L'allaitement maternel est la modalité idéale d'alimentation du nourrisson. Les propriétés anti-infectieuses du lait maternel sont bien documentées. L'allaitement maternel protège les nourrissons contre les infections intestinales et respiratoires. L'allaitement maternel exclusif est recommandé pendant les 6 premiers mois, principalement parce que le lait maternel satisfait de façon optimale à tous les besoins nutritionnels et hydriques du nourrisson. Les nouvelles infections périnatales par le VIH dans les pays riches ont presque été éliminées grâce à la combinaison du dépistage prénatal du VIH, à la prophylaxie antirétrovirale de la mère et de l'enfant, à la césarienne élective et l'évitement de l'allaitement maternel. Bien que les interventions efficaces soient disponibles pour réduire la transmission in utero et intrapartum dans les pays à ressources limitées, la transmission postnatale du VIH par l'allaitement demeure un enjeu de santé publique. L'acquisition du VIH par l'allaitement maternel est responsable d'environ 40% des nouvelles infections en Afrique subsaharienne. Les études effectuées au cours de cette thèse faisaient partie d'un programme d'intervention qui a porté sur l'utilisation des différentes formes d'alimentation du nourrisson dans un environnement rural, à Umkhanyakude, dans le nord du KwaZulu-Natal, en Afrique du Sud. Les femmes ont été incluses dans cette étude avant le début de l'accès universel aux antirétroviraux en Afrique du Sud (2005). Le travail de doctorat visait à acquérir une meilleure compréhension de la transmission postnatale du VIH-1 par l'allaitement maternel, indispensable pour atteindre l'objectif de l'Organisation mondiale de la Santé de réduire toutes les formes de transmission du VIH de la mère à l'enfant (TME) à moins de 5% d'ici la fin de 2015. Dans la première étude, nous apportons la preuve que l'exposition cumulative à l'ARN VIH-1 par le lait maternel est un facteur de risque associé à la transmission postnatale de la mère à l'enfant, indépendamment du taux de CD4 maternels et de la charge virale plasmatique du VIH-1. Ces données fournissent une meilleure évaluation du risque de transmission mère-enfant du VIH-1 et de la charge virale dans le compartiment mammaire. Dans la seconde étude, nous confirmons que la charge virale associée aux cellules dans le lait maternel est un meilleur facteur prédictif du risque de TME postnatale précoce que la charge virale libre. En revanche, la charge virale libre est un facteur prédictif de transmission postnatale tardive (au-delà de 6 mois). Dans la troisième étude, nous avons étudié l'impact sur la TME du VIH-1 du cytomégalovirus (CMV) et du virus d'Epstein-Barr (EBV) dans le lait maternel des mères infectées par le VIH. Des niveaux élevés de CMV sont excrétés dans le lait maternel, et un niveau significatif de l'EBV est fréquemment observé. Les mères dont le lait maternel contient des niveaux élevés de CMV étaient jusqu'à deux fois et demi plus susceptibles de transmettre le VIH-1 à leur enfant par l'allaitement maternel comparativement aux femmes ayant un faible niveau de réplication de CMV. Nous apportons donc la preuve d'une association, indépendante de la charge virale du VIH-1, entre l'excrétion du CMV dans le lait maternel et la transmission postnatal du VIH-1. Chez les femmes allaitantes infectées par le VIH-1 et sous traitement antirétroviral, le risque de transmission résiduelle par l'allaitement est expliqué en partie par la persistance du virus associé aux cellules dans le lait maternel. D'autres études sont nécessaires pour approfondir les connaissances sur le mécanisme du VIH-1 transmission pendant l'allaitement, et les facteurs associés à l'excrétion compartimentée du VIH-1 dans le lait maternel, et pour aider à développer des médicaments plus efficaces pour une utilisation dans les populations à ressources limitées où l'évitement de l'allaitement maternel est souvent impossible. / Breastfeeding is a most valuable source of nutrition for infants. The anti-infective properties of breast milk are well documented and breastfeeding protects infants against gastrointestinal and respiratory illnesses. There is no disagreement that breastfeeding is the best form of nutrition for all infants everywhere. Exclusive breast-feeding for 6 months is recommended for the general population primarily because human milk can satisfy all of an infants' nutritional and hydration needs. New perinatal HIV infections in resource-rich countries have nearly been eliminated with the combination of universal, opt-out antenatal HIV testing, antiretroviral prophylaxis of the mother and infant, elective cesarean delivery, and avoidance of breastfeeding. Although effective interventions are available to reduce in utero and intrapartum transmission in resource-limited settings, postnatal transmission of HIV through breastfeeding has remained a significant problem. Acquisition of HIV through breast milk accounts for an estimated 40% of new infections in sub-Saharan Africa, where more than 90% of perinatal infection occurs. The studies performed during this PhD were part of a larger intervention program in KwaZulu-Natal that focused on the use of different forms of infant feeding within a rural setting. The Umkhanyakude district in northern KwaZulu-Natal, South Africa, is one of the areas worst affected by the HIV and AIDS pandemic, and has some of the highest prevalence figures in the world. Women were enrolled into this study prior to commencement of the South African national antiretroviral roll-out in 2005. This PhD research forms part of efforts to gain a better understanding of postnatal transmission of HIV-1 via breastfeeding, and to support the World Health Organization in their goal to reduce all forms of mother-to-child transmission (MTCT) to below five percent by the end of 2015. In the first study performed, we provide for the first time evidence that cumulative exposure to HIV-1 RNA in breast milk is a key risk factor associated with postnatal mother-to-child transmission, independent of maternal CD4 and plasma HIV-1 viral load. This data provides a better evaluation of the risk of HIV-1 MTCT and intra-breast viral load. In the second study we confirm that cell-associated virus load in breast milk is a stronger predictor of the risk of early postnatal MTCT than cell-free virus, independent of HIV-1 replication in blood and breast milk. In contrast, cell-free virus load is a stronger predictor of later postnatal HIV-1 transmission. We provide evidence that the HIV-1 reservoir is a main risk factor for post-natal MTCT of HIV-1. In the third study performed, we investigated the significance and impact of Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in breast milk from HIV-infected mothers, and MTCT of HIV-1. High levels of CMV is shed in breast milk, and frequently a significant level of EBV is shed in HIV-infected women. Hence, mothers whose breast milk contained high levels of CMV, were up to two and a half times more likely to transmit HIV-1 to her infant via breastfeeding compared to women with low levels. This is the first evidence of an association, independent of HIV-1 viral load, between CMV in breast milk and postnatal MTCT of HIV-1. In contemporary breastfeeding populations with access to antiretroviral prophylaxis, the residual HIV-1 transmission risk, especially in the early postpartum period, is explained in part by the persistence of cell-associated virus in breast milk. More studies are needed to further knowledge on the mechanism of HIV-1 transmission during lactation, and factors associated with compartmentalized shedding of HIV-1 in breast milk, and to help develop more effective drugs for use in resource-limited populations where avoidance of breastfeeding is almost impossible.

