Full text of DOI:10.1007/BF03345099

J. Endocrinol. Invest. 25: 670-674, 2002
Presence of bovine leptin in edible commercial milk and
infant formula
M. Lage**, R. Baldelli*, J.P. Camiña*, J. Rodriguez-Garcia***, A. Peñalva*,
C. Dieguez**** and F.F. Casanueva*
*Endocrinology Service, Regina Elena Cancer Institute, Rome, Italy, **Department of Medicine,
Endocrinology Section, ***Department of Biochemistry and ****Department of Physiology, Santiago de
Compostela University and Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain
ABSTRACT. Leptin is a hormone secreted by the
adipocytes that contribute to the control of ener-
gy balance, and circulating leptin levels reflect the
amount of adipose tissue in the body, helping to
regulate food intake and energy expenditure.
Since it has been shown that human milk contains
immunoreactive leptin, which is identical to intact
human leptin, we decided to investigate the pos-
sible presence of immunoreactive bovine leptin in
different kinds of common commercial milk. To de-
termine the presence or absence of immunoreac-
tive leptin in bovine milk for human consumption,
81 samples (66 commercial pasteurized milk and
15 artificial formulae for new-born babies) of the
most common Spanish commercial types of milk
were studied. All samples were evaluated before
and after centrifugation, and leptin levels were
measured by RIA. Leptin was detected in all sam-
ples and RIA standard curves were not perturbed
when centrifuged and non-centrifuged milk re-
placed the buffer. Mean values of leptin in full-
cream, semi-skimmed and skimmed samples,
were: 5.7 0.3 ng/ml, 4.1 0.1 ng/ml, 3.7 0.1
ng/ml (significantly different). Leptin values were
reduced after centrifugation. A significant correla-
tion was observed between leptin levels and lipid
content (p<0.0005, r=0.67) while no correlation
was observed with respect to carbohydrate and
protein levels. Interestingly, some preparations of
infant formulae present very high leptin values
reaching up to 18.9 ng/ml. In conclusion, leptin is
present in significant and variable concentrations in
edible commercial bovine milk, with higher con-
centrations in infant formula.
(J. Endocrinol. Invest. 25: 670-674, 2002)
©2002, Editrice Kurtis
INTRODUCTION
and insulin stimulate leptin synthesis from the adi-
pose tissue, whereas adrenergic activation and T
induce inhibition (16-19). Our group reported that
human leptin was present in milk and colostrum
from nursing mothers (20), a relevant finding con-
sidering that after delivery milk replaces the pla-
centa in providing the new-born with critical nu-
trients and growth factors. In rats, leptin is trans-
ferred from the mother’s milk to the pup’s stom-
ach and then to the pup’s serum (20-24). This sug-
gests that during the neonatal period, leptin may
be absorbed without degradation by the intesti-
nal system and plays a role in the regulation of
neonatal food intake and intestinal maturation
(20, 25, 26). In the present work, the presence of
leptin in edible bovine milk (commercial pasteur-
ized samples and infant formulae) was analyzed.
The targets were twofold: 1) to observe whether
leptin was also present in bovine milk and 2) to
observe whether different milk brands differ in
leptin content.
Leptin, the ob gene product, is a 167 amino acid
hormone that is secreted by the adipocytes,
which acts as an afferent satiety hormone regu-
lating appetite, weight gain, and body fat stores
(1-12). Leptin plasma levels correlate mainly with
the adipose tissue stores, but not in a simple re-
lationship, as other factors and hormones partic-
ipate in its regulation (13). The participation of
leptin in new physiological and pathological pro-
cesses unrelated to metabolic functions has been
described (13-15). In addition, glucocorticoids
Key words: Leptin, commercial milk, infants formula.
Correspondence: Prof. F.F. Casanueva, Department of Medicine,
Endocrine Unit, San Francisco Street s.n., PO Box 563. E-15780 Santiago
de Compostela, Spain.
E-mail: endocrine@usc.es
Accepted March 3, 2002.
