Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

Super-Resolution Stimulated Raman Scattering Microscopy with Graphical User Interface-Supported A-PoD.

Raman microscopy is a vibrational imaging technology that can detect molecular chemical bond vibrational signals. Since this signal is originated from almost every vibrational mode of molecules with different vibrational energy levels, it provides spatiotemporal distribution of various molecules in living organisms without the need for any labeling. The limitations of low signal strength in Raman microscopy have been effectively addressed by incorporating a stimulated emission process, leading to the development of stimulated Raman scattering (SRS) microscopy. Furthermore, the issue of low spatial resolution has been resolved through the application of computational techniques, specifically image deconvolution. In this article, we present a comprehensive guide to super-resolution SRS microscopy using an Adam-based pointillism deconvolution (A-PoD) algorithm, complemented by a user-friendly graphical user interface (GUI). We delve into the crucial parameters and conditions necessary for achieving super-resolved images through SRS imaging. Additionally, we provide a step-by-step walkthrough of the preprocessing steps and the use of GUI-supported A-PoD. This complete package offers a user-friendly platform for super-resolution SRS microscopy, enhancing the versatility and applicability of this advanced microscopy technique to reveal nanoscopic multimolecular nature. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Super-resolution stimulated Raman scattering microscopy with graphical user interface-supported A-PoD Support Protocol: Deuterium labeling on cells with heavy water for metabolic imaging.

Evaluation of Deuterium Oxide Deposition Velocity over a Forest Environment.

ABSTRACT: Field experiments were performed to evaluate the deposition velocity of tritium oxide within a forest environment at the Savannah River Site near Aiken, SC. Field releases were designed to guide selection of deposition velocity values for use in safety-basis modeling. Six releases of deuterium oxide were conducted in 2020 and 2021 with corresponding air samples during and following each release. Samples were analyzed to determine the deuterium-to-hydrogen ratio in water and converted to concentrations of deuterium in the air during the experiment. Measurements were compared to prior model simulations to evaluate model performance and deposition velocity estimates. Field releases demonstrated vertical and horizontal mixing of a plume in a forest. Predicted deposition velocities ranged from 2.4 to 5.4 cm s -1 on average. In all cases, model simulations underpredicted deuterium concentration by 1 to 2 orders of magnitude, indicating the model does not sufficiently mix the plume into the forest. While the model underestimated the transfer of material downward through the forest, it does suggest that the model's estimates are conservative for making downwind dose estimates because of lower plume depletion, leading to higher concentration and dose estimates. While the field releases do not cover all possible meteorological conditions, we conclude it is appropriate to use a non-zero deposition velocity when performing safety-basis modeling of tritium oxide based on conservatism within the model. A recommendation of 1.0 cm s -1 as a deposition velocity is made, which is beyond the 95 th percentile value estimated from the prior modeling study.

Subcutaneous deuterated substrate administration in mice: An alternative to tail vein infusion.

PURPOSE: Tail-vein catheterization and subsequent in-magnet infusion is a common route of administration of deuterium (2 H)-labeled substrates in small-animal deuterium (D) MR studies. With mice, because of the tail vein's small diameter, this procedure is challenging. It requires considerable personnel training and practice, is prone to failure, and may preclude serial studies. Motivated by the need for an alternative, the time courses for common small-molecule deuterated substrates and downstream metabolites in brain following subcutaneous infusion were determined in mice and are presented herein. METHODS: Three 2 H-labeled substrates-[6,6-2 H2 ]glucose, [2 H3 ]acetate, and [3,4,4,4-2 H4 ]beta-hydroxybutyrate-and 2 H2 O were administered to mice in-magnet via subcutaneous catheter. Brain time courses of the substrates and downstream metabolites (and semi-heavy water) were determined via single-voxel DMRS. RESULTS: Subcutaneous catheter placement and substrate administration was readily accomplished with limited personnel training. Substrates reached pseudo-steady state in brain within ∼30-40 min of bolus infusion. Time constants characterizing the appearance in brain of deuterated substrates or semi-heavy water following 2 H2 O administration were similar (∼15 min). CONCLUSION: Administration of deuterated substrates via subcutaneous catheter for in vivo DMRS experiments with mice is robust, requires limited personnel training, and enables substantial dosing. It is suitable for metabolic studies where pseudo-steady state substrate administration/accumulation is sufficient. It is particularly advantageous for serial longitudinal studies over an extended period because it avoids inevitable damage to the tail vein following multiple catheterizations.

