Role

K. M.

and significance
2

of enzymes
A. Friend

in human

Shahani,

A. J. Kwan,

and

Beverly

ABSTRACT
milk, levels milk detail. probably cells; synthetase stimulated in growth procedures made.
3)

Although
definitive milk summarized. spilled lactate are and and Am. I lipase, into and synthesized diastase, nutrition. activity Clin.

human

milk

generally milk studied views

contains concerning and that human

1)

higher their levels milk lipoprotein is spilled role

levels or

of enzymes significance. colostrum are and the discussed

than The versus

bovine enzyme normal in more are lactose epithelial

little are

information as compared The is presented the milk malate few

is available to bovine most from widely the blood; the

in human Evidence

in human enzymes

to support dehydrogenases, in the protease, Consideration mammary and that

lipase from

ribonuclease and and

4)

2) lysozyme glucose-6-phosphate gland in response are present

secretory

dehydrogenase, to hormonal in sufficient the between stimuli; quantities various various

Downloaded from ajcn.nutrition.org by guest on January 17, 2013

bile

salt assay

lysozyme must meaningful 1980.

to aid enzyme studies

infants could be

be given

to standardizing

units Nutr.

so 33:

comparisons

1861-1868,

Human milk is physiologically adapted for the promotion of health and nutrition in the newborn infant. In contrast to processed cows milk, human milk is a live secretion containing active enzymes, hormones and essential nutrients presumably in the proper forms and proportions for the infant. Although human milk generally contains considerably higher levels of enzymes than bovine milk, little definitive information is available concerning their role or significance. Shahani et al. (1) reviewed the enzymes in bovine milk and suggested that they are probably normal constituents of the secretory epithelial cells where they participate in cell metabolism and the biosynthesis of milk constituents. During the milking process the cells rupture and spill the enzymes into the milk. In addition, some enzymes may be secreted directly into the milk to benefit the newborn infants who have incomplete digestive systems. The current paper will review the physiological significance of some human milk enzymes and will present evidence that these enzymes perform roles similar to those suggested for the bovine system. Table 1 shows the relative concentration of enzymes in human milk as compared to bovine milk. The number in parenthesis refers to the enzyme classification numbers sugThe American JournalofClinical Nutrition 33: AUGUST

gested by the Committee on Enzymes of the International Union of Biochemistry (2). By and large, the levels of various enzymes are higher in human milk than those in bovine milk. Information on the more significant enzymes will be presented later. As shown in Table 2, the stage of lactation significantly affects the concentration of enzymes in human milk. In general, the majority of the enzymes are present in significantly higher concentrations soon after parturition and gradually decline thereafter. In addition, the manner and form of nutrient intake by lactating women as well as the time of sampling may also influence the levels ofthe milk enzymes. In a study with 60 lactating women, Karmarker and Ramakrishnan (3) found that dietary fat intake up to 72 g/day increased the levels of lipase, esterase, and alkaline phosphatase but decreased acid phosphatase. In a later study, they concluded that lipase, esterase and alkaline phosphatase play a role in the digestion, assimilation, and metabolism of fat, but are not influenced by protein and
I From the Department of Food Science and Technology, University of Nebraska, Lincoln, Nebraska 68583. 2 Published as Paper no. 5751, Journal Series, Nebraska Agricultural Experiment Station. Research reported was conducted under Project no. 16-026.

1980, pp. 1861-1868.

Printed in U.S.A.

I 861

1862 TABLE Enzyme 1 levels

SHAHANI

ET

AL.

in human

milk

versus

bovine

milk
Relative content Reference Human milk Bovine milk

Enzyme

Adenosine (3.6.1.3)

triphosphatase

23x

(39)

(ATP

phosphohydrolase)
aminotransferase 2 oxoglutarate aminotransferase) 400x (21)

Alanine (2.6.1.2) (L-Alanine: Aldolase (4. 1.2. 13) (Fructose-l,6 a-Amylase (3.2.1.1) (a-1,4-Glucan 18-Amylase (3.2.1.2)

5x
diphosphate (diastase) 4-glucanohydrolase) 40x D-glyceraldehyde-3-phosphatelyase) 25x

(21)

(8)

Downloaded from ajcn.nutrition.org by guest on January 17, 2013

(21)