670

Leptin in milk
MATERIALS AND METHODS
of buffer (Fig. 1). With respect to the different milk
subtypes, a significant difference was found in full-
cream milk (5.7 0.3 ng/ml) vs semi-skimmed
(4.1 0.1 ng/ml), and skimmed milk (3.7 0.1 ng/ml)
(p<0.0005). All values were significantly reduced af-
ter centrifugation (p<0.0005) (Fig. 2). Leptin levels
in the different brands of milk ranged from 2.6 to
8.5 ng/ml (Fig. 3). Levels of leptin detected in in-
fant milk ranged from 1.9 up to strikingly high lev-
els of 18.9 ng/ml (Fig. 3). The correlation study
showed a significant correlation between leptin lev-
els and lipid concentrations (p<0.0001, r=0.67) but
not with proteins or carbohydrates (Fig. 4).
In order to determine the presence of immunoreactive bovine lep-
tin levels in edible milk, samples of the most widely used Spanish
commercial milk samples were collected (no.=81). In particular 66
types of milk were normal pasteurised commercial milk from dif-
ferent brands (1=Asturiana®, 2=Campobueno®, 3=Celta®,
4=Consumer®, 5=Dia®, 6=Feiraco®, 7=Froiz®, 8=Hypercor®, 9=La
Vaquera®, 10=Lagisa®, 11=Larsa®, 12=Leader Price®, 13=Leyma®,
14=Oro del Valle®, 15=Pascual®, 16=President®, 17=Puleva®,
18=Ram®, 19=Reny Picot®, 20=Rio®, 21=Super®, 22=Vega de
Oro®). All were analyzed as full-cream, semi-skimmed or skimmed
samples before and after centrifugation at 3000 x g, 30 min, 4 C
after which the fat layer was removed. Fifteen samples were of the
kind used for artificial new-born lactation (infant formulae)
(23=Adapta-1®, 24=Adapta-2®, 25=Adapta-peg®, 26=Almiron-1®,
27=Aptamil-1®, 28=Aptamil-2®, 29=Blemil-plus®, 30=Enfalac®,
31=Nativa-1®, 32=Nativa-2®, 33=Nidina-1®, 34=Nidina-2®,
35=Nutriben®, 36=O-Lac®, 37=President®). Milk lipids, proteins
and carbohydrate levels data reported by manufacturers.
Leptin milk levels were measured on several occasions for each
brand in duplicate by multi-species leptin RIA kit (Linco Research
Inc, St. Charles, U.S.A.) The limit of sensitivity was 0.5 ng/ml, the
interassay coefficient of variation was 8.7% and the intrassay co-
efficient of variation was 3.6%.
Data are presented as mean SE. The Anova test, and the t test
for paired and unpaired data were used when appropriate. The
correlation study was evaluated with a simple regression test.
p<0.05 was considered significant. Statistical analyses were car-
ried out using a Statview 5 software for Windows (SAS Institute
INC., Cary, U.S.A., 1999).
DISCUSSION
Human milk as well as the milk of several mam-
malian species contains, besides major nutrients,
anti-infectious and immunocompetent substances,
biologically active substances such as hormones,
peptides and growth factors which can directly in-
fluence the physiology of new-born babies after in-
testinal absorption (27, 28). The function of these
bioactive peptides and steroids in milk remains to
A
7
6
5
4
3
2
*
RESULTS
Leptin was detected in all milk samples analyzed
and the RIA validation test indicated that milk did
not interfere with the assay. RIA standard curves
were not altered when 100 μl of centrifuged and
non-centrifuged milk substituted the same volume
No centrifuged
Centrifuged
1.2
1.0
0.8
0.6
0.4
0.2
0.0
RIA buffer
Milk
B
7
6
5
4
3
2
1
0
*
*
Full-cream
Semi-skimmed
Skimmed
Fig. 2 - A) Leptin concentration in non-centrifuged (no.=66) and
centrifuged (no.=66) bovine milk; (p<0.0005). The error bars
represent the 10-90th range, the box represents the 25-75th
range and the line represents the median; B) leptin concentra-
tion in full-cream (no.=22), semi-skimmed (no.=22) and skimmed
(no.=22) milk. p<0.0005.
0
1
2
5
10
20
50
Leptin (ng/ml)
Fig. 1 - RIA standard curves of human leptin in the absence or
presence of 100 ml of commercial milk which substituted the
assay buffer. Milk dilutions always paralleled standard curves.