Heavy water coupling gel for short-wave infrared photoacoustic imaging.

Significance: Changes in lipid, water, and collagen (LWC) content in tissue are associated with numerous medical abnormalities (cancer, atherosclerosis, and Alzheimer's disease). Standard imaging modalities are limited in resolution, specificity, and/or penetration for quantifying these changes. Short-wave infrared (SWIR) photoacoustic imaging (PAI) has the potential to overcome these challenges by exploiting the unique optical absorption properties of LWC>1000 nm. Aim: This study's aim is to harness SWIR PAI for mapping LWC changes in tissue. The focus lies in devising a reflection-mode PAI technique that surmounts current limitations related to SWIR light delivery. Approach: To enhance light delivery for reflection-mode SWIR PAI, we designed a deuterium oxide (D2O, "heavy water") gelatin (HWG) interface for opto-acoustic coupling, intended to significantly improve light transmission above 1200 nm. Results: HWG permits light delivery >1 mJ up to 1850 nm, which was not possible with water-based coupling (>1 mJ light delivery up to 1350 nm). PAI using the HWG interface and the Visualsonics Vevo LAZR-X reveals a signal increase up to 24 dB at 1720 nm in lipid-rich regions. Conclusions: By overcoming barriers related to light penetration, the HWG coupling interface enables accurate quantification/monitoring of biomarkers like LWC using reflection-mode PAI. This technological stride offers potential for tracking changes in chronic diseases (in vivo) and evaluating their responses to therapeutic interventions.

Investigating the Metabolism of Plants Germinated in Heavy Water, D2O, and H218O-Enriched Media Using High-Resolution Mass Spectrometry.

Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, Lepidium sativum, germinated in D2O and H218O-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.

A Novel Demulsifier with Strong Hydrogen Bonding for Effective Breaking of Water-in-Heavy Oil Emulsions.

In the heavy petroleum industry, the development of efficient demulsifiers for the effective breaking of interfacially active asphaltenes (IAA)-stabilized water-in-heavy oil (W/HO) emulsions is a highly attractive but challenging goal. Herein, a novel nitrogen and oxygen containing demulsifier (JXGZ) with strong hydrogen bonding has been successfully synthesized through combining esterification, polymerization and amidation. Bottle tests indicated that JXGZ is effectual in quickly demulsifying the IAA-stabilized W/HO emulsions; complete dehydration (100%) to the emulsions could be achieved in 4 min at 55 °C using 400 ppm of JXGZ. In addition, the effects of demulsifier concentration, temperature and time on the demulsification performance of JXGZ are systematically analyzed. Demulsification mechanisms reveal that the excellent demulsification performance of JXGZ is attributed to the strong hydrogen bonding between JXGZ and water molecules (dual swords synergistic effect under hydrogen bond reconstruction). The interaction of the "dual swords synergistic effect" generated by two types of hydrogen bonds can quickly break the non-covalent interaction force (π-π stacking, Van der Waals force, hydrogen bonds) of IAA at the heavy oil-water interface, quickly promote the aggregation and coalescence of water molecules and finally achieve the demulsification of W/HO emulsions. These findings indicate that the JXGZ demulsifier shows engineering application prospects in the demulsification of heavy oil-water emulsions, and this work provides the key information for developing more efficient chemical demulsifiers suitable for large-scale industrial applications.

D2O as an Imperfect Replacement for H2O: Problem or Opportunity for Protein Research?

D2O is commonly used as a solvent instead of H2O in spectroscopic studies of proteins, in particular, in infrared and nuclear-magnetic-resonance spectroscopy. D2O is chemically equivalent to H2O, and the differences, particularly in hydrogen-bond strength, are often ignored. However, replacing solvent water with D2O can affect not only the kinetics but also the structure and stability of biomolecules. Recent experiments have shown that even the mesoscopic structures and the elastic properties of biomolecular assemblies, such as amyloids and protein networks, can be very different in D2O and H2O. We discuss these findings, which probably are just the tip of the iceberg, and which seem to call for obtaining a better understanding of the H2O/D2O-isotope effect on water-water and water-protein interactions. Such improved understanding may change the differences between H2O and D2O as biomolecular solvents from an elephant in the room to an opportunity for protein research.

