Piotr P Karwasz

Jordan-Hölder sequences and self-adjoint Ôa, bÕ-modules

Keywords: Mathematics Subject Classification (2010): 32S25, 32S40, 32S50

Given the lack of uniqueness of the Jordan-Hölder composition series in the theory of Ôa, bÕ-modules we are interested whether the particularities of certain Ôa, bÕ-modules can be transmitted to their composition series. This article will focus on the properties of Jordan-Hölder composition series of self-adjoint Ôa, bÕ-modules. In particular we will prove that a self-adjoint composition series always exists for self-adjoint Ôa, bÕ-modules.

Introduction

The Brieskorn module of a germ of holomorphic function f : C n 1 C with an isolated singularity at the origin introduced by E Brieskorn in [START_REF] Brieskorn | Die Monodromie der isolierten Singularitäten von Hyperflächen[END_REF] can be formally completed for the operation b : df d ¡1 . The result is called Ôa, bÕ-module and can be defined in an abstract way as: Definition 1.1. An Ôa, bÕ-module is a free CÖÖb××-module E of finite rank over the ring of formal power series in b, endowed with a C-linear endomorphism 'a' which satisfies:

ab ¡ ba b 2
We recall some basic classification of this object: a sub-Ôa, bÕ-module F of E is a sub-CÖÖb××-module of E stable by 'a' and the Ôa, bÕ-structure passes onto the quotient CÖÖb××-module EßF which satisfies all the properties of an Ôa, bÕ-module except that it has possibly a b-torsion. The sub-Ôa, bÕ-module F is called normal if EßF is free on CÖÖb××.

Since the completion of Brieskorn modules generates regular Ôa, bÕ-modules ([Bar93]), i.e. Ôa, bÕ-modules that can be embedded as a sub-Ôa, bÕ-module into an Ôa, bÕ-module E satisfying aE bE, we'll limit our inquiries to this subclass of objects.

All regular Ôa, bÕ-modules of rank 1 are generated by an element e λ satisfying ae λ λbe λ for a complex number λ. We will refer to them as elementary Ôa, bÕ-modules of parameter λ and note them with E λ .

¦ Institut Élie Cartan Nancy UMR 7502

Nancy-Université, B.P. 70230, 54506 Vandoeuvre-lès-Nancy, France E-mail: piotr.karwasz@iecn.u-nancy.fr

A basic result shows that a regular Ôa, bÕ-module E admits Jordan-Hölder composition series 0 F 0 F 1 ¤ ¤ ¤ F n E with the F i normal in E and for a regular Ôa, bÕ-module the quotients F i ßF i¡1 are elementary Ôa, bÕ-modules E λ of parameter λ.

The isomorphism classes of such quotients vary from a Jordan-Hölder composition series to another and a quotient E λ may appear as E λ j in another sequence, with j È Z.

At a first approach we studied the behaviour of such sequences under the duality functor (cf. [START_REF] Barlet | Théorie des Ôa, bÕ-modules. II. Extensions[END_REF]): Definition 1.2. Let E be an Ôa, bÕ-module and E 0 the elementary Ôa, bÕ-module of parameter 0, then we may define upon the CÖÖb××-module Hom CÖÖb×× ÔE, E 0 Õ an Ôa, bÕ-module structure given by: Ôa ¤ ϕÕÔxÕ aϕÔxÕ ¡ ϕÔaxÕ This module is called the dual Ôa, bÕ-module and noted E ¦ . However, as it was proven by R. concerning the symmetry of Jordan-Hölder composition series, we will prefer the study of self-adjoint Ôa, bÕ-modules for the greater interest they play in the theory of singularities.

In the context of Ôa, bÕ-modules, the conjugate itself is defined in a way borrowed from the complex vector spaces:

Definition 1.3. Let E be an Ôa, bÕ-module, we call conjugate Ôa, bÕ-module and note it Ȇ, the set E endowed with the Ôa, bÕ-structure given by reversing the signs of both 'a' and 'b':

a ¤ Ȇ v ¡a ¤ E v and b ¤ Ȇ v ¡b ¤ E v.
In particular we call adjoint the conjugate of the dual Ôa, bÕ-module E ¦ and we call self-adjoint an Ôa, bÕ-module E which is isomorphic to Ȇ¦ .

When working with isomorphisms between an Ôa, bÕ-module E and its adjoint Ȇ¦ , it is often useful to look at it as a CÖÖb××-bilinear map between E ¢ Ȇ and E 0 . Such a perspective brought us ( [Kar]) to give the following definition of Ôa, bÕ-bilinear map and Ôa, bÕ-hermitian forms: Definition 1.4. Let E, F and G be Ôa, bÕ-modules and Φ a CÖÖb××-bilinear map between E ¢ F and G. We say that Φ is an Ôa, bÕ-bilinear map (or form if An Ôa, bÕ-bilinear map between E¢F and E 0 is non-degenerate if it induces an isomorphism between E and F ¦ .

G E 0 ) if: aΦÔv,
An Ôa, bÕ-module that admits a non-degenerate hermitian form will be called hermitian.

The hermitian Ôa, bÕ-bilinear forms on an Ôa, bÕ-module E induce, as in the case of complex vector spaces, an isomorphism Φ : E Ȇ¦ which is equal to its image Φ¦ under the adjonction functor.