(a-1,4-Glucan
Arylesterase (3.1.1.2) (Aryl-ester Aspartate (2.6.1.1) (L-Aspartate:

maltohydrolase)

4x
hydrolase) aminotransferase 2 oxoglutarate aminotransferase) lOx

(39)

(21)

Catalase (1.11.1.6) (Hydrogen-peroxide: Cholinesterase (3.1.1.8) (Acylcholine

lOx
hydrogen peroxide oxidoreductase) 9-l6x acyl-hydrolase) isomerase ketol-isomerase) 20-30x glucuronohydrolase)
+

(39)

(39)

Glucosephosphate (5.3.1.9) (D-Glucose-6-phosphate $-Glucuronidase (3.2.1.31) (/J-D-GlucurOnide

2x

(21)

(49)

Inorganic pyrophosphatase (3.6.1.1) (Pyrophosphate phosphohydrolase) Lactate dehydrogenase (1.1.1.27) (L-Lactate: NAD oxidoreductase) Lactose synthetase (2.4.1.22) (UDP galactose: Lipase (3.1.1.3) (Glycerol-ester
a

(39)

3.5x

(5,

18)

lOx D-glucose, I-galactosyltransferase) 8-lOx hydrolase) reported as diastase is most likely a-amylase but may also contain some $-amylase.

(14)

(16,

50)

The enzyme

ENZYMES TABLE

IN

HUMAN

MILK

1863

1 (Continued)
Relative content Reference Human milk Bovine milk

Enzyme

Lysozyme (3.2.1.17) (Mucopeptide

3000x N-acetylmuramythydrolase) l-5x

(33)

Malate dehydrogenase (1.1.1.37) (L-Malate NAD oxidoreductase) Peroxidase (1.11.1.7) (Donor:

(5,

18)

lOOx hydrogen-peroxide acid phosphohydrolase) alkaline monoester phosphohydrolase) 5x peptide hydrolase) 3.5x pyrimidine-nucleotide-2-transferase) 40x oxidoreductase) Same

39, 51

Phosphatase, (3. 1.3. 16) (Phosphoprotein Phosphatase, (3.1.3.1) (Orthophosphoric Protease (3.4.4-) (Peptidyl Ribonuclease (4.7.7.16) (Ribonucleate

(21)

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(21,

38)

(39,

40)

(42,

43)

Xanthine
(1.2.3.2)

oxidase
oxygen oxidoreductase)

lOx

(5,46-48)

(Xanthine:

vitamin supplementation (3A). Sklavunu-Zurukzoglu et al. (4) reported the administration of vitamins A, B6, B12, C, and K to lactating women did not have any influence on the level of glucose-6-phosphate dehydrogenase. Similarly, Deodhar et al. (5) demonstrated daily vitamin intake had no effect on xanthine oxidase, lactic acid dehydrogenase, and malic acid dehydrogenase activities in human milk. Belavady (6, 7), however, noted that protein supplementation for three to four weeks increased alkaline phosphatase (6) and xanthine oxidase (6, 7). The following represent the most widely studied enzymes of human milk. Diastase Diastase catalyzes the hydrolysis of starch to maltose, and, therefore, is most likely the enzyme a-amylase but may refer to a mixture of a and $ amylases. Hanafy et al. (8) noted a sharp increase in the level of diastase in

colostrum at the middle of pregnancy, a rapid fall during the first 6 months of lactation, followed by a gradual decline until a constant level was reached at 6 months. These authors suggested that the diastase in the milk may be beneficial to newborn infants who are deficient in pancreatic diastase. This would also explain the cli ically observed lack of digestive disturbances in breast-fed infants offered starch formula early-a situation which occurs particularly in poor classes. Glucose-6-phosphate dehydrogenase

Glucose-6-phosphate dehydrogenase catalyzes the oxidation of glucose-6-phosphate (2). Sklavunu-Zurukzoglu et al. (4) studied 17 nursing mothers with normal red cell glucose-6-phosphate dehydrogenase and found their milk rich in the enzyme. Two mothers with complete absence of the enzyme in red cells also had complete absence ofthe enzyme in the milk. Since the large quantity of glu-

1864

SHAHANI
2 levels normal
Enzyme

ET

AL.

