Kenneth E. Hundrieser

Changes in the lipids of human milk from 2 to 16 weeks postpartum.

Changes in total lipid, fatty acids, total cholesterol, free cholesterol, and lipid phosphorus in mature milk with time were investigated. Milk samples were collected from 10 mothers at 2, 6, 12, and 16 weeks postpartum. During 1 day, each mother donated two complete breast expressions. Expressions were taken using an electric breast pump 1 h after the previous a.m. and p.m. nursing. For each mother, the a.m. and p.m. samples were pooled for analysis. It was observed that the amount of total lipid increased significantly (p less than 0.05) from 3.9 g/100 ml at 2 weeks to 5.2 g/100 at 16 weeks postpartum. The total fatty acid composition remained uniform during the investigation. Average total cholesterol and free cholesterol in the milk were 10.3 mg/100 ml and 8.3 mg/100 ml, respectively. These concentrations did not change significantly with time postpartum. Average lipid phosphorus was 3.9 mg/100 ml and also remained constant throughout. We conclude that the fatty acid pattern, lipid phosphorus, total cholesterol, and free cholesterol of mature milk to 16 weeks postpartum remains relatively constant while total lipid concentration increases.

A comparison of methods for determination of total lipids in human milk

Abstract A modified Folch extraction, a Celite-Na 2 SO 4 column elution, a spectrophotometric method and the creamatocrit method for total lipid analysis of human milk were compared against the Roese-Gottlieb method. The constant, proportional and random errors associated with each method were determined. Pooled milk was centrifuged to separate milk lipid and skim milk. Varying concentrations of milk lipid were reintroudced into the skim milk to provide 25 samples for analysis with a range of lipids from approximately 0.5 to 8.0 percent. There was a high correlation (r>0.977) between the Roese-Gottlieb method and all methods evaluated. A least squares equation was developed between the Roese-Gottlieb method and each method tested. The greatest constant error, as estimated by the least squares y-intercept, was observed with the Celite Na 2 SO 4 column procedure and spectrophotometric method. The greatest proportional error as estimated by the slope was observed with the Celite-Na 2 SO 4 column procedure. Random error based on the least squares standard error of estimate in the y direction was greatest for the spectrophotometric method. While there are differences between methods, with careful standardization all the methods tested could provide satisfactory results for analysis of total lipids in human milk.

Distribution of trans-octadecenoic acid in the major glycerolipids of human milk

The intramolecular distributions of fatty acids in triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine of human milk were determined. Milk donated from 10 mothers during their 2nd week of lactation was analyzed. Based on pancreatic lipase digestion of milk triacylglycerols, the 14:0 and 16:0 fatty acids were observed predominantly in the sn-2 position, while 18:0, 18:1, and 18:2 were observed predominantly in the sn-1,3 positions. The average trans-18:1 content of the triacylglycerols was 3.4% and was greater in the sn-1,3 positions. Phosphatidylcholine and phosphatidylethanolamine were digested by phospholipase A2. As in other tissues, saturated fatty acids were esterified at the sn-1 position and polyunsaturated fatty acids at the sn-2 position of the phospholipids. The proportion of trans-18:1 averaged 2.5% in phosphatidylcholine and 3.7% in phosphatidylethanolamine. The proportion of trans-18:1 was slightly greater in the sn-2 position of the phospholipids. In conclusion, there appeared to be a reverse positional distribution of saturated and polyunsaturated fatty acids in triacylglycerol compared to phosphatidylcholine and phosphatidylethanolamine. When compared to other mammalian tissues, there were only small differences observed in trans-18:1 content between positions within a glycerolipid class and between glycerolipids of human milk.