We should remark that not every self-adjoint Ôa, bÕ-module is hermitian and there are even examples of Ôa, bÕ-modules that only admit an anti-hermitian non-degenerate form. In the general case every regular self-adjoint Ôa, bÕ-module can be decomposed into a hermitian part and an anti-hermitian part (not necessarily in a unique way) ( [Kar]).

Self-adjoint composition series

In view of Belgrade's result we will work with the adjonction functor. All proofs however should be valid with only minor modifications if we work with the duality functor.

Consider a regular Ôa, bÕ-module E of rank n È N and a Jordan-Hölder decomposition of itself:

0 F 0 F 1 . . . F n E.
with F j ßF j¡1 E λj , the elementary Ôa, bÕ-module of parameter λ j . We say that the sequence is self-adjoint if λ n¡j 1 ¡λ j for all 1 j n and the Ôa, bÕ-module F n¡j ßF j is self-adjoint for all 0 j Önß2×.

We shall prove the following theorem for regular hermitian Ôa, bÕ-modules and we will extend it successively to all self-adjoint Ôa, bÕ-modules. Theorem 2.1. Let E be a regular hermitian Ôa, bÕ-module then it has a Jordan-

Hölder sequence 0 F 0 F 1 . . . F n E which is self-adjoint.
Before proving the theorem we shall introduce a couple of lemmas.

Lemma 2.2. Let E be a regular hermitian Ôa, bÕ-module and Φ : E Ȇ¦ a hermitian isomorphism.

If there exists F 1 normal sub-Ôa, bÕ-module isomorphic to E λ such that

ΦÔF 1 ÕÔF 1 Õ 0, then there exists a normal sub-Ôa, bÕ-module F n¡1 of rank n ¡ 1 such that EßF n¡1 F1
¦ and F n¡1 ßF 1 is hermitian.

Proof. Let e λ be the generator of F 1 and H the hermitian form associated to Φ by HÔx, yÕ ΦÔyÕÔxÕ and consider the annihilator of this form under H:

F n¡1 :
Øx È E HÔe λ , xÕ 0Ù. We remark that the condition HÔe λ , e λ Õ 0 gives us F 1 F n¡1 and F n¡1 is normal, because it is the kernel of a morphism.

/ / F 1 / / E / / y i W I B 6 EßF n¡1 O O 1 1 1 / / 0 F n¡1 1 1 ' ' EßF 1 1 1 1 F F | Ð Ô Ø 0 O O / / F n¡1 ßF 1 @ @ q t x } / / 0
Let us consider the following exact sequence:

0 F 1 E EßF 1 0
from which we can pass to the adjoint sequence:

0 ÔEßF 1 Õ ¦ Ȇ¦ π F ¦ 1 0.
Since π is the restriction morphism of forms on E to the sub-Ôa, bÕ-module F 1 , the kernel of π, K : Ker π can be described as follows:

K Øϕ È Ȇ¦ ϕÔF 1 Õ 0Ù.
The adjoint sequence being exact, we can identify from now on ÔEßF 1 Õ ¦ with K, i.e. sub-Ôa, bÕ-module of Ȇ¦ whose elements annihilate F 1 .

If we consider the restriction of the map Φ to F n¡1

Φ Fn¡1 : F n¡1 Ȇ¦
and the fact that by definition ΦÔxÕÔe λ Õ 0 for all x È F n¡1 , we obtain that ΦÔF n¡1 Õ ÔEßF 1 Õ ¦ . On the other side for all ϕ È ÔEßF 1 Õ ¦ the element y Φ ¡1 ÔϕÕ verifies ΦÔyÕÔe λ Õ 0, therefore we have also ÔEßF 1 Õ ¦ ΦÔF n¡1 Õ. It follows that ΦÔF n¡1 Õ ÔEßF 1 Õ ¦ and since Φ is an isomorphism, F n¡1 is isomorphic to its image by Φ: ÔEßF 1 Õ ¦ . Let us look now at the following exact sequence:

0 ÔF n¡1 ßF 1 Õ ÔEßF 1 Õ ÔEßF n¡1 Õ 0 and its adjoint sequence: 0 ÔEßF n¡1 Õ ¦ i ÔEßF 1 Õ ¦ π ÔF n¡1 ßF 1 Õ ¦ 0.
π designates the restriction application on the forms of ÔEßF 1 Õ ¦ . Ker π is thus the forms of ÔEßF 1 Õ ¦ that annihilate ÔF n¡1 ßF 1 Õ ¦ or with the convention of the previous paragraph, the forms of ̦ that annihilate F n¡1 and F 1 F n¡1 :

Ker π Øϕ È Ě¦ s.t. ϕÔF n¡1 Õ 0Ù
We note that the hermitianity of Φ gives us ΦÔe λ ÕÔF n¡1 Õ ΦÔF n¡1 ÕÔe λ Õ 0 and therefore we have ΦÔF 1 Õ Ker π. An easy calculation shows that Ker π is of rank 1. Since ΦÔF 1 Õ is normal, of rank 1 and included into Ker π, they must be equal.