Heavy Water Reduces the Electronic Conductance of Protein Wires via Deuteron Interactions with Aromatic Residues.

Proteins are versatile, self-assembling nanoelectronic components, but their hopping conductivity is expected to be influenced by solvent fluctuations. The role of the solvent was investigated by measuring the single molecule conductance of several proteins in both H2O and D2O. The conductance of a homologous series of protein wires decreases more rapidly with length in D2O, indicating a 6-fold decrease in carrier diffusion constant relative to the same protein in H2O. The effect was found to depend on the specific aromatic amino acid composition. A tryptophan zipper protein showed a decrease in conductance similar to that of the protein wires, whereas a phenylalanine zipper protein was insensitive to solvent changes. Tryptophan contains an indole amine, whereas the phenylalanine aromatic ring has no exchangeable protons, so the effect of heavy water on conductance is a consequence of specific D- or H-interactions with the aromatic residues.

Heavy water (D2O) induces autophagy-dependent apoptotic cell death in non-small cell lung cancer A549 cells by generating reactive oxygen species (ROS) upon microtubule disruption.

OBJECTIVE: Deuterium oxide (D2O) or heavy water is known to have diverse biological activities and have a few therapeutic applications due to its limited toxicity to human subjects. In the present study, we investigated the mechanism of D2O-induced cytotoxicity in non-small cell lung cancer A549 cells. RESULTS: We found that D2O-treatment resulted in cytotoxicity, cell cycle arrest, and apoptosis in A549 cells in a dose-dependent fashion. In contrast, limited cytotoxicity was observed in lung fibroblasts WI38 cells. Moreover, D2O-treatment resulted in the disruption of the cellular microtubule network, accompanied by the generation of ROS. On further investigation, we observed that the intracellular ROS triggered autophagic responses in D2O-treated cells, leading to apoptosis by inhibiting the oncogenic PI3K/ Akt/ mTOR signaling. D2O-treatment was also found to enhance the efficacy of paclitaxel in A549 cells. SIGNIFICANCE: D2O induces autophagy-dependent apoptosis in A549 cells via ROS generation upon microtubule depolymerization and inhibition of PI3K/ Akt/ mTOR signaling. It augments the efficacy of other microtubule-targeting anticancer drug taxol, which indicates the potential therapeutic importance of D2O as an anticancer agent either alone or in combination with other chemotherapeutic drugs.

Simple Self-Powered Sensor for the Detection of D2O and Other Isotopologues of Liquid Water.

Distinguishing between heavy water and regular water has been a continuing challenge since these isotopologues of water have very similar physical and chemical properties. We report the development and evaluation of a simple, inexpensive sensor capable of detecting liquid D2O and other isotopologues of liquid water through the measurement of electrical signals generated from a nanoporous alumina film. This electrical output, consisting of a sharp voltage pulse followed by a separate broad voltage pulse, is present during the application of microliter volumes of liquid. The amplitude and temporal characteristics of these pulses have been combined to enable four diagnostic parameters for sensing D2O and H218O. The sensing mechanism is based on different modification effects on the alumina surface by H2O and D2O, spatially localized variations in the surface potential of alumina induced by isotopically substituted water molecules, combined with the effect of isotopic composition on charge transfer. As a proof-of-concept demonstration, a sensing system has been developed that provides real-time detection of liquid D2O in a stand-alone system.

A large-scale LC-MS dataset of murine liver proteome from time course of heavy water metabolic labeling.

Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies. Several algorithms and tools have been developed to determine the turnover rates of peptides and proteins from time-course stable isotope labeling experiments. The availability of benchmark mass spectrometry data is crucial to compare and validate the effectiveness of newly developed techniques and algorithms. In this work, we report a heavy water-labeled LC-MS dataset from the murine liver for protein turnover rate analysis. The dataset contains eighteen mass spectral data with their corresponding database search results from nine different labeling durations and quantification outputs from d2ome+ software. The dataset also contains eight mass spectral data from two-dimensional fractionation experiments on unlabeled samples.

The T1R3 subunit of the sweet taste receptor is activated by D2O in transmembrane domain-dependent manner.

