INHIBITORS OF ANGIOTENSIN-I CONVERTING ENZYME (ACEI)

Figure 1. Change in diastolic blood pressure (DBP) after oral administration of 20 g casein hydrolysate per day for 4 weeks in mildly hypertensive volunteers. Each point represents the mean of 18 subjects and vertical bars show SEM. ** and ***: significantly different from ‘0 week’ with P<0.01 and P<0.001, respectively.12

Angiotensin-I converting enzyme (ACE) is a key enzyme involved in the regulation of blood pressure. Due to its activity two amino acids are removed from angiotensin-I, yielding the octapeptide angiotensin-II, which stimulates vasoconstriction and renal retention of water and salts. Inhibition of the synthesis of angiotensin-II thus lowers blood pressure. Various casein derived peptides have been identified and IC50 values (concentration leading to 50% inhibition) have been reported using in vitro assays. Tryptic hydrolysates of casein were effective when administered intravenously, intraperitoneally and orally in spontaneously hypertensive rats¹¹. The latter suggests that these peptides can pass the intestinal tract and after absorption inhibit the production of angiotensin-II in the blood. In mildly hypertensive volunteers (n=18 ; average diastolic blood pressure = 99 mm Hg ; average systolic pressure = 141 mm Hg) a 4 weeks daily administration of 20g casein hydrolysate resulted in reductions of systolic (SBP) and diastolic (DBP) blood pressure of 4.6 and 6.6mm Hg, respectively. After cessation of the intake of the hydrolysate, blood pressure tended to return to pretreatment values again (Figure 1). A recently completed placebo-controlled study at the University of Pennsylvania with casein hydrolysates further enriched in the active peptides, showed a dose-response effect on both DBP and SBP with 1.7 and 3.4g of hydrolysate (manuscript in preparation). Blood pressure is widely recognised as an established risk factor for cardiovascular disease. In western countries, the prevalence of hypertension is estimated to be in one in four adults. In the USA alone more than 50 million adults suffer from mild to severe hypertension. Thus it seems that casein derived antihypertensive peptides have a commercial outlook for both nutritional supplements and functional foods.

WHEY PROTEINS AND CYSTEINE

Studies with rats have shown that dairy proteins may offer protection to the host against carcinogens.¹³ The whey proteins were particularly effective. The mechanism of the protective action was not elucidated, but in the large intestine a positive correlation was found between faecal fat concentration and the tumours per group, suggesting that free fatty acids and bile acids might be involved in hyper-proliferation of colonic epithelial cells.¹⁴ Also the liver concentrations of glutathione (GSH), a natural antioxidant, were higher in the dairy groups, which should be attributed to the higher amounts of the sulphur containing amino acid cysteine, the rate-limiting amino acid for the GSH biosynthesis.¹⁵ Clinical evidence shows that GSH depletion is predictive of poor survival in HIV patients and cysteine supplementation is useful in combination with disease-specific treatments and detoxification of xenobiotics, chemical agents entering the human body and compromising cellular integrity^16,17,18. Liver GSH plays a pivotal role in preventing or restoring these unfavourable health conditions.

Whey protein mixtures like concentrates (WPC) or isolates (WPI) typically contain about 2.4-2.5% of cysteine, whereas extensively purified whey proteins like -lactalbumin and bovine serum albumin may contain up to 6% of cysteine on protein.² In order to allow easier formulation of products with doses of cysteine known to enhance liver glutathione (3 grams of product / per day), whey derived cysteine enriched hydrolysates with about 7% of cysteine on protein have been developed for liver health.

MILK GROWTH FACTORS: TRANSFORMING GROWTH FACTOR β2

Growth factors are proteins or peptides involved in cell-to-cell communication. They act by binding to specific receptors on the cell surface. In general, every growth factor has its specific type of receptor. By binding to the receptor it triggers a complex series of intracellular signals, often involving an array of different proteins. Endpoints of growth factor induced events may be increased or decreased cell growth, cell differentiation, activation of white blood cells, production of other growth factors, or even cell death. Growth factors may act locally or at a remote location, after transport through the systemic circulation.

Milk contains more than 50 growth factors and hormones. Their concentrations in milk (< 0.001 g/l) are much lower than those of e.g. immunoglobulins (0.8 g/l) or lactoferrin (0.02-0.2 g/l), yet their concentration needed for activity is in the order of μg or even ng.

Figure 2. Proliferation of NRK cells (DNA label incorporation) in function of TGF in various fraction (AST34-44)

The bovine growth factors have received interest since Howarth et al¹⁹ showed that oral administration of a growth factor extract from cheese whey was able to reduce small bowel damage in methotrexate treated rats. Methotrexate is a drug used to treat tumours in cancer patients. Cell and animal studies have subsequently shown that transforming growth factor β2 (TGFβ2) is likely one of the factors responsible for the protective effect. This is due to its ability to arrest healthy gut cells in their growth cycle while tumour cells are not affected and consequently are damaged by the chemo- or radio-therapy^.20,21,22 Thus side-effects of treatment, like nausea, vomiting and sores, may be reduced and compliance to treatment increased. Human studies are underway. The sequence of bovine transforming growth factor β2 (TGFβ2) is 100% identical to its human counterpart. In the milk it is bound to a latent binding protein. After acid activation a biologically active TGFβ2 dimer of 224 amino acids (about 26.000 Daltons) is released.

