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Department of Pediatrics, Children’s Nutrition Research Center, U.S. Department of Agriculture/Agricultural Research Service, Baylor College of Medicine, Houston, Texas 77030
Address all correspondence and requests for reprints to: Morey W. Haymond, M.D., CNRC, 1100 Bates Street, Houston, Texas 77030. E-mail: mhaymond{at}bcm.tmc.edu
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Abstract |
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Using the ratios of [13C6] enrichments of glucose in lactose and plasma glucose and that of galactose in lactose and plasma glucose, we determined that 98 ± 3% of glucose and 68 ± 7% of galactose in lactose were derived from plasma glucose in the fed state, and 72 ± 4 and 51 ± 3%, respectively, after a 24-h fast. Virtually identical results (97 ± 6 and 64 ± 4%, respectively) were obtained during the glucose feeding study. On the basis of the [13C1] enrichment of glucose and galactose in lactose (derived from [2-13C]glycerol), glycerol contributes to the production of galactose but not glucose within the breast.
Thus, plasma glucose is an important source of lactose, but significant amounts of glucose and galactose in lactose are generated within the breast, a process denoted hexoneogenesis. In this process, glycerol is a precursor for milk galactose but not glucose.
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Introduction |
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On the basis of studies in ruminants, the primary source of milk lactose is thought to be plasma glucose. Other than a study on the impact of glucose and insulin on milk production (5), to our knowledge, the precursor substrates that are used in lactose synthesis in humans have not been explored. Studies of plasma glucose uptake across the mammary gland in ruminants demonstrate that sufficient amounts of glucose are taken up to account for nearly all of the carbon required for lactose synthesis (1). Using [U-14C]glucose, direct evidence was provided that plasma glucose is the primary precursor for lactose synthesis in cows (1, 6, 7) but provided only 80% of the lactose carbon in perfused goat mammary gland (1, 6, 7). In these latter studies, all of the glucose in lactose was derived from plasma glucose, but only 60–80% of the galactose. Thus, the galactose appearing in milk in ruminants is most likely the result of conversion of plasma-derived glucose to galactose via UDP glucose phosphorylase and UDP galactose 4 epimerase (8). However, some portion of galactose is reported to be derived from acetate and glycerol carbon (1, 9). These data from ruminants raise the provocative concept that a significant portion of the galactose used in lactose synthesis is derived from a gluconeogenic-like process within the breast.
Furthermore, indirect lines of evidence from studies in mice and ruminants support the hypothesis that the lactating breast has the enzyme capacity for converting three carbon substrates to glucose-6-phosphate (10, 11, 12, 13). In contrast to humans, ruminants produce and subsequently use short-chain fatty acids for glucose production. Therefore, it is difficult to extrapolate from ruminant studies to human physiology.
As a result, the present studies were performed to determine the source(s) of carbon for the glucose and galactose in milk lactose under both the fed and short-term fasting conditions. To determine the source(s) of milk lactose in humans requires the use of stable isotope tracers. To our knowledge, no published method exists for the independent measurement of isotopomer distribution in milk glucose and galactose. Therefore, a method of analysis was developed. We hypothesize that: 1) during nutrient absorption dietary carbohydrate, via plasma glucose, is the primary source of lactose carbon; but 2) in the fasted state, the human breast is capable of de novo synthesis of both glucose and galactose.
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Materials and Methods |
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Lactase [Aspergillus oryzae, 5,500 o-nitrophenyl ß-D-galactopyranoside U/g] was obtained from ICN Biochemicals, Inc. (Irvine, CA), and ß-galactosidase (A. oryzae, 9,600 U/g, and Escherichia coli, grade VIII, 740,000 U/g) was obtained from Sigma-Aldrich Corp. (St. Louis, MO). BaOH (0.3 N), ZnSO 4 (0.3 N), D-(+)glucose (dextrose, corn sugar), D-(+)galactose, and D-lactose (monohydrate) were purchased from Sigma-Aldrich Corp.. All organic solvents were HPLC grade. [1-13C]galactose (99 atom %) and deuterium oxide (99 atom %) were purchased from Cambridge Isotope Laboratory (Andover, MA).
Glucose concentrations were determined using a Biochemical Analyzer, model 2700 Select (Yellow Springs Instruments, Yellow Springs, OH). Galactose concentrations were determined using a Glucose and L-Lactate Analyzer, model 2300 Stat+, retrofitted to read galactose (Yellow Springs Instruments).