We obtain ÔEßF n¡1 Õ ¦ Ker π F 1 . Now we know that Φ sends F n¡1 onto ÔEßF 1 Õ ¦ and F 1 onto Ker π, so starting with the following exact sequence:

0 Ker π ÔEßF 1 Õ ¦ π ÔF n¡1 ßF 1 Õ ¦ 0
we can obtain another by substituting Ker π with F 1 and ÔEßF 1 Õ ¦ with F n¡1 :

0 F 1 F n¡1 ÔF n¡1 ßF 1 Õ ¦ 0.
or in other terms ÔF n¡1 ßF 1 Õ ¦ ÔF n¡1 ßF 1 Õ. Note that the isomorphism is given by x ΦÔxÕ Fn¡1 and is therefore hermitian. The proof may be summarized by the graph of interwoven exact sequences presented in figure 1.

Remark 2.3. If ae λ λbe λ and 2λ Ê N, then HÔe λ , e λ Õ 0. In fact HÔe λ , e λ Õ È E 0 must satisfy: aHÔe λ , e λ Õ HÔae λ , e λ Õ HÔe λ , ¡ae λ Õ HÔλbe λ , e λ Õ HÔe λ , ¡λbe λ Õ 2λbHÔe λ , e λ Õ which has non-trivial solutions in E 0 only if 2λ È N. The double inversion of signs in the second factor are due to the hermitian nature of the form.

Lemma 2.4. If E is a regular hermitian Ôa, bÕ-module and there exist λ È C such that E contains two distinct normal elementary sub-Ôa, bÕ-modules F and G of parameters f g λ mod Z then there exists F 1 F n¡1 two normal sub-Ôa, bÕ-modules of rank 1 and n ¡ 1 respectively such that ÔEßF n¡1 Õ ¦ F 1 and F n¡1 ßF 1 is hermitian.

Proof. We will denote by H an hermitian form on E. Let e f and e g be generators of F and G and suppose without loss of generality that f ¡g 0. We will show that there exists a normal elementary sub-Ôa, bÕ-module F 1 of E whose generator e È E satisfies HÔe, eÕ 0.

By the fundamental property ab¡ba b 2 of Ôa, bÕ-modules we have ab f ¡g e g Ôg f ¡ gÕb ¤ b f ¡g e g . Let's pose e 1 b f ¡g e g . Consider now the complex vector space:

V : Øαe f βe 1 α, β È CÙ Note that every v È V satisfies av f bv. The b-linearity of H and the definition of the action of a gives us:

Ôa ¡ 2f bÕHÔv, vÕ 0 which has in E 0 only solutions of the form αb 2f e 0 , α È C. There exists therefore an application B from V ¢ V to C such that:

HÔv, wÕ BÔv, wÕb 2f e 0 v, w È V

The bilinearity and hermitianity of H imply that B is in fact a C-bilinear symmetric or anti-symmetric form on a 2 dimensional complex vector space, depending whether 2f is even or odd. In the anti-symmetric case every vector will be isotropic, in the symmetric case we have:

BÔe f xe 1 , e f xe 1 Õ a 0 a 1 x a 2 x 2
for some complex numbers a i . The vector space V has therefore an isotropic vector e 0 such that BÔe, eÕ 0, and hence HÔe, eÕ 0. By eventually dividing e by a certain power of b, operation that does not change the relation HÔe, eÕ 0, we can assume that e Ê bE, hence the module F 1 generated by e is normal.

We can now conclude by applying lemma 2.2

Lemma 2.5. Let E be a regular Ôa, bÕ-module and:

0 . . . F i¡1 F i F i 1 . . . E
be a Jordan-Hölder composition series with F i ßF i¡1 E λi for all i and suppose there is a j such that λ j 1 λ j mod Z.

Then we can find another Jordan-Hölder composition series that differs only in the j-th term F ½ j such that F ½ j ßF j¡1 E λ ½ j and F j 1 ßF ½ j E λ ½ j 1 with λ j λ ½ j 1 mod Z and λ j 1 λ ½ j mod Z, i.e. we can permute the quotients up to an integer shift of the parameters.

Proof. Let consider G : F j 1 ßF j¡1 and the canonical projection π : E EßF j¡1 . G is a rank two module. Using the classification of regular Ôa, bÕ-modules of rank 2 given by D. Barlet in [START_REF] Barlet | Theory of Ôa, bÕ-modules. i[END_REF] we see that the only two possibilities for G are:

G E λj E λj 1 in which case we take F ½ j π ¡1 ÔE λj 1 Õ or G E λj 1 1,λj
generated by y and t satisfying: ay λ j by at λ j 1 bt y that has also another set of generators: t and x : y Ôλ j 1 ¡ λ j 1Õbt which satisfy:

ax Ôλ j 1Õbx at Ôλ j ¡ 1Õbt x.
In this case we take F ½ j π ¡1 Ô x Õ. Lemma 2.6. Let λ be either 0 or 1ß2 and E be a regular hermitian Ôa, bÕ-module. Suppose that there is an unique normal elementary sub-Ôa, bÕ-module of parameter equal to λ modulo Z and suppose moreover that every Jordan-Hölder sequence contains at least 2 elementary quotients of parameter equal to λ modulo Z.

Then there exists F 1 F n¡1 two normal sub-Ôa, bÕ-modules of rank 1 and n ¡ 1 respectively such that ÔEßF n¡1 Õ ¦ F 1 and F n¡1 ßF 1 is hermitian.