MSCPNT / Department of Nutrition / Introduction: Iodine is an essential nutrient required by humans for the synthesis of thyroid hormones, which are vital for normal growth and development. Objective: The primary aim of the study was to describe the iodine status of lactating mothers and infants aged from 0 to 6 months in the Vhembe and Mopani Districts. Methods: A cross-sectional study conducted on 246 infant-mother pair, from the Mopani and the Vhembe Districts. Data was gathered using a questionnaire. Breastmilk, mother urine, infant urine, household salt and drinking water were collected to be analysed for iodine content. Results: The median of breastmilk iodine concentration level amongst lactating mothers in the Vhembe District was 101.4 µg/l (IQR 62.9 – 175.1 µg/l) and 154.4 µg/l (IQR 92.6 – 211.8 µg/L) in Mopani. The median UIC of mothers in Vhembe was 98.5 (IQR 57.66 – 153.93), whereas in the Mopani District the median UIC of mothers was 126.08 µg/l (IQR 69.89 – 206.71 µg/L). The median UIC of infants in Vhembe was 220 (IQR 106.67 – 418.43 µg/l) and in the Mopani District was 321.94 µg/l (IQR 167.96 – 482.66 µg/l). Conclusion: The BMIC in the study signifies iodine sufficiency in both the Vhembe and the Mopani Districts. The results of this study suggest that the BMIC be included in studies assessing iodine status in lactating mothers since the UIC only reflects iodine that was consumed recently. The UIC may under estimate the maternal iodine status if it is not complemented by the BMIC data. / NRF