671

M. Lage, R. Baldelli, J.P. Camiña, et al.
Edible milk
10
5
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Infant formula
30
25
20
15
10
5
Fig. 3 - Leptin levels in commercial sam-
ples from different brands of pasteurised
full-cream edible milk (no.=22; upper
panel). Leptin levels in commercial sam-
ples of infant-formulae milk brands (low-
er panel).
0
23 24 25 26 27 28 29 30
31 32
33 34 35 36 37
Commercial milk brands
be elucidated, although it has been proposed that
these substances could be related to the develop-
ment of the neonatal small intestine, or play im-
portant and different roles in immune function (29).
The human neonate has a reduced immune system
and needs the support of the mother with the
transplacentally passage of IgG antibodies (29). In
rats, the presence of leptin in milk and the transfer
from milk to the pup’s stomach and blood (20)
raised the possibility that maternal leptin may exert
biological effects on the infant at a time in which
both the adipose tissue and the appetite regulato-
ry systems are immature (30). Many bioactive com-
pounds have been shown to be transferred from
milk to the neonate blood, and although the trans-
fer of leptin has only been demonstrated for ex-
perimental animals, it is likely that it also occurs in
man (31, 32). As humans ingest a considerable
amount of bovine milk and more in the neonatal
period, in this work the presence of leptin in edible
milk was analyzed. To the best of our knowledge,
this is the first study providing evidence of variable
leptin levels in normal commercial milk. Our analy-
ses revealed that leptin concentrations in non-cen-
trifuged milk were higher than in centrifuged milk;
an explanation for this clear difference could be due
to the fact that a part of leptin could be associated
with the milk fat droplet, as previously shown (26).
Although detectable, the leptin concentration mea-
sured in commercial milk is unlikely to be able to
alter circulating leptin levels, if one considers the
limited amount of milk ingested in the adult peri-
od, plus the fact that proteins are rapidly digested
and destroyed in the gastrointestinal tract. Of more
biological relevance, however, are the high leptin
concentrations observed in infant formulae. In fact,
in some infant preparations leptin levels were ex-
tremely high with respect to the previous ones
found in the milk of lactating women (20) and one
must be aware of the high milk intake and the re-
peated daily ingestion for the low plasma volume
of neonates. In a recent work, leptin concentrations
were determined in the circulation and milk of sows
differing in body condition at farrowing and in feed
consumption during lactation. The highest serum
concentrations of leptin at farrowing and weaning
were detected in sows exhibiting the greatest
amount of backfat; leptin was detected in both
skimmed and whole milk throughout lactation, but
levels were not correlated with backfat thickness or
circulating leptin concentration (33). As leptin from
different species can be transferred from intestine to
blood in the neonatal period (20, 33), biologically
active leptin may be significantly increased at reg-
ular intervals in the new-born plasma due to the
high levels found in artificial milk. Whilst leptin has
been found to inhibit food intake in adult animals,
neonatal ones need to maximize food intake and
also maintain high thermo-regulatory metabolic
rates to maintain body temperature. This suggests
that leptin may function differently in the neonate
compared with adult animals. Currently, it is not
possible to ascertain whether this hypothetical
bovine leptin rise may play a regulating role in the
672

Leptin in milk
8
7
6
5
4
3
2
milk. Leptin determination in milk samples may help
to control the elaboration process as well as to pre-
vent the alterations in edible brands. The latter point
deserves further examination.
In conclusion, the present work provides evidence
for the presence of immunoreactive bovine leptin
levels in commercial milk and infant formulae.
Moreover, the presence of extremely high leptin lev-
els in infant formulae types of milk suggests a po-
tential link between milk–borne leptin and neonatal
developing of gastrointestinal function or in the reg-
ulation of food intake. Further work is needed in or-
der to ascertain the possible absorption of leptin in
humans, and whether this milk-borne leptin exerts a
regulatory role on appetite in the suckling infant.
r=0.67; p<0.0001
3.0 3.5 4.0
0
0.5
1.0
1.5
2.0
2.5
Lipids (g/100 ml)
8
7
6
5
4
3
2
ACKNOWLEDGMENTS
This work was supported by grants from Fondo de Investigacion
Sanitaria, Spanish Ministry of Health, and Secretaria Xeral de
Investigación e Desenvolvemento, Conselleria de Educacion,
Xunta de Galicia and DGICYT.
r=0.05; p<0.75
3.8 4.0
2.8
3.0
3.2
3.4
3.6
Proteins (g/100 ml)
8
7
6
5
4
3
2
REFERENCES
1. Zhang Y., Proenca R., Maffei M., Barone M., Leopold L.,
Friedman J.M. Positional cloning of the mouse obese gene
and its human homologue. Nature 1994, 372: 425-432.