Proof. Let F 1 E µ be the elementary sub-Ôa, bÕ-module the hypothesis and ØF i Ù a J-H sequence beginning with F 1 and such that EßF n¡1 is of parameter µ ½ equal to λ mod Z. We can find such a sequence by using repeatedly the previous lemma. Consider the exact sequence:

0 F n¡1 E ÔEßF n¡1 Õ 0
and the adjoint sequence:

0 ÔEßF n¡1 Õ ¦ i ̦ π F ¦ n¡1 0.
The image of i is a normal elementary sub-Ôa, bÕ-module of ̦ of parameter equal to ¡λ mod Z (since ÔEßF n¡1 Õ ¦ E ¡µ ½). But λ ¡λ mod Z and E ̦ so by the uniqueness of F 1 given in the hypothesis Im

¡ ÔEßF n¡1 Õ ¦ © ΦÔF 1 Õ, thus ÔEßF n¡1 Õ ¦ F 1 .
By replacing ̦ by E and ÔEßF n¡1 Õ ¦ by F 1 in the sequence we obtain:

0 F 1 i E F ¦ n¡1 0
which is exact and i is the inclusion of sub-Ôa, bÕ-modules, so F ¦ n¡1 ÔEßF 1 Õ or equivalently F n¡1 ÔEßF 1 Õ ¦ . Note that the first isomorphism is given by Φ ¡1 , while the second by the restriction of Φ.

Consider the following sequence and its adjoint: 0

F n¡1 ßF 1 EßF 1 EßF n¡1 0 0 ÔEßF n¡1 Õ ¦ ÔEßF 1 Õ ¦ ÔF n¡1 ßF 1 Õ ¦ 0 by replacing ÔEßF n¡1 Õ ¦ and ÔEßF 1 Õ ¦ with F 1 and F n¡1 we obtain: 0 F 1 ϕ F n¡1 π ÔF n¡1 ßF 1 Õ ¦ 0
for the uniqueness of F 1 , ϕ can only be (up to multiplication by a complex number) the inclusion F 1 F n¡1 and hence ÔF n¡1 ßF 1 Õ ¦ ÔF n¡1 ßF 1 Õ. Note that π is the restriction of Φ to F n¡1 , so the isomorphism is hermitian.

We can now prove the theorem.

Proof of theorem 2.1. We will prove the theorem by induction on the rank of the Ôa, bÕ-module. For rank 0 and 1 the theorem is obvious.

Suppose we proved the theorem for every rank n and let's prove it for rank n. Let find F 1 F n¡1 of rank 1 and n ¡ 1 such that ÔEßF n¡1 Õ ¦ F 1 and F n¡1 ßF 1 is hermitian. We can have different cases which are exhaustive:

(i) We can find G, a normal elementary sub-Ôa, bÕ-module of E of parameter λ not equal to 0 or 1ß2 mod Z. Then ΦÔGÕÔGÕ 0 by remark 2.3 and we can apply lemma 2.2.

We still need to prove the induction step for Ôa, bÕ-modules whose only normal elementary sub-Ôa, bÕ-modules have parameter λ 0 or λ 1ß2 modulo Z.

(ii) For λ 0 or λ 1ß2 there are two distinct normal elementary sub-Ôa, bÕ-modules of parameter equal to λ mod Z. We apply lemma 2.4. The Ôa, bÕ-modules that were not included in the previous points have an unique normal elementary sub-Ôa, bÕ-module of parameter equal to 1ß2 modulo Z and an unique normal elementary sub-Ôa, bÕ-module with an integer value of the parameter.

(iii) There is only one normal elementary sub-Ôa, bÕ-module of parameter equal to λ mod Z, where λ 0 or 1ß2, but at least two quotients of a J-H sequence are of parameter equal to λ mod Z. We apply lemma 2.6.

Only modules of rank at most 2 (one for each possible value of λ) still need to be checked.

(iv) The rank of E is 2 and one quotient of a J-H sequence is equal to 0 mod Z, the other equal to 1ß2 mod Z. By the classification of rank 2 modules this case is impossible. In fact with the notations of [START_REF] Barlet | Theory of Ôa, bÕ-modules. i[END_REF]:

ÔE λ E µ Õ ¦ E ¡λ E ¡µ ̦ λ,µ E 1¡λ,1¡µ
so if λ 0 mod Z and µ 1ß2 mod Z the Ôa, bÕ-module is not self-adjoint. By induction hypothesis F n¡1 ßF 1 has a J-H composition series that verifies the theorem and by taking the inverse image by the canonical morphism F n¡1 F n¡1 ßF 1 and adding 0 and E we find a J-H sequence of E that satisfies the theorem.

Since for an anti-hermitian form A we have AÔe, eÕ 0 for every e È E, by using an anti-hermitian version of lemma 2.2 alone and proceeding by induction, we can prove theorem 2.1 in the anti-hermitian case.

We wish now to extend the result to all regular self-adjoint Ôa, bÕ-modules. We have proven in [Kar] that every regular Ôa, bÕ-module E can be decomposed into a direct sum of hermitian or anti-hermitian Ôa, bÕ-modules. We can hence prove the following theorem: Theorem 2.7. Let E be a self-adjoint regular Ôa, bÕ-module. Then it admits a self-adjoint Jordan-Hölder composition series.