TABLE
Enzyme versus

in human milk

milk

colostrum

Colostrum

Normal . milk +

Reference

Adenosine triphosphatase Alanine amino transferase Aldolase a-Amylase (diastase) fJ-Amylase Aspartate aminotransferase Catalase Cholinesterase Glucose-6-phosphate dehydrogenase Glucose-phosphate isomerase Inorganic pyrophosphatase Lactic dehydrogenase Lipase Lysozyme Lysozyme Malic dehydrogenase Peroxidase Phosphatase, acid Phosphatase, alkaline Protease Xanthine oxidase
a

(39) (21) (21) (8) (21) (21) (39) (39) (4) (21) (39) (5, 18) (21) (47) (33) (5, 18) (39, 51) (21) (21) (39) (5, 46, 47) likely a-

2x
+ +

1.5x 2x 2x
+

No l.5x
+

change

+
+

No +
+

change

3x 4x 2x
+

The

enzyme but may

reported also contain

as diastase some

is most fl-amylase.

amylase

cose-6-phosphate dehydrogenase in the milk is related to the increased rate of carbohydrate metabolism in the mammary gland during lactation, these authors suggested that the great variation of glucose-6-phosphate dehydrogenase found in the same individual is due to the same neurohormonal influence which regulates the production and secretion of miLk. Lactose synthetase

conversion of UDP galactose and N-acetylglucosamine to N-acetyllactosamine and UDP. In the presence of a-lactalbumin, the latter reaction is partially inhibited and lactose formation is favored (9, 12). Andrews (13) showed that a-lactalbumin, in the presence of glucose at normal tissue concentration, acts as a competitive inhibitor of the A protein catalyzed synthesis of the Nacetyllactosamine. In another study Andrews (14) found that the A protein does possess slight lactose synthetase activity even in the absence of a-lactalbumin. Fitzgerald et al. (15) suggested that a-lactalbumin lowers the Km of glucose so that it can be used maximally in lactose biosynthesis. Andrews (14) observed in a crude preparation of the A protein from human milk that the Km for glucose was lowered from 60 mM in the absence to 3 mt in the presence of a-lactalbumin. Klee and Klee (16) reported in the presence of a-lactalbunun, the normal in vivo concentration of glucose is optimal for lactose synthesis. Brew et al. (12) found that as the mammary gland developed during pregnancy, the concentration of A protein and, to a lesser extent the concentration ofa-lactalbumin, increased with time. At parturition, however, when lactation commenced, the level of a-lactalbumin increased significantly and provided the proper conditions for lactose synthesis. Turkington et al. (17) further observed that the prolactin induced synthesis of A protein and a-lactalbumin in mouse mammary gland cell cultures occurs specifically in daughter cells formed in the presence of insulin and hydrocortisone. This control mechanism for lactose biosynthesis by the A protein and ct-lactalbumin insures that lactose is synthesized only in the mammary gland in response to specific hormones. Lactic and malic acid dehydrogenases

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Lactose synthetase, an enzyme present in the mammary gland and in milk, catalyzes the synthesis of lactose from uridine diphosphate (UDP) galactose and glucose (28). The lactose synthetase enzyme has two components-an A protein which is a glycoprotein containing hexose, hexosamine and sialic acid (9, 10) and a B protein which is the milk protein a-lactalbumin (9, 1 1). Separated A protein acts as a galactosyl transferase in the

Kjellberg and activity of lactic milk is high in milk, and highest They also noted tween body size lactic dehydrogenase the highest lactic demonstrated in

Karisson (18) found that the dehydrogenase in human colostrum, lower in mature at the end of lactation. an inverse relationship beof a particular species and activity in milk. Thus, dehydrogenase activity was the mouse, followed by man,