Study and protocol design
Subjects. The study was approved by the Institutional Review Board for Human Subject Research at Baylor College of Medicine (Houston, TX). Twelve normal lactating women were recruited for this study after written consent was obtained from each of them. They are the same lactating subjects reported Tigas et al. (14). The subjects were studied in two protocols (see below). Before being accepted in the study, they underwent a general physical examination and had normal hemoglobin and screening tests for liver and renal function and a negative pregnancy test. The ages of the subjects averaged 27 ± 3 yr (protocol 1) and 30 ± 2 yr (protocol 2); the weights were 69 ± 5 and 67 ± 3 kg, and body mass index was 26 ± 3 and 25 ± 3 kg/m2, respectively. The women were between 6 wk and 3 months postpartum. All infants were healthy, born at term, and exclusively breast-fed at the time of the study. Each lactating woman was provided iron supplementation over the course of these studies, if not already taking iron as part of her postpartum management prescribed by her obstetricians.
Protocol 1: Sustacal feeding and short-term fasting. In protocol 1, each woman was studied on two occasions, in random order, once in the fed state and once during a 24-h fast. The studies were separated by 2–4 wk. Each woman and her infant were admitted to the Metabolic Research Unit of the Children’s Nutrition Research Center (CNRC) or the General Clinical Research Center on the evening before study. At 1800 h on the evening of admission, two iv catheters were introduced in the antecubital fossae or forearm veins under EMLA (Astra Pharmaceuticals, Wayne, PA) cream analgesia, one for isotope infusion and the other (in the contralateral arm) for blood sampling. On both study occasions, subjects were fed a supper meal of 10 kcal/kg at 1800 h and were subsequently fasted (except for water) overnight. At 0600 h, after the overnight fast, baseline milk and blood samples (5 ml) were obtained, and the subjects received the following: 1) an oral dose of deuterium oxide (0.1 g/kg) to measure the rapidity of equilibration of maternal body water with milk water to estimate the existence, if any, of dead volume of milk in the breast; 2) a primed constant rate infusion of [U-13C]glucose to measure the fraction of galactose and glucose in lactose derived from the plasma glucose space; and 3) a primed constant rate infusion of [2-13C]glycerol to measure the fraction of glucose and galactose in milk lactose derived from plasma glycerol (14). In the fed protocol, the women drank 90 kcal (90 ml) of Sustacal (Mead Johnson Nutritionals, Evansville, IN) every 30 min between 0600 h and 1800 h, providing a total of approximately 2160 kcal and approximately 33 µmol·kg-1·min-1 of carbohydrate. During the fasting study, the women drank an equal volume of water but received no calories from 0600 h until 1800 h on the day of study.
In addition to the baseline samples, blood and milk samples (6–10 ml) were collected at 3-h intervals. The infants were breast-fed approximately every 3 h (from both breasts at each nursing), and milk samples were collected before and immediately after each feeding.
In protocol 1, we attempted to maintain energy neutrality and used a commercially available preparation containing unlabeled complex carbohydrate. As a result, we were unable to estimate the contribution of dietary glucose to milk production. Thus, protocol 2 was designed to address this issue.
Protocol 2: Oral glucose load. Each woman was studied on one occasion using a protocol identical to that outlined above except: 1) she received oral glucose enriched with [1-13C]glucose every 15 min at a rate of 33 µmol·kg-1·min-1 instead of Sustacal but no 2H2O, [U-13C]glucose, or [2-13C]glycerol was administered; and 2) the duration of study was 9 h.
Plasma analysis
Enrichments of [1-13C]glucose, [U-13C]glucose, and the glucose isotopomers, were measured by gas chromatography mass spectrometry (GCMS) using the penta-acetate derivative and of [2-13C]glycerol using the triacetate derivative as previously described (15). Deuterium enrichments in both plasma and milk (defatted) water were measured using isotope ratio mass spectrometry (16). All measurements were made in the Stable Isotope Core Laboratory of the CNRC.
Milk analyses
Defatting milk. A homogeneous sample of milk (1 ml) is pipetted into microcentrifuge tubes (1.5-ml conical, screw-capped tubes, CEL Associates Inc., Houston, TX). The tube is capped and placed upside down in a 20-ml scintillation vial and centrifuged for 10 min at 3,000 rpm (900x g) at 4 C in a refrigerated centrifuge (Beckman Coulter model J-6B, Beckman Coulter Instruments, Inc., Palo Alto, CA). Without disturbing the layer of milk fat, the supernatant is carefully decanted into a new microcentrifuge tube.
Deproteinizing milk. To a 1.5-ml microcentrifuge tube, 50 µl defatted milk, 450 µl deionized water, 500 µl BaOH, and 500 µl ZnSO4 are added. The tubes are capped tightly, vortexed, and centrifuged for 10 min at 3,000 rpm (900x g) at 4 C. The defatted, deproteinized supernatant is used for lactose hydrolysis.