Proof. Let decompose E into E m i 1 H i
where m is an integer, while the H i are either indecomposable self-adjoint or of the form G Ǧ¦ , where G is indecomposable non self-adjoint Ôa, bÕ-module.

Each term of this sum admits a self-adjoint composition series. In fact if H i is indecomposable self-adjoint, then it is hermitian or anti-hermitian. We can therefore apply the previous theorem 2.1.

On the other hand if H i is the sum G Ǧ¦ of a module and its adjoint, we can easily find a self-adjoint Jordan-Hölder composition series. Take in fact any Jordan-Hölder series of G, Then the following composition series of G Ǧ¦ is self-adjoint:

0 G 0 ¤ ¤ ¤ G n G.
0 G 0 G 1 . . . G G ÔGßG n Õ ¦ G ÔGßG n¡1 Õ ¦ ¤ ¤ ¤ G ÔGßG 0 Õ ¦ G Ǧ¦ .
We will now prove the theorem on induction on m. The case m 1 was already proven. Suppose now m 2 and let E ½ : H 1 and F : m i 2 H i . We have therefore E E ½ F , and E ½ and F are both self-adjoint. By the remark above we can find a self-adjoint composition series of E ½ :

0 E ½ 0 ¤ ¤ ¤ E ½ r E ½
while by induction hypothesis, we can find a self-adjoint composition series of F :

0 F 0 ¤ ¤ ¤ F s F.
Then the following composition series is self-adjoint:

0 E ½ 0 E ½ 1 ¤ ¤ ¤ E ½ Örß2× E ½ Örß2× F 1 ¤ ¤ ¤ E ½ Örß2× F Ösß2× Ö¤ ¤ ¤× E ½ ÖÔr 1Õß2× F ÖÔs 1Õß2× E ½ ÖÔr 1Õß2× F ÖÔs 1Õß2× 1 ¤ ¤ ¤ E ½ ÖÔr 1Õß2× F E ½ ÖÔr 1Õß2× 1 F ¤ ¤ ¤ E ½ F,
where depending on the parity of r and s, Ö¤ ¤ ¤× stands for (i) the sign if r and s are both even.

(ii) the sign if one is even and the other odd. Ôa, bÕ-modules of rank 1. They are therefore isomorphic to E 0 .

Figure 1 :

 1 Figure 1: Modules in symmetric positions with respect to the dotted line are each other's adjoint.

and consider the adjoint series 0

 0 ÔGßG n Õ ¦ ÔGßG n¡1 Õ ¦ . . . ÔGßG 0 Õ ¦ Ǧ¦ .

(

  iii) the subsequence E ½ Örß2× F ÖÔs 1Õß2× This case needs a short verification. If r and s are odd, then the two central quotients of the series are isomorphic to E ½ ÖÔr 1Õß2× ßE ½ Örß2× and F ÖÔs 1Õß2× ßF Ösß2× . Since E ½ i and F i are self-adjoint series both quotients are self-adjoint

  wÕ ΦÔav, wÕ ΦÔv, awÕ for every v È E and w È F .An Ôa, bÕ-bilinear map between E ¢ Ȇ and E 0 is called an Ôa, bÕ-hermitian È E and SÔbÕ È CÖÖb××. Ôa, bÕ-anti-hermitian forms satisfy the same equation with SÔ¡bÕ replaced by ¡SÔ¡bÕ.

	form if moreover it satisfies
	ΦÔv, wÕ SÔbÕe 0	ΦÔw, vÕ SÔ¡bÕe 0
	with v, w


Juan Antonio

Hernandez Bort

Beate Stern

Nicole Borth
email: nicole.borth@boku.ac.at

Juan A Hernández Bort

CHO-K1

Keywords: Mammalian Biotechnology Secondary Metabolomics cell sorting, glutamine, host cell optimisation glutamine, ammonia, CHO-K1 cells, cell sorting, assisted evolution, glutamine-free medium

teaching and research institutions in France or abroad, or from public or private research centers.

INTRODUCTION

Mammalian cells, and especially Chinese hamster ovary (CHO) cells, are widely used in biomedical research and the pharmaceutical industry for production of recombinant proteins.

Thus, the improvement of cellular properties to increase productivity and product quality is of major interest.

During the last decades many studies were carried out in order to improve growth media.