2. Pelleymounter M.A., Cullen M.J., Baker M.B., et al. Effects
of the obese gene product on body weight regulation in
ob/ob mice. Science 1995, 269: 540-543.
r=0.07; p<0.62
5.6 5.8 6.0
4.2
4.4
4.6
4.8
5.0
5.2
5.4
3. Halaas J.L., Gajiwala K.S., Maffei M., et al. Weight-reduc-
ing effects of the plasma protein encoded by the obese
gene. Science 1995, 269: 543-546.
Carbohydrates (g/100 ml)
Fig. 4 - Scatter analysis of leptin levels vs total lipids, protein
and carbohydrate levels, in different brands of pasteurized ed-
ible milk.
4. Campfield L., Smith F., Guisez Y., Devos R., Burn P. Recom-
binant mouse OB protein: evidence for a peripheral sig-
nal linking adiposity and central neural networks. Science
1995, 269: 546-549.
5. Considine R.V., Considine E.L., Williams C.J., Hyde T.M.,
Caro J.F. The hypothalamic leptin receptor in humans:
identification of incidental sequence polymorphisms and
absence of the db/db mouse and fa/fa rat mutations.
Diabetes 1996, 45: 992-994.
post-natal period; nevertheless, the point deserves
consideration as this mechanism could lead to a
possible role of leptin in neonatal development of
the gastrointestinal tract, as suggested by the pres-
ence of leptin receptors in these tissues, and im-
mune function, or more relevant in the regulation
of food intake in neonates. This could explain the
intriguing observation that children with ob gene
mutation develop obesity only after stopping lac-
tation while in those children with db gene muta-
tion obesity starts immediately after birth (Farooqi
I.S., personal communication).
The amount of leptin in a given milk brand was quite
constant over time (data not shown), an observation
with potential industrial applications. In fact, there
is no easy explanation for the striking divergence in
leptin content among the different brands of edible
6. Considine R.V., Nyce M.R., Kolaczynski J.W., et al. Dexa-
methasone stimulates leptin release from human adi-
pocytes: unexpected inhibition by insulin. J. Cell. Biochem.
1997, 65: 254-258.
7. Considine R.V., Sinha M.K., Heiman M.L., et al. Serum im-
munoreactive-leptin concentrations in normal-weight and
obese humans. N. Engl. J. Med. 1996, 334: 292-295.
8. Ahima R.S., Prabakaran D., Mantzoros C., et al. Role of lep-
tin in the neuroendocrine response to fasting. Nature 1996,
382: 250-252.
9. al Shoumer K.A., Anyaoku V., Richmond W., Johnston D.G.
Elevated leptin concentrations in growth hormone-deficient
hypopituitary adults. Clin. Endocrinol. 1997, 47: 153-159.
673

M. Lage, R. Baldelli, J.P. Camiña, et al.
10. Houseknecht K.L., Mantzoros C.S., Kuliawat R., Hadro E.,
Flier J.S., Kahn B.B. Evidence for leptin binding to proteins
in serum of rodents and humans, modulation with obesity.
Diabetes 1996, 45: 1638-1643.
22. Aoki N., Kawamura M., Matsuda T. Lactation-dependent
down regulation of leptin production in mouse mammary
gland. Biochim. Biophys. Acta 1999, 1427: 298-306.
23. Smith-Kirwin S.M., O’Connor D.M., De Johnston J., Lancey
E.D., Hassink S.G., Funanage V.L. Leptin expression in hu-
man mammary epithelial cells and breast milk. J. Clin.
Endocrinol. Metab. 1998, 83: 1810-1813.
11. Klein S., Coppack S.W., Mohamed Ali V., Landt M.
Adipose tissue leptin production and plasma leptin kinet-
ics in humans. Diabetes 1996, 45: 984-987.
12. Sinha M.K., Opentanova I., Ohannesian J.P., et al. Evi-
dence of free and bound leptin in human circulation.
Studies in lean and obese subjects and during short-term
fasting. J. Clin. Invest. 1996, 98: 1277-1282.