Lactose hydrolysis. Defatted, deproteinized milk (150 µl) and 50 µl (0.55 U) ß-galactosidase in a final buffer concentration of 93 mM sodium phosphate (pH 7.3), 1 mM MgCl2, and 112 mM 2-mercaptoethanol are added to 1.5-ml microcentrifuge tubes. The reaction mix is incubated at 37 C for 1 h.
Derivatization of hydrolyzed products. Fifty microliters of each hydrolyzed sample are aliquotted into 4-ml vials and taken to dryness under nitrogen at room temperature. Fifty microliters of acetic anhydride/pyridine (2:1) are added to each sample, and the samples are heated to 60 C for 10 min or allowed to sit overnight at room temperature. Then, the samples are dried under nitrogen at room temperature, reconstituted in 100 µl ethyl acetate, and transferred to autosampler vials.
GCMS analysis.
The derivatized samples are analyzed using a Hewlett-Packard Co. GCMS (GC 6890, MS 5973) with a SP-17 column (30 m x 0.25 mm x 0.25 µm; Supelco, Inc., Bellefonte, PA). The conditions for the GC are the following: injector, 250 degree (splitless injection of samples); oven, 70 C for 1.0 min; ramp, 30 C/min to 280 C; hold at 280 C for 5 min. The positive ionization mode is employed with methane as the reagent gas and selected monitoring of m/z 331–337 for glucose and galactose. The SP-17 column separates the 
- and ß-anomers of glucose and galactose. We have used the -anomers of both sugars for the analyses. These peaks are very well separated, with a difference in retention time of approximately 0.25 min.
Calculations
Standard product precursor relationships were calculated. The percentage of product, i.e. glucose and galactose in milk lactose, that was derived from the precursor pool (plasma glucose), i.e. percentage milk sugar 
plasma glucose, was calculated using the following equation:
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[13C6] enrichments are derived from the [U-13C]glucose tracer (protocol 1) and [13C1] enrichments from the [1-13C]glucose tracer (protocol 2).
The contribution from glycerol to milk glucose and galactose produced from other sources than plasma glucose via processes within the breast (% milk sugar glycerol) was calculated using the following equation:
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Statistical analysis
All data are expressed as mean ± SE. The product/precursor relationships were compared using a paired t test.
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Results |
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Reproducibility of the lactose analysis
A variety of methodologies for milk hydrolysis were attempted, including commercial over-the-counter lactase preparations. Initial results were always promising, but over time the enrichments in glucose would decrease, indicating dilution by unlabeled glucose. Eventually, we identified that these products were stabilized with starch (and reacted to iodine). The procedure described above (using ß-galactosidase from E. coli) was found to be stable and reproducible over time. The coefficient of variation for preparation and GCMS analyses of 10 aliquots of a sample was 5% for glucose and 6% for galactose. The SP-17 GC column separates the - and ß-anomers of glucose and galactose, respectively, in the following order: -galactose, -glucose, ß-glucose, and ß- galactose (Fig. 1
). Figure 1 depicts the GCMS total ion chromatogram of a sample of glucose and galactose standards and a sample of hydrolyzed milk, demonstrating a complete separation of the glucose and galactose moieties.
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Figure 2 depicts the results of the time course of deuterium enrichment in plasma and breast milk water during the fed and fasted conditions. Within 3 h of the oral administration of the 2H2O, the plasma and milk water enrichments of 2H were essentially identical and remained so throughout the study period, thus eliminating the possibility of any significant pools of preformed milk.
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Analysis of milk samples obtained before and after each feeding gave essentially identical values (data not shown). The values obtained after the feedings were used because, theoretically, they would contain the most recently synthesized lactose. Table 1 provides the enrichments of [13C1] (from [2-13C]glycerol) and [13C6] (from [U-13C]glucose) in plasma glucose, milk glucose, and milk galactose between 9–12 h of Sustacal feeding or 21–24 h of fasting. The ratios between [13C6] milk glucose (galactose) and [13C6] plasma glucose as well as the [13C1] ratios were calculated for each individual subject. On the basis of the means of these ratios, we demonstrated that, during feeding, the enrichment of [13C6] in glucose derived from the hydrolysis of lactose was 98 ± 3% of the [13C6]glucose enrichment in plasma (Fig. 3). The enrichment of [13C6]galactose was 68 ± 7% that of the plasma [13C6]glucose. During short-term fasting (24 h), the fractions of glucose and galactose derived from plasma glucose were 72 ± 4 and 51 ± 3%, respectively (Fig. 3). Thus, collectively, plasma glucose contributes 83% of the substrate for lactose synthesis in the fed condition but only 62% during short-term fasting.