Glucose and glutamine concentrations are usually overdosed in mammalian cell culture media to achieve high growth rates and biomass concentration. However, such high concentrations lead to higher consumption rates of these components [START_REF] Butler | The effects of glutamine utilisation and ammonia production on the growth of BHK cells in microcarrier cultures[END_REF][START_REF] Miller | A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH[END_REF][START_REF] Glacken | Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells[END_REF]. On the one hand, entry of glucose into the glycolytic pathway leads to pyruvate formation, which can either be shuttled into the tricarboxylic acid (TCA) cycle or converted into lactate. Due to the high flux of glucose to pyruvate and the inefficient coupling between glycolysis and TCA cycle, lactate accumulates during cultures, which has a negative impact in cells [START_REF] Glacken | Reduction of waste product excretion via nutrient control: Possible strategies for maximizing product and cell yields on serum in cultures of mammalian cells[END_REF][START_REF] Hassell | Growth inhibition in animal cell culture. The effect of lactate and ammonia[END_REF][START_REF] Ozturk | Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production[END_REF]. On the other hand, glutamine is the amino acid consumed with the highest rate and its importance for an optimal growth in cell culture has been known since the 50s [START_REF] Eagle | Nutrition needs of mammalian cells in tissue culture[END_REF]. Glutamine is an alternative energy source to glucose for rapidly dividing cells, as it is mostly metabolized via a truncated form of the TCA cycle [START_REF] Aledo | Glutamine breakdown in rapidly dividing cells: waste or investment?[END_REF]. Feeding of excess glutamine induces a high ammonium production in cell cultures [START_REF] Butler | The effects of glutamine utilisation and ammonia production on the growth of BHK cells in microcarrier cultures[END_REF][START_REF] Ljunggren | Glutamine limited fed-batch culture reduces the overflow metabolism of amino acids in myeloma cells[END_REF], which reduces viable cell density and productivity [START_REF] Hassell | Growth inhibition in animal cell culture. The effect of lactate and ammonia[END_REF][START_REF] Hansen | Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture[END_REF][START_REF] Cruz | Effects of ammonia and lactate on growth, metabolism, and productivity of BHK cells[END_REF][START_REF] Gawlitzek | Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells[END_REF] and alters glycoforms of proteins [START_REF] Andersen | The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by chinese hamster ovary cells[END_REF][START_REF] Borys | Ammonia affects the glycosylation patterns of recombinant mouse placental lactogen-I by chinese hamster ovary cells in a pH-dependent manner[END_REF][START_REF] Valley | Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins[END_REF][START_REF] Grammatikos | Intracellular UDP-Nacetylhexosamine pool affects N-glycan complexity: a mechanism of ammonium action on protein glycosylation[END_REF]. Moreover, it was also described that ammonia concentrations of 10mM will prevent growth of fibroblasts [START_REF] Visek | Ammonia effects in cultures of normal and transformed 3T3 cells[END_REF]. Although ammonia is derived mainly from deamidation of glutamine [START_REF] Eagle | Nutrition needs of mammalian cells in tissue culture[END_REF], the catabolism of the other amino acids present in the cultivation medium is also responsible for its overproduction [START_REF] Mclimans | Ammonia loading in cell culture systems[END_REF]. When ammonium diffuses across the cell membrane, intracellular pH (pH i ) tends to decrease and electrochemical gradients are perturbed [START_REF] Schneider | The importance of ammonia in mammalian cell culture[END_REF][START_REF] Martinelle | Mechanisms of ammonia and ammonium ion toxicity in animal cells: transport across cell membranes[END_REF]. To overcome this problem, glutamine has been substituted by other less ammoniagenic carbon substrates like glutamate or asparagine F o r P e e r R e v i e w [START_REF] Christie | The adaptation of BHK cells to a non-ammoniagenic glutamatebased culture medium[END_REF][START_REF] Huang | Improvement of Vero cell growth in glutamate-based culture by supplementing ammoniagenic compounds[END_REF]. In addition, sophisticated methods, including microporous hollow fiber modules or media filtration with cation exchangers, have been developed for waste product reduction in culture supernatants, in order to maximize the final product concentration and yield [START_REF] Capiaumont | Methods for reducing the ammonia in hybridoma cell cultures[END_REF][START_REF] Bibila | In pursuit of the optimal fed-batch process for monoclonal antibody production[END_REF][START_REF] Chen | Engineering of a mammalian cell line for reduction of lactate formation and high monoclonal antibody production[END_REF].

Substitution of glucose and glutamine by other energy substrates successfully reduced lactate and ammonia, respectively, but resulted in considerably decreased viable cell density [START_REF] Altamirano | Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine[END_REF].

However, it was also described that control of glutamine at low levels can improve cell densities [START_REF] Lee | Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production[END_REF].

For several years now, growth media for CHO cells have been optimised to a degree that allows growth in protein-free media [START_REF] Sinacore | Adaptation of mammalian cells to growth in serum-free media[END_REF][START_REF] Schneider | Optimisation of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum-and protein-free culture medium[END_REF][START_REF] Cleveland | Routine large-scale production of monoclonal antibodies in a protein-free culture medium[END_REF][START_REF] Schröder | Serum-and protein-free media formulations for the Chinese hamster ovary cell line DUKXB11[END_REF][START_REF] Keen | Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line[END_REF]. In addition, cells have even been subjected to "Bioreactor evolution" [START_REF] Prentice | Improving performance of mammalian cells in fedbatch processes through "bioreactor evolution[END_REF] to improve their process relevant properties. All in all, increases in volumetric productivity during the last 10 years were mainly achieved by increases in the integral viable cell density (IVCD), by optimising process design and feeding strategies [START_REF] Durocher | Expression systems for therapeutic glycoprotein production[END_REF][START_REF] Khattak | Feed development for fedbatch CHO production process by semisteady state analysis[END_REF][START_REF] Xie | Integrated approaches to the design of media and feeding strategies for fed-batch cultures of animal cells[END_REF]. Additional improvements were obtained by prolonging cell survival at the end of the culture by decreasing cell sensitivity to apoptotic stimuli. This strategy was tried by expression of anti-apoptotic genes in monoclonal antibody producing CHO cells [START_REF] Figueroa | Enhanced cell culture performance using inducible anti-apoptotic genes E1B-19K and Aven in the production of a monoclonal antibody with Chinese hamster ovary cells[END_REF][START_REF] Mastrangelo | Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults[END_REF][START_REF] Meents | Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-x(L) on productivity, cell survival, and mitochondria number in CHO-DG44 grown in suspension and serum-free media[END_REF][START_REF] Astley | The role of Bcl-2 and its combined effect with p21CIP1 in adaptation of CHO cells to suspension and protein-free culture[END_REF].