Deuterium oxide (D2O) is water in which the heavier and rare isotope deuterium replaces both hydrogens. We have previously shown that D2O has a distinctly sweet taste, mediated by the T1R2/T1R3 sweet taste receptor. Here, we explore the effect of heavy water on T1R2 and T1R3 subunits. We show that D2O activates T1R3-transfected HEK293T cells similarly to T1R2/T1R3-transfected cells. The response to glucose dissolved in D2O is higher than in water. Mutations of phenylalanine at position 7305.40 in the transmembrane domain of T1R3 to alanine, leucine, or tyrosine impair or diminish activation by D2O, suggesting a critical role for T1R3 TMD domain in relaying the heavy water signal.

Hydrogen-rich water treatment targets RT1-Db1 and RT1-Bb to alleviate premature ovarian failure in rats.

Background: Premature ovarian failure (POF) is defined as the cessation of ovarian function before the age of 40 years, imposing a significant health burden on patients. However, effective etiological therapy for POF is scarce. Thus, we aimed to explore the protective role and targets of hydrogen-rich water (HRW) in POF. Methods: Based on cyclophosphamide (CTX)-induced POF rat models, the protective role of HRW treatment was mainly determined through serum 17-ß-estradiol (E2), follicle-stimulating hormone (FSH), anti-mullerian hormone (AMH) levels, ovarian histomorphological analysis, and TUNEL assay. Tandem mass tag (TMT)-based quantitative proteomic analysis was then conducted on ovarian tissues, and the targets of HRW in POF were identified integrating differential expression analysis, functional enrichment analysis, and interaction analysis. Results: In HRW treatment of POF rats, the serum AMH and E2 levels significantly increased, and FSH level significantly reduced, indicating the protective role of HRW. After TMT quantitative proteomic analysis, a total of 16 candidate differentially expressed proteins (DEPs) were identified after the cross analysis of DEPs from POF vs. control and POF+HRW vs. POF groups, which were found to be significantly enriched in 296 GO terms and 36 KEGG pathways. The crucial targets, RT1-Db1 and RT1-Bb, were finally identified based on both protein-protein interaction network and GeneMANIA network. Conclusions: The HRW treatment could significantly alleviate the ovarian injury of POF rats; RT1-Db1 and RT1-Bb are identified as two crucial targets of HRW treatment in POF rats.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Utilization of a kinetic isotope effect to decrease decomposition of ceftriaxone in a mixture of D2O/H2O.

The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant organisms, were major breakthroughs in the development of ß-lactam antibiotics. Although cephalosporins are more stable with respect to hydrolytic degradation than penicillins, they still experience a variety of chemical transformations. The present study offers an insight into the rates and mechanisms of ceftriaxone degradation at the therapeutic concentration in water, a mixture of water and deuterium oxide, and deuterium oxide itself at the neutral pH. Specific ceftriaxone degradation products were observed in aged samples (including a previously unreported dimer-type species), and by comparing the degradation rates in H2O and D2O, the observation of a kinetic isotope effect provided some valuable insight as to the nature of the initial ceftriaxone degradation. The effect of protium to deuterium isotope change on the degradation kinetics of ceftriaxone was evaluated using the method of initial rates based on HPLC analysis as well as by quantitative 1H NMR spectroscopy. Moreover, computational analysis was utilized to get a molecular insight into chemical processes governing the ceftriaxone degradation and to rationalize the stabilizing effect of replacing H2O with D2O.

Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide.

Fatty acid synthesis is a complex and highly energy demanding metabolic pathway with important functional roles in the control of whole-body metabolic homeostasis and other physiological and pathological processes. Contrary to other key metabolic pathways, such as glucose disposal, fatty acid synthesis is not routinely functionally assessed, leading to incomplete interpretations of metabolic status. In addition, there is a lack of publicly available detailed protocols suitable for newcomers in the field. Here, we describe an inexpensive quantitative method utilizing deuterium oxide and gas chromatography mass spectrometry (GCMS) for the analysis of total fatty acid de novo synthesis in brown adipose tissue in vivo. This method measures the synthesis of the products of fatty acid synthase independently of a carbon source, and it is potentially useful for virtually any tissue, in any mouse model, and under any external perturbation. Details on the sample preparation for GCMS and downstream calculations are provided. We focus on the analysis of brown fat due to its high levels of de novo fatty acid synthesis and critical roles in maintaining metabolic homeostasis.

Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution.

Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic B. cereus spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37 °C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

Application of elemental analysis-coupled isotope ratio mass spectrometry for protein turnover analysis using deuterium labeling: Purification and analysis of hydrogen isotope ratio of non-derivatized protein-bound alanine.

RATIONALE: Heavy water can be used as a tracer for the evaluation of protein turnover. By adding heavy water (D2 O) to the precursor pool, nonessential amino acids, including alanine, can be isotopically labeled in vivo. Protein turnover can then be quantified by measuring the hydrogen isotope ratio of protein-bound alanine. METHODS: In this study, we constructed a novel method to apply deuterium labeling of alanine to the evaluation of protein turnover using elemental analysis-coupled isotope ratio mass spectrometry (EA-IRMS). We established a preparative high-performance liquid chromatography method to isolate alanine from protein hydrolysates. EA-IRMS was then used to determine the hydrogen isotope ratio of alanine isolated from hydrolysates of protein from mouse myoblast C2C12 cells that had been treated with D2 O over the course of 72 h. RESULTS: In cells treated with 4% D2 O, the deuterium enrichment of alanine increased to approximately 0.9% over time, while that of cells treated with 0.017% D2 O increased to approximately 0.006%. The rate of protein synthesis calculated by fitting the increase of deuterium excess to rise-to-plateau kinetics was similar regardless of the concentration of D2 O. When C2C12 cells treated with insulin and rapamycin were analyzed 24 h after the addition of 0.017% D2 O, protein turnover was found to be accelerated by insulin, but this effect was offset by co-treatment with rapamycin. CONCLUSION: The derivative-free measurement of the hydrogen isotope ratio of protein-bound alanine using EA-IRMS can be applied to the evaluation of protein turnover. The proposed method is an accessible option for many laboratories to perform highly sensitive IRMS-based evaluations of protein metabolic turnover.

HIV-Infected and HIV-Uninfected Western Kenyan Women Produce Equivalent Amounts of Breast Milk at 6 Wk and 6 Mo Postpartum: A Prospective Cohort Study Using Deuterium Oxide Dose-to-Mother Technique.

BACKGROUND: Regardless of their HIV serostatus, mothers are advised to exclusively breastfeed infants ≤6 mo postpartum. How this guidance impacts breast milk intake among HIV-exposed infants in varied contexts needs to be better understood. OBJECTIVES: The objective of this study was to compare breast milk intake of HIV-exposed and HIV-unexposed infants at 6 wk and 6 mo of age, as well as the associated factors. METHODS: In a prospective cohort design, which we followed from a western Kenya postnatal clinic, 68 full-term HIV-uninfected infants born to HIV-1-infected mothers (HIV-exposed) and 65 full-term HIV-uninfected infants born to HIV-uninfected mothers were assessed at 6 wk and 6 mo of age. Breast milk intake of infants (51.9% female) weighing 3.0-6.7 kg (at 6 wk of age) was determined using the deuterium oxide dose-to-mother technique. Student t test for independent samples compared the variations in breast milk intake between the 2 groups. Correlation analysis detected the associations between breast milk intake and maternal and infant factors. RESULTS: Daily breast milk intakes by HIV-exposed and HIV-unexposed infants were not significantly different at either 6 wk (721 ± 111 g/d and 719 ± 121 g/d, respectively) or 6 mo (960 ± 121 g/d and 963 ± 107 g/d, respectively) of age. Maternal factors that significantly correlated with infant breast milk intake were FFM at both 6 wk (r = 0.23; P < 0.05) and 6 mo (r = 0.36; P < 0.01) of age and weight at 6 mo postpartum (r = 0.28; P < 0.01). Infant factors that significantly correlated at 6 wk were birth weight (r = 0.27; P < 0.01), present weight (r = 0.47; P < 0.01), length-for-age z-score (r = 0.33; P < 0.01), and weight-for-age (r = 0.42; P > 0.01). At 6 mo, they were infant length-for-age (r = 0.38; P < 0.01), weight-for-length (r = 0.41; P > 0.01), and weight-for-age (r = 0.60; P > 0.01). CONCLUSIONS: Full-term breastfeeding infants born to HIV-1-infected and HIV-1-uninfected women attending standard Kenyan postnatal care clinics ≤6 mo of age in this resource-poor setting consume comparable amounts of breast milk. This trial was registered at clinicaltrials.gov as PACTR201807163544658.