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). The means of the individual ratios differ slightly from those that can be calculated using the mean enrichments provided in Tables 1
and 2.
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During ingestion of labeled glucose alone, the ratios between the [13C1] enrichment (from the [1-13C]glucose derived from the meal) in milk glucose/plasma glucose and in galactose/plasma glucose were virtually identical (97 ± 6 and 64 ± 4%, respectively) to those observed with [U-13C]glucose described above. We have previously demonstrated that in these women 94% of glucose appearing in plasma was derived from the meal glucose (based on the [1-13C] enrichments in plasma glucose and that of the glucose drinks) (14). The above milk data demonstrate that 83% of lactose is derived from plasma glucose. Thus, during the continuously fed condition, we would estimate that dietary carbohydrate contributes 78% (0.94 x 0.83 x 100) of the substrate necessary to produce lactose.
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Discussion |
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Several explanations might be considered for the dilution of the plasma glucose label measured in the milk glucose and galactose. The first is the possibility of hidden pools of preformed milk within the breast. This seems unlikely because of the turnover of milk with each feeding. In addition, using a small oral dose of deuterated water, we demonstrated that plasma and milk water were in complete equilibrium after 3 h. The dilution in galactose, in both the fed and short-term fasted states, suggests that under both conditions a substantial amount of the carbon is derived from nonglucose substrates, presumably entering the breast from the plasma. Although we cannot delineate the contributions of amino acids, lactate, and pyruvate to this processes, we have demonstrated that glycerol contributes substantially to the galactose formed by hexoneogenesis and that this contribution is most likely from direct uptake of the glycerol into the breast rather than the uptake of maternal glucose derived from glycerol via gluconeogenesis in the mother’s liver and/or kidney. Comparing the ratios of the enrichments of [13C1] (derived from [2-13C]glycerol) and [13C6] (derived from [U-13C]glucose) between milk galactose and plasma glucose (in protocol 1), we demonstrated that under fed and fasted conditions, glycerol accounted for about 24 and 14%, respectively, of the galactose formed within the mammary gland from other sources than plasma glucose. This would suggest that about 76 and 86%, respectively, of the galactose generated via hexoneogenesis must be derived from other substrates than glycerol.
In contrast to galactose, it is of great interest that the glycerol contributed little or nothing to the glucose found in lactose. This again is consistent with studies in bovine mammary gland incubation studies and perfused goat mammary gland (9, 12). However, it is important to note that during fasting the glucose in lactose was also derived from substrates other than maternal plasma glucose. We presumed that the substrates available for the formation of glucose-1-phosphate for the subsequent conversion to galactose would have been the same pool of glucose-1-phosphate available as a precursor of glucose. The glycerol labeling of galactose, but not glucose in milk lactose, demonstrates that these hexoses are being derived from at least two separate pools, presumably within the breast. This is both reassuring and disturbing at the same time; it is reassuring in that little or no glucose-6-phosphatase is present in at least bovine (8) and rat ( 17) mammary tissue and disturbing in the sense that we have no clear idea as to the precursor pool for glucose produced within the breast for lactose synthesis. One consideration is that of intramammary gland glycogen. However, to our knowledge no such pool exists in sufficient quantity to dilute the glucose in lactose over the time of these studies. Elucidation of the source(s) of this unlabeled glucose in lactose formed within the mammary gland will require further study.
In the fed state (under the conditions of these studies), we conclude that oral glucose is an important source of substrate for both the glucose and galactose moieties in human milk. If one can extrapolate from the ingestion of oral glucose (labeled with [1-13C]glucose) to that of a normal mixed meal, our data would suggest that up to 75% of the milk lactose is derived directly from the meal carbohydrate. In conclusion, the results from these studies demonstrate that although adequate carbohydrate supply is important for lactogenesis, processes independent of glucose availability are required to maintain lactose production in both the fed and short-term fasted conditions.
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Acknowledgments |
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Footnotes |
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This work is a publication of the USDA/Agricultural Research Service, CNRC, Department of Pediatrics, Baylor College of Medicine, Houston, TX. The contents of this publication do not necessarily reflect the views of policies of the USDA, nor does mention of trade names, commercial products, or organizations imply endorsement from the U.S. Government.
Abbreviations: CNRC, Children’s Nutrition Research Center; GCMS, gas chromatography mass spectrometry; USDA, U.S. Department of Agriculture.
Received July 13, 2001.
Accepted October 15, 2001.
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