In an attempt to obtain an optimised host cell line to be used for recombinant biologic production, we explored the possibility to achieve comparable growth and viability in a cell line independent of glutamine in the culture medium. In the present report we present a new approach to easily and rapidly evolve cells towards improved growth properties using cell sorting assisted evolution. Cells surviving a progressive stress treatment were sorted out of the parent population based on their cellular state and viability and typically showed enhanced robustness, a prolonged life span and higher viable cell density with concomitant improvement in IVCD.

MATERIALS AND METHODS

Cell line and media

The original CHO-K1 adherent cell line obtained from the American Type Culture Collection (ATCC) was adapted within three weeks to growth in suspension in CD CHO, a chemically defined, serum-free medium (Gibco, Invitrogen, Carlsbad, CA, USA) and supplemented with 8mM L-glutamine. Studies were carried out in spinner flasks with a working volume of 100ml at 37°C in a humidified atmosphere containing 7% carbon dioxide and with constant stirring at 50rpm.

Progressive reduction of glutamine and Assisted Evolution

Cells were seeded in a concentration of 10 7 cells/ml incubated at 37°C and surviving cells were sorted out with either Fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Separation (MACS) when viability declined to 10% after 20-24 hours. Surviving cells were expanded and seeded into a spinner flask with a starting concentration of 1x10 5 cells/ml. The batch was monitored until viability dropped below 10%. Surviving cells were sorted out again and transferred into medium with reduced glutamine concentration. This procedure (Fig. 1) was repeated reducing glutamine supplement into culture medium after each completed cycle from 8mM to 4mM, and from 4mM to 2mM. Finally, cells adapted to 2mM glutamine were transferred into culture medium without glutamine supplementation.

Viability and cell concentration

Cell viability was estimated with a Neubauer hemocytometer using the trypan blue exclusion method. Total cell number was determined by counting cell nuclei with a Multisizer™ 3 Coulter Counter (Beckman Coulter, Fullerton, CA, USA) after lysis of the cell membrane by incubation of samples for two hours in 0.1M citric acid containing 2% Triton X-100.

Cell sorting

Viable cells were sorted out from cell culture bulk using either a FACS Vantage™ (Becton Dickinson, Franklin Lakes, NJ, USA), equipped with a Sort Enhancement Module, or a Dead Cell Removal kit in combination with a MACS column (Miltenyi Ltd, Germany). MACS protocol was performed according to supplier instructions. For FACS, a combination of a SSC-H / FSC-H gate and a gate for cells that exclude DAPI (4,6-diamidino-2-phenylindole) was used. DAPI is taken up and stains DNA only of damaged or dead cells. The intact sorted cells were deposited into a 6-well plate containing 3ml growth medium pre-incubated at 37°C. Both cell sorting systems were tested and found to give similar results, with the MACS protocol faster and easier to handle. The FACS protocol is more time consuming, but due to its multiparameter character allows a finer discrimination between healthy cells and cells, that although still with an integral membrane, are already compromised.

Analysis of glucose, lactate and ammonia.

Concentration of glucose and lactate in the cell culture supernatant were determined using a YSI 7100 MBS Bioanalytical System (YSI Life Sciences, USA). Ammonia was measured by photometer with an Ammonia Assay Kit (Megazyme, Ireland) according to supplier´s instructions.

Amino acid analysis

A HPLC Agilent 1200 Serie System equipped with a fluorescence detector (Ex:340nm; Em:450nm) was used for amino acids determination of the cell culture supernatant harvested

Plasmid and transient transfection

For transient transfection, 10 7 cells were co-transfected with 5pmol each of two plasmids bearing a human IgG1 heavy chain and lambda light chain under the control of a cmvpromoter. For the calculation of molar plasmid concentrations, the molecular weight average of a nucleotide pair was assumed to be 660 pg/pmol. Only cells with viability greater than 95 % were used for transfection. According to Amaxa AG protocol, cell pellet was resuspended in 100µl pre-mixed Solution kit V + Supplement (both included in Amaxa kit V), mixed with plasmids, and immediately nucleofected with the Amaxa Nucleofector (programm H-14).