ENZYMES

IN

HUMAN

MILK

1865

storage and then the cow. The activity of malic dehydrogenase shows the same principal variations during lactation and appears to exist in a similar inverse relationship with body weight as shown by lactic dehydrogenase. This is expected since metabolic rates are known to be higher in cells from small animals than large. These authors also demonstrated that although the lactic and malic dehydrogenase isozymes in milk show characteristic electrophoretic patterns at different stages of lactation, the blood serum patterns show no such variation. These results suggest that lactic dehydrogenase and malic dehydrogenase are synthesized in the mammary gland rather than transported from the blood stream and that lactic and malic dehydrogenase levels in the milk of various species probably reflect mammary gland metabolism. Lipase Lipase catalyzes the hydrolysis of glycerol esters (fats and oils) in emulsion (1). Hernell and Olivecrona (19, 20) have isolated a serum stimulated lipoprotein lipase from the cream and a bile salt stimulated lipase from the skim portion ofhuman milk. The serum stimulated lipase has no apparent physiological function in the milk and probably results from mammary gland leakage (19). The bile salt stimulated lipase is present in much higher activity than the serum stimulated lipase (19) and has been shown to resemble pancreatic lipase (21). In bovine milk, the bile salt stimulated lipase appears to be pancreatic in origin and is transported to the mammary gland to participate in the synthesis of milk fat (22). The bile salt stimulated lipase in human milk, however, appears to play a significant role in the digestion of milk triacylglycerols in the intestine of infants fed human milk (23, 24). It has sufficient activity at low pH and at intestinal bile salt concentrations to augment the low levels of lipid resorption in the newborn (25). This is particularly important since free fatty acids are an important energy source for the infant (26). It is interesting to note that human milk lipases, in contrast to bovine milk, may be activated at refrigerated temperatures. For this reason Tarassuk et al. (27) recommended pasteurization of human milk intended for ity. Lysozyme

in order

to prevent

hydrolytic

rancid-

Lysozyme catalyzes the hydrolysis of f? (-1,4-) linkage between N-acetylglucosamine and N-acetylmuramic acid in the bacterial cell wall (30). The enzyme lyses mostly gram positive and a few gram negative bacteria and has been postulated to play a role in the inherent antibacterial activity of milk (31). In 1931, Rosenthal and Lieberman (32) demonstrated that human milk lysozyme plays a significant role in the development of the intestinal flora of breast-fed infants. Unlike the mixed intestinal flora in infants on cows milk, there is a prevalence of Lactobacillus bfidus in the intestine of healthy breastfed infants (26). Chandan et al. (33) reported that the lysozyme content of human milk is about 3000 times greater than that in bovine milk. Isolation and characterization of the human milk lysozyme revealed a MW of 15,000 and an activity 3.5 times that of egg white lysozyme and 100 times that of bovine milk lysozyme (34). This low level of lysozyme activity in bovine milk and absence in prepared infant formulas (29) may represent a significant problem in nutrition of infants fed these products as their sole diet (35). Eitenmiller et al. (36) determined that the human milk lysozyme is antigenically and serologically different from the bovine enzyme. Hyslop et al. (37) observed that the relationship between lysozyme and total protein content remains relatively constant in both colostrum and milk. Since lysozyme did not increase with the increase in total milk secretion after parturition, these authors suggested that the appearance of lysozyme in milk may be considered a spillover product from the breast epithelial cells. Phosphatases Acid phosphatase, which is not a phosphomonoesterase (1), has practically the same activity in both human and bovine milk (21). The phosphomonoesterase alkaline phosphatase, however, has an activity in human milk which is 40 times lower than that in bovine milk (21).

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1866

SHAHANI

ET

AL.

Stewart et al. (38) determined that the alkaline phosphatase concentration appears to be related to the fat concentration in human milk. Karmarkar and Ramakrishnan (3) also determined that alkaline phosphatase activity increases with an increase in fat concentration, while acid phosphatase does not. Stewart et al. (38) also found no correlation between either nitrogen content or total solids other than fat and alkaline phosphatase activity. These authors did note a tendency for alkaline phosphatase to increase as lactation progressed, while Heyndrickx (21) reported a fourfold lower activity in normal milk than colostrum. Protease Protease catalyzes the hydrolysis of proteins (2). Since there are higher levels of proteolytic enzymes in human milk compared to cows milk (39, 40), Storrs and Hull (40) suggested that these enzymes may provide the breast-fed infant with significant digestive assistance immediately after birth. Studies by Eigel (41) indicate that in bovine milk plasmm is the protease responsible for in vivo production of minor casein fractions. However, no such protease or plasmin has been reported for human milk. Ribonuclease Ribonuclease catalyzes the hydrolysis of ribonucleic acid (1). Bingham and Zittle (42) isolated and characterized ribonuclease A from bovine milk and found it to be identical to bovine pancreatic ribonuclease A with respect to amino acid composition, electrophoretic behavior and immunological properties. These authors suggested that pancreatic ribonuclease enters the milk via the blood stream. Dalaly et al. (43) purified human milk ribonuclease and found it possesses an absolute specificity toward pyrimidine and ribose moieties. In this respect, it appears to resemble the bovine pancreatic ribonuclease A. However, the human milk ribonuclease differs considerably from the bovine enzyme in amino acid composition. Xanthine Xanthine of purines, oxidase oxidase pynmidines catalyzes the oxidation and aldehydes (1). In