24. Laud K., Gourdou I., Belair L., Keisler D.H., Djiane J. De-
tection and regulation of leptin receptor mRNA in ovine
mammary epithelial cells during pregnancy and lactation.
FEBS Lett. 1999, 463: 194-198.
13. Casanueva F.F., Diéguez C. Neuroendocrine regulation
and actions of leptin. Front. Neuroendocrinol. 1999, 20:
317-363.
25. Trottier G., Koski K.G., Brun T., Toufexis D.J., Richard D.,
Walker C.D. Increased fat intake during lactation modifies
hypothalamic-pituitary-adrenal responsiveness in devel-
oping rat pups: a possible role for leptin. Endocrinology
1998, 139: 3704-3711.
14. Sierra-Honigmann M.R., Nath A.K., Murakami C., et al.
Biological action of leptin as an angiogenic factor. Science
1998, 281: 1683-1686.
26. Smith-Kirwin S.M., O’Connor D.M., Johnston J., De Lancey
E., Hassink S.G., Funage V.L. Leptin expression in human
mammary epithelial cells and breast milk. J. Clin. Endo-
crinol. Metab. 1998, 83: 1810-1813.
15. Jin L., Zhang S., Burguera B.G., Couce M.E., et al. Leptin
and leptin receptor expression in rat mouse pituitary cells.
Endocrinology 2000, 141: 333-339.
27. Grosvenor C.E., Picciano M., Baumruscker C.R. Hormones
and growth factors in milk. Endocr. Rev. 1992, 14: 710-
728.
16. Hamilton B.S., Paglia D., Kwan A.Y., Deitel M. Increased
obese mRNA expression in omental fat cells from mas-
sively obese humans. Nature Med. 1995, 1: 953-956.
28. Koldovsky O., Illnerova H., Macho L., Strbak V., Stepankova
R. Milk-borne hormones: possible tools of communication
between mother and suckling. Physiol. Res. 1995, 44: 349-
351.
17. Montague C.T., Prins J.B., Sanders L., Digby J.E., O’Rahilly
S. Depot- and sex-specific differences in human leptin
mRNA expression: implications for the control of regional
fat distribution. Diabetes 1997, 46: 342-347.
29. Hanson L.A., Ceafalau L., Mattsby-Baltzer I., et al. The
mammary gland-infant intestine immunologic dyad. Adv.
Exp. Med. Biol. 2000, 478: 65-76.
18. Leal-Cerro A., Considine R.V., Peinó R., et al. Serum im-
munoreactive-leptin levels are increased in patients with
Cushing’s syndrome. Horm. Metab. Res. 1996, 28: 711-713.
30. Smas C.M., Sul H.S. Control of adipocyte differentiation.
19. Peinó R., Piñeiro V., Gualillo O., et al. Cold exposure in-
hibits leptin secretion in vitro by a direct and non-specific
action on adipose tissue. Eur. J. Endocrinol. 2000, 142:
195-199.
Biochem. J. 1995, 309: 697-710.
31. Berseth C.L. Enhancement of intestinal growth in neonatal
rats by epidermal growth factor in milk. Am. J. Physiol.
1987, 253: 662-665.
20. Casabiell X., Pineiro V., Tomé M.A., Peinó R., Dieguez C.,
Casanueva F.F. Presence of leptin in colostrum and /or
breast milk from lactating mothers: a potential role in the
regulation of neonatal food intake. J. Clin. Endocrinol.
Metab. 1997, 82: 4270-4273.
32. Dvorak B., McWilliam D.L., Williams C.S., et al. Artificial
formula induces precocious maturation of the small intes-
tine of artificially reared suckling rats. J. Pediatr. Gastro-
enterol. Nutr. 2000, 31: 162-169.
21. Houseknecht K.L., McGuire M.K., Portocarrero C.P., McGuire
M.A., Beerman K. Leptin is present in human milk and is re-
lated to maternal plasma leptin concentration and adiposity.
Biochem. Biophys. Res. Commun. 1997, 240: 742-747.
33. Estienne M.J., Harper A.F., Barb C.R., Azain M.J. Con-
centrations of leptin in serum and milk collected from lac-
tating sows differing in body composition. Domest. Anim.
Endocrinol. 2000, 19: 275-280.
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