For most of human history, breastfeeding has been the optimal source of nutrition for infants. Human milk contains a variety of important nutritional sources including vitamins, fats, proteins, and immunological components. With the rise of artificial infant formulas, however, breastfeeding as a whole has decreased around the world. Preterm infants are especially susceptible to diseases such as necrotizing enterocolitis in the first few weeks of life. Therefore, they have the most to gain from the extra immunological and nutritional support that is present in human milk. Within the last few decades, donor human milk has been viewed as the next best option if mothers own milk is not available. Donor human milk contains many of the same beneficial milk properties as regular human milk including immunoglobulins and human milk oligosaccharides. Studies have shown decreases in preterm cases of NEC and fewer deaths in infants who received DHM. One argument against the use of DHM is that pasteurization can reduce the beneficial enzymes and immunoglobulins present in samples. However, the increased use of human milk fortifiers has been able to significantly decrease the nutrient gap between regular human milk and donor milk. Overall, DHM along with proper fortification serves as the best and most cost effective way to feed preterm infants if mother’s milk is unavailable.

Human breast milk is a biofluid produced by a woman’s body during pregnancy. Breast milk contains necessary nutrition to a growing infant as well as xenobiotics--including drugs of abuse-- consumed by the woman which diffuse into the breast milk from the bloodstream. Since breast milk is recommended to be part of all infants’ diets, being able to detect any toxic components--such as drugs--in the matrix is critical. However, despite the ease and noninvasive nature of collection, human breast milk is a difficult matrix to analyze due to its high fat and protein content. Thus far, no literature has been published on the analysis of breast milk through direct analysis in real time mass spectrometry (DART-MS). Adapting DART-MS to detect drugs of abuse in human breast milk will allow for quick and timely identification of drugs present in an individual’s breast milk, as well as aid in research regarding the potential harmful effects of drugs--both licit and illicit--on an infant who is breastfeeding. Forensically, this method could potentially allow toxicologists to use breast milk as a matrix to determine if drugs played a role in a woman’s or breastfed child’s death. Using both C18 biocompatible solid-phase microextraction (BIO-SPME) fibers and QuickStrip™ cards, a DART-MS method was developed to be able to detect drugs of abuse in human breast milk. Four drugs of abuse (cocaine, codeine, morphine, and delta-9-tetrahydrocannabinol (Δ9-THC))--all of which are either commonly abused during the postpartum period or are of particular danger to breastfeeding women--were chosen to be studied. The drugs of abuse were extracted from either whole or pre-filtered human breast milk using either liquid-liquid extraction or C18 BIO-SPME fibers and detected with DART-MS using parameters suggested by IonSense, Incorporation (Inc.). Mass spectral results indicated that macromolecules in whole breast milk did not hinder extraction or detection and that a larger amount of the analytes were ionized/desorbed when using the BIO-SPME fibers. Thus, a BIO-SPME method adopted from IonSense, Inc. utilizing C18 fibers and SPME DART-MS parameters (with temperature and rail time adjustments) can be used to quickly detect cocaine, codeine, morphine, and Δ9-THC in human breast milk, indicating that this method may be used for the detection of other drugs of abuse in breast milk. In addition, BIO-SPME fibers can be used to quantify the concentration of cocaine in breast milk between a range of 50 and 200 nanograms per milliliter as demonstrated by a matrix-matched calibration curve created using various concentrations of cocaine. Despite its benefits, the BIO-SPME and DART method cannot be used on samples containing more than one drug of abuse (based upon the drug concentrations utilized in this study) due to competitive adsorption and competitive ionization, respectively, as not all drugs could be detected when this method was applied to breast milk samples containing numerous drugs.

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Mature human milk composition was determined from monthly samples collected from five, healthy, Caucasian, lactating women. A 72-hour dietary record was kept monthly from the twelfth week of pregnancy to the sixth month postpartum. Nutrient content of milk samples was similar to values reported in the literature. Calcium and zinc concentrations of human milk decreased significantly during the five month study. Moisture, energy, total lipids, protein, and magnesium levels remained fairly constant over the course of lactation. Dietary intake during pregnancy was not significantly correlated with nutrient concentration in human milk. For dietary intake during lactation, a significant correlation was observed between caloric intake and energy content of human milk for the second month of lactation. A significant correlation was observed between protein intake and protein content in the milk for the sixth month of lactation. Zinc concentration was significantly correlated with dietary intake during the fourth month of lactation. No other significant correlation was observed between nutrient content of human milk and dietary intake during the lactation period. / M.S.

