47) but this is not a universal finding(48, 49).

The effect on cardiac function from in utero exposure to HIV and cART is unclear. Infants exposed to HIV and cART had significantly higher heart rate and lower systolic contractile function (fractional shortening) 1-month after birth. Higher heart rate in infants exposed to HIV and cART has been previously reported(50, 51) however these infants were perinatally infected with HIV where infants in the current study were not. Lipshultz et al.(51) showed increased fractional shortening in infants exposed HIV/cART at birth; contrary to the current study’s findings. The current study suggests a hemodynamically significant effect of HIV and cART exposure on infant cardiac function. This intrinsic effect might be a continuum of cardiac dysfunction later in life, evidenced by pre-adolescents exposed to HIV/cART in utero having higher resting heart rate and lower diastolic function when compared to their unexposed peers(10). Well-characterized follow up cardiac function studies in adolescents and adults exposed to HIV/cART are warranted to fully determine the effect of HIV and cART in utero exposure on long-term cardiovascular health and heart function.

Mechanisms for adverse infant outcomes in children exposed to HIV and cART have not been fully elucidated. The relationship between altered maternal amino acid metabolism and infant cardiac abnormalities has been previously demonstrated in non-HIV animal models(13, 14). However, we were not able to confirm this in HIV(+) human pregnancy as maternal leucine kinetics were not associated with infant birth weight or cardiac function. There tended to be a relationship between lower maternal leucine concentration and lower fractional shortening in the infants but in the absence of abnormal maternal kinetic rates or cord blood leucine concentrations, we cannot comment on the significance of this finding. Future studies that examine both maternal and fetal amino acid levels and kinetics and their relationship to body composition and cardiac function in HIV and cART exposed infants are warranted.

Limitations

Maternal substrate kinetics were measured during the post-absorptive state thus the findings cannot be extrapolated to other conditions (fed, insulin-stimulated, exercise). Although pre-study meal composition was identical, habitual maternal diet was not measured. There was no difference between smoking prevalence between groups in the current study however smoking has been associated with altered protein metabolism(52) and low birth weight(53). We did not statistically control for the effect of smoking and thus small differences between the groups in smoking prevalence might have influenced outcomes. Maternal diet was not recorded and maternal physical activity was not objectively measured (e.g. accelerometry) rather by self-report. Umbilical cord blood was not collected thus cord blood levels of leucine, IGF-1, insulin and other metabolites were not measured which may have assisted in the interpretation of the effect of maternal substrate metabolism on infant outcomes. Participants were not stratified by cART composition or HIV duration so the independent effects of HIV and cART on protein metabolism could not be identified. Apart from birth weight, other obstetric outcomes were not analyzed but would be important to examine in larger, future studies.

Conclusions

Maternal leucine metabolism during late pregnancy is mildly altered in HIV(+) women however the clinical importance of this is not clear. Well-controlled viremia and comprehensive pre-natal care during HIV(+) pregnancy might be important to minimize abnormalities in maternal metabolism and adverse infant outcomes. Larger, well-characterized studies examining maternal substrate metabolism during different conditions and the effect on infant outcomes are needed in order to quantify maternal nutritional needs during pregnancy in both well-nourished and food insecure environments.

Acknowledgments

We thank the nursing staff at the Washington University Institute for Clinical and Translational Sciences Clinical Research Unit and AIDS Clinical Trials Unit for their hard work and altruism. We thank Bettina Mittendorfer, PhD and Kevin Yarasheski, PhD for their assistance with interpretation of the data and editing of the manuscript. We also acknowledge our funding sources: NIH DK074343, P30DK056341, DK020579, AI069495, P41 GM103422 and RR024992 from the National Center for Research Resources (NCRR) and NIH Roadmap for Medical Research.

Footnotes

Contributions: WTC, GKS, MRH, DNR, AS researched data, wrote/edited manuscript. AGC and RP reviewed/edited manuscript. ETO and KB researched data, reviewed/edited the manuscript. The authors declare no conflicts of interest.

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