It is feasible to enrich these growth factors to levels of about 100-2000 g per gram protein for industrial use. ELISA and growth factor specific bioassays are used to follow specific activity during purification. Figure 2 shows an example of such a bioassay, using normal rat kidney (NRK) cells, with various fractions obtained during purifications aiming at enrichment for TGFβ2 compared to a raw material.

REFERENCES

1. European Journal of Nutrition 42, Suppl 1, 2003, PASSCLAIM - Process for the assessment of scientific support for cliams on foods.
2. Schaafsma GJ & Steijns JM (2000), Dairy Ingredients as a source of functional foods, In: Essentials of Functional Foods (eds. Schmiddle MK & Labuza TP), ASPEN Publishers, Maryland, 181-204.
3. Schlimme, E and Meisel, H (1995), Bioactive peptides derived from milk proteins. Structural, physiological and analytical aspects. Die Nahrung 39 (1), 1-20.
4. Steijns JM (2001) , Milk ingredients as nutraceuticals. International Journal of Dairy Technology 54 (3), 81-88.
5. Bostwick EF, Steijns JM & Braun S (2000), Lactoglobulins. In Natural Food Antimicrobial Systems, pp 133-158, (ed. AS Naidu) CRC Press, Boca Raton.
6. Steijns JM & Hooijdonk v ACM (2000), Occurrence, structure, biochemical properties and technological characteristics of lactoferrin. British Journal of Nutrition 84 Suppl. 1, S1-S8.
7. Waard de R & Belzen v. N (2003), The anticarcinogenic potential of lactoferrin. AGROFood industry hi-tech, March-April issue, 35-39.
8. Wit de JN & Hooydonk ACM (1996), Structure, functions and applications of lactoperoxidase in natural antimicrobial systems. Netherlands Milk & Dairy Journal 50, 227-244.
9. Evans ME, Brown JM & McIntosh MK (2002), Isomer-specific effects of conjugated linoleic acid (CLA) on adiposity and lipid metabolism. Journal of Nutritional Biochemistry 13, 508-517.
10. Merrill AH, Schmelz E-M, Cameron Sullards M. & Dillehay DL (2001), Shingolipids: novel inhibitors of colon carcinogenesis. Bulletin of the IDF 363, 27-29.
11. Karaki H , Doi K, Sugano S, Uchiwa H, Sugai R, Murakami U & Takemoto S (1990) Antihypertensive effect of tryptic hydrolysate of milk casein in spontaneously hypertensive rats. Comparative Biochemistry and Physiology 96C, 367-371.
12. Sugai R (1998) ACE inhibitors and functional foods. Bulletin of the IDF 336,17-20.
13. McIntosh GH , Regester GO , Le Leu RK, Royle PJ & Smithers GW (1995), Dairy proteins protect against dimethylhy-drazine-induced intestinal cancers in rats. Journal of Nutrition 125, 809-816.
14. Meer v.d. R, Govers MJAP, Lapr&eacute; JA & Kleibeuker JH (1994) , Dietary calcium as a possible anti-promotor of colorectal carcinogenesis. In: Dairy products in human health and nutrition (eds. Serrano Rios et al), Balkema, Rotterdam, 439-448.
15. Sen CK (1997), Nutritional biochemistry of cellular glutathione. Journal of Nutritional Biochemistry 8, 660-672.
16. Friedman M (1994), Improvement in the safety of foods by SH-containing amino acids and peptides: a review. Journal Agricultural Food Chemistry 42, 3-20.
17. Dr&ouml;ge W & Holm E (1997), Role of cysteine and glutathione in HIV infection and other diseases associated with muscle wasting and immunological dysfunction. FASEB Journal 11, 1077-1089.
18. Herzenberg LA, De Rosa SC, Dubs JG, Roederer M , Anderson MT , Ela SW, Deresinski SC & Herzenberg LA (1997), Glutathione deficiency is associated with impaired survival in HIV disease. Proceedings National Academy of Sciences USA 94, 1967-1972.
19. Howarth GS , Francis GF , Cool JC, Xu X, Byard RW & Read LC (1996), Milk growth factors enriched from cheese whey ameliorate intestinal damage by methotrexate when administered orally to rats. Journal of Nutrition 126, 2519-2530.
20. Playford R, Macdonald CE & Johnson WS (2000), Colostrum and milk-derived peptide growth factors for the treatment of gastrointestinal disorders. American Journal Clinical Nutrition 72, 5-14.
21. Taylor, VL, Goddard C & Read LC (2001), A milk growth factor extract reduces chemotherapeutic drug toxicity in epithelial cells in vitro. In Vitro Cellular Developmental Biology-Animal 37, 310-318.
22. Land v. &rsquo;t B, Meijer HP, Frerichs J, Koetsier M, Jager D, Smeets RL, M&rsquo;Rabet L & Hoijer M (2002), Transforming growth factor β2 protects the small intestine during methotrexate treatment in rats possibly by reducing stem cell cycling. British Journal of Cancer 87, 113-118

BIOGRAPHY

Dr. Jan an Steijns studied biology at Nijmegen university and obtained his PhD at Utrecht University after studying flavonoid biosynthesis in plants. Further employment included development of self-instruction tools for biochemistry classes of laboratory workers and product development on rapid techniques for industrial microbiology. Since 1990 Dr. Steijns has worked with DMV International in several R&D positions, including product analysis, product development, technical sales service and external research. From 2000 onwards Dr. Steijns is responsible for the Center of Expertise Nutrition, located in Wageningen and serving both DMV International and the Campina consumer divisions on nutrition consultancy, concept development, clinical and external research.