Lectin affinity chromatography has been applied to the separation of the sialyloligosaccharides of human milk. A human milk sialyloligosaccharide fraction was tritium labeled and applied to a highly substituted WGA-agarose column (20 mg/ml). Only a single component from the complete sialyloligosaccharide fraction was retarded in the WGA-agarose column. The WGA-bound fraction when applied to paper chromatography migrated with identical mobility as the sialylhexasaccharide fraction (S-5) of human milk, previously isolated and described by Kobata and Ginsburg in 1972 [Arch. Biochem. Biophys., 150:273-281]. A purified sialylhexasaccharide fraction (S-5), isolated according to the method of Kobata and Ginsburg, was radiolabeled and applied to the WGA-agarose column. The WGA-bound (60%) and WGA-unbound (40%) sialylhexasaccharide fractions were isolated. The WGA-bound sialylhexasaccharide fraction was subjected to neuraminidase digestion to remove sialic acid, and the resulting neutral oligosaccharide had more affinity for the WGA-agarose column. Sequential exoglycosidase digestion of the asialo derivative of the WGA-bound fraction with jack bean β -galactosidase and β -hexosaminidase demonstrated the presence of a lacto-N-neohexaose core. The position of sialic acid in the sialyllacto-N-neohexaose was determined by simultaneous digestion of the sialylhexaose with jack bean β -galactosidase and β -hexosaminidase, which removed the non-sialylated branch from the sialylhexaose and produced a sialyltetraose. The sialyltetraose was found to be sialyltetrasaccharide c as demonstrated by its elution time on HPLC and direct binding to monospecific anti-sialyltetrasccharide c serum. The structural data indicated that the WGA-bound sialylhexaose is a sialyl derivative of lacto-N-neohexaose with sialic acid linked to the 3 branch of this core structure which represents a previously undescribed sialyloligosaccharide in human milk. The structure of the WGA-bound sialylhexaose is, / M.S.

The percent in-vitro digestibility of proteins in human milk was determined by three different methods and compared with the in-vitro digestibility of proteins in Enfamil, Similac and Isomil. In-vitro digestibility was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), pH drop and a simultaneous dialysis method. Significant differences (P<0.05) were observed in the in-vitro protein digestibility of human milk and the three infant formulas. Estimated percent in-vitro protein digestibility of each of the samples was also found to be significantly affected by the method of determination. The in-vitro protein digestibility of all four samples estimated by simultaneous dialysis was lower than results obtained by SDS-PAGE and pH drop methods. Except for Isomil, results obtained by the pH drop method were lower than those determined by SDS-PAGE. The in-vitro protein digestibility of Enfamil and Similac was found to be greater than that of human milk by all three methods. Except for results obtained by SDS-PAGE, the in-vitro digestibility of proteins in Isomil was found to be greater than for the proteins in human milk. These results indicate that the proteins in human milk are not as extensively hydrolyzed in-vitro as the proteins in powdered infant formulas. / Master of Science

The influence of vitamin B-6 intake on vitamin B-6 status and the concentration of B-6 vitamers in milk of 21 white lactating women (21 to 35 years) was examined at 3 to 7 months postpartum. None of the women met the RDA for lactating women of 2.5 mg/day when considering vitamin B-6 intakes from food sources alone. All subjects taking vitamin B-6 supplements had adequate vitamin B-6 status as determined by coenzyme stimulation of erythrocyte alanine aminotransferase activity; all subjects not taking vitamin B-6 supplements had inadequate vitamin B-6 status. Plasma pyridoxal 5-phosphate values were significantly higher for subjects in the supplemented than in the nonsupplemented group. Pyridoxal, pyridoxamine, pyridoxine, and total vitamin B-6 concentrations in milk were higher, sometimes significantly, in the supplemented than in the unsupplemented group as determined by microbiological assay and HPLC. There were significant correlations between data obtained by the microbiological and HPLC analyses for pyridoxal and total vitamin B-6 concentrations. Pyridoxal was the predominant B-6 vitamer found in human milk. Distribution of the B-6 vitamers appeared to stay relatively constant despite vitamin B-6 status. / Master of Science