1946, Rodkey and Ball (44) reported that human milk is devoid of xanthine oxidase, while cows milk is rich in this enzyme, even after commercial pasteurization. These authors suggested that the xanthine oxidase activity may be used to distinguish human milk and cows milk and, in particular, could be used to test for the dilution of human milk samples with cows milk. Owen and coworkers (45) also reported that xanthine oxidase is absent in human milk. However, in 1960, Bradley and Gunther (46), using a more sensitive assay technique, found small amounts of xanthine oxidase in human milk. On the basis ofthese fmdings, Owen and Hytten (47) reexamined human milk samples and found xanthine oxidase. The activity of xanthine oxidase peaks on the 3rd day after birth and decreases with the progress of lactation (46, 47). Zikakis et al. (48) also showed that human milk xanthine oxidase differs from bovine milk xanthine oxidase and it is not of bacterial origin. Although vitamin supplementation has no effect on the activity of xanthine oxidase (5), the concentration of protein can be correlated with xanthine oxidase activity (6, 7). Conclusion Human milk is a highly complex fluid containing numerous active enzymes. These enzymes in milk may serve specific functions or have no known role. However, the enzyme activity levels in human milk are affected by factors such as the sensitivity of the assay, the duration of milk sampling, the time of lactation for sampling, and variations within individuals. Also, the significance of each enzyme in human milk as a reflection of the nutritional status of the mother, its possible origin, and its nutritive value to the infant is not absolutely clear. But, evidence has been presented to support the views that in human milk 1) lipoprotein lipase and ribonuclease are possibly spilled into the milk via the blood stream from the pancreas; 2) lysozyme is spilled into the milk from the secretory epithelial cells; 3) lactate and malate dehydrogenases, glucose-6-phosphate dehydrogenase, and lactose synthetase are synthesized in the mammary gland in response to hormonal stimuli during pregnancy and lactation; and 4) bile salt stimulated lipase, diastase, pro-

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ENZYMES

IN

HUMAN

MILK
of microsomal lactose

1867 syn-

tease, and lysozyme are present in human milk in significant quantities to aid newborn breast-fed infants in the development of their proper growth, health, and nutrition. It is also important to note that there is no uniformity among the assay procedures, substrates used or activity units expressed for the same enzyme reported by different workers. Consideration must be given to standardizing these parameters so that activities reported by various workers can be readily compared. Calculation of specific activities in terms of moles substrate per minute per standard amount of milk could be of value; El References
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nents and solubilization thetase. J. Biol. Chem. 12.

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BREW, K., T. C. VANAMAN AND HILL. The role of a-lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction. Proc. Natl. Acad. Sci. U.S.A. 59: 491, 1968. ANDREWS, P. Purification of lactose synthetase A protein from human milk and demonstration of its interaction with a-lactalbumin. FEBS Lett. 9: 297, 1970. ANDREWS, P. Lactose synthetase A protein from human milk. Biochem. J. 1 1 1: 14P, 1969. FITZGERALD, MAWAL,

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milk ribonuclease and lysozyme. Anal. Biochem. 37: 208, 1970. RODKEY, F. L., AND E. G. BALL. A rapid test for distinguishing human milk from cows milk based upon a difference in their xanthine oxidase content. J. Lab. Chin. Med. 31: 354, 1946. MODI, V. V., E. C. OWEN AND R. PROUDFOOT. Species differences in the occurrence of xanthine oxidase in milk. Proc. Nutr. Soc. 18: 1, 1959. BRADLEY, P. L., AND M. GUNTHER. The xanthine oxidase of human milk and colostrum. Biochem. J. 74: iSP, 1960.
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