The identity of the cholesteryl esters in human milk

Milk samples were collected from 11 mothers who were at least 4 weeks postpartum. The amounts of fat and the fatty acid compositions of cholesteryl esters (CE) and triacylglycerols (TG) in the milk were determined. The mean concentration of total milk lipid was 3.01 gm/100 ml of milk±.42 SD. The major fatty acids esterified with CE and TG were 16∶0,cis 18∶1 and 18∶2. The patterns were similar except for a greater proportion ofcis 18∶1 in the CE. The majortrans fatty acid detected was the 18∶1 isomer which accounted for 4.48% of the TG fatty acids and 2.96% of the CE fatty acids.

A lack of correlation among fatty acids associated with different lipid classes in human milk

The fatty acids associated with triacylglycerol, cholesteryl ester, phosphatidylethanolamine, phosphatidylcholine and sphingomyelin in human milk were compared. Ten milk samples were selected for lipid class analysis based on their total lipid polyunsaturated/saturated fatty acid ratio (P/S ratio). The P/S ratio of the selected milk samples ranged from 0.3 to 0.8. Linoleic acid was the predominant fatty acid in milk to affect the P/S ratio. The percentage of linoleic acid in milk triacylglycerol was correlated (r=0.84,P<0.05) with the total milk lipid P/S ratio. Linoleic acid esterified to cholesterol was not correlated with total milk lipid P/S ratio but was correlated (r=−0.66,P<0.05) with the quantity of lipid in the milk. Linoleic acid in the phospholipid classes did not correlate with shift in P/S ratio of the total milk lipid or linoleic acid content of other lipid classes.

Changes in cholesteryl esters of human milk with total milk lipid.

Twenty-five milk samples with a wide range of total lipid and degree of fatty acid saturation were chosen for this study. The milk samples were analyzed for total lipid, total cholesterol, and free cholesterol. The cholesteryl esters and triacylglycerols in these samples also were isolated and the fatty acids associated with each lipid class determined. Total cholesterol averaged 13.5 ± 3.1 mg/dl of milk and was significantly correlated (r = 0.60, p < 0.05) with total lipid. Free cholesterol averaged 10.9 ‡ 2.3 mg/dl of milk and also was significantly correlated (r = 0.72, p < 0.05) with total lipid. As total milk lipid increased, the fatty acids in cholesteryl esters became more saturated. The fatty acids most affected were 18:2 and 20:4. Total milk lipid and 18:2 in cholesteryl esters were inversely related (r = −0.49, p < 0.05). There was also a negative correlation (r = −0.51, p < 0.05) between 20:4 in cholesteryl esters and total lipid. The fatty acids in triacylglycerol were not correlated with total lipid. From these results it appears that the fatty acids esterified to cholesterol and triacylglycerol in milk may be drawn from different substrate pools.

Bile salt-stimulated lipase in huma milk from 2 to 16 weeks postpartum

Our objectives were to determine the change in bile salt-stimulated lipase (BSSL) activity in mature milk with time postpartum and to determine if BSSL activity was related to total lipid in the milk. Milk samples were collected from 12 mothers at 2,6,12 and 16 weeks postpartum. A significant (P<.05) decrease in BSSL activity with time was observed. The average values expressed as μmol free fatty acids released/min/ml milk were 5.3, 4.5, 4.1 and 3.6 at 2,6, 12 and 16 weeks postpartum. Total lipid in the milk increased significantly (P <.05) from 4.1 g/dl at 2 weeks to 5.4 g/dl at 16 weeks postpartum but was not significantly correlated with BSSL activity.

The effect of temperature and length of storage on bile salt-stimulated lipase and esterase in human milk

Abstract Five milk samples were analyzed for bile salt-stimulated lipase (BSSL) and bile-salt stimulated esterase (BSSE) activities immediately after collection. The samples then were divided into aliquotes and stored at 25, 4, −20 and −70 C. After 6, 12 and 24 hours of storage BSSL and BSSE activities were determined. Activities were also determined in milk after 2 and 4 weeks of storage at −20 and −70 C. There was no significant change in BSSL activity due to storage. The average activity of BSSL was 1.54 μmole free fatty acid /released/min/ml milk ±0.46 (SD). Activity from BSSE was significantly (P