Directly after the electroporation procedure, cells were transferred into a culture flask with growth medium pre-warmed to 37°C at a concentration of 2x10 5 cells/ml. Samples were harvested to determine viability and starting cell number. Final cell density and viability were determined 48 hours after transfection. The protocol was previously evaluated to yield transfection efficiencies better than 90 and to have a standard deviation of +/-15% [START_REF] Pichler | Selection of CHO Host Cell Subclones with Increased Specific Antibody Production Rates by Repeated Cycles of Transient Transfection and Cell Sorting[END_REF]

IgG heavy chain determination by ELISA

Microtiter Nunc MaxiSorp were coated with goat-anti-human-IgG-gamma-chain-specific (#I3382; Sigma) diluted 1:1000 in 0.1M sodium bicarbonate buffer (pH=9.6) at 4°C overnight. The plates were washed and then incubated with serial dilutions of a self-purified IgG standard and culture supernatant samples for 1 hour in a dilution buffer containing 1% BSA at room temperature. After a wash step, the bound IgG was incubated with horse radish peroxidase (HRP) conjugated with a goat-anti-human-IgG (#62-8420, Zymed) diluted 1:1000 in dilution buffer for 1 hour. For the detection of the antigen-antibody reaction, 200µl of a 0.4mg/ml o-phenyldiamine dihydrochlorid (OPD) solution was added as substrate after a wash step, and incubated for 5 minutes at room temperature. The OPD reaction was stopped with 50µl of 2M H 2 SO 4 and read at 492nm. Calculation was carried out using a 4 parameter calibration curve with a detection range from 0 to 200ng/ml IgG. Deviation of standard, control and sample replicates was less than 15%.

RESULTS

Extension of culture life span

The rationale for this work arose from the concept that cells submitted to an iterative but not drastic change of milieu conditions should be able to evolve towards a more efficient metabolism and better adaption to the new conditions. When glutamine supplement was reduced in CHO-K1 culture medium without adaptation, both growth rate and viable cell density decreased by approximately 20% (data not shown). This reduction became even worse during further routine passage in medium with the reduced glutamine content for a period of at least 6 weeks. In order to quickly restore cellular growth properties, cells were To achieve this, CHO-K1 cells were seeded at high density (10 7 cells/ml) and incubated at 37°C until viability declined to 10%. This typically occurred after 20-24h. Surviving cells were sorted out with FACS or MACS and expanded in a T flask. This was necessary to remove dead cells and debris, which at high concentrations is toxic and would have killed the few remaining viable cells. In the second part of this approach, a batch with the expanded cells was started by seeding cells at 1x10 5 cells/ml in a spinner flask. The batch was monitored until viability dropped below 10%. Surviving cells were sorted out. The process was repeated, while reducing glutamine supplement after each completed cycle from 8mM to 4mM, and from 4mM to 2mM. Finally, cells adapted to 2mM glutamine were transferred into culture medium without glutamine supplementation and showed the same growth profile as cells supplemented with 2mM glutamine. Thus for the 0mM glutamine adapted cell line no additional cycle in assisted evolution was required. The different adapted cell lines were maintained in the medium with the respective glutamine concentration in routine culture (on total now for over one year, without loss of phenotypic properties, data not shown).

In order to compare changes in cellular properties, batch cultures with these newly established CHO-K1 cell lines were started with the respective L-glutamine concentrations (8mM, 4mM, 2mM and 0mM) in spinner flasks with a working volume of 100ml and an initial concentration of 1x10 5 cells/ml, incubated at 37°C and 7% CO 2 with constant stirring at 50rpm. Results presented are the mean of two independent replicate batch cultures.

The 2mM and 0mM adapted cell line was able to increase the life span by approximately 30% relative to the parental cells and to maintain a viability of greater than 95% for 10 days (Fig. 2). Moreover, the integral of viable cells was increased by 22% for the 2mM culture and by 13% for the 0mM culture (Fig. 3).

Cellular Metabolism

To evaluate the changes in metabolism that occurred upon assisted evolution, the adapted cell lines were cultivated in batch cultures in duplicate and the nutrient consumption of amino acids and glucose, as well as production of lactate (Fig. 4) and ammonium was analysed. The cells adapted to 2mM and 0mM glutamine, although lacking an important energy source, did not increase their glucose uptake rate nor did they shift towards mmore efficient utilization of glucose, as the ratio of lactate produced per glucose consumed remained the same. Instead, these cells compensated for carbon and nitrogen source by increasing the consumption of glutamate, aspartate and to a lesser degree asparagine (Fig. 5). Aspartate is produced in the cultures grown at 8 and 4mM, while it is consumed in those at 2 and 0mM. Uptake of other amino acids was slightly lower, while the production rate of alanine and to a lesser degree glycine was lower in these cultures than in the cultures grown at 8 and 4mM glutamine.

Except for glutamine, no amino acid was completely depleted at the end of batch cultures.

The low alanine secretion and ammonium production (Fig. 6) in culture supernatant by cells adapted to reduced-glutamine media during growth phase agrees with the conclusions of some authors, who reported that excess of ammonium production may be the cause of high alanine excretion after activation of transamination pathways [START_REF] Ozturk | Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production[END_REF][START_REF] Hansen | Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture[END_REF][START_REF] Bonarius | Activity of glutamate dehydrogenase is increased in ammonia-stressed hybridoma cells[END_REF].

Final ammonia concentration in CHO-K1 2mM grown at 0mM glutamine was comparable to CHO-K1 8mM and CHO-K1 4mM (Fig. 6), in spite of the absence of glutamine in growth medium. On the other hand, a reduction in ammonia production was observed during the exponential phase of batches. Comparison between adapted and non-adapted cell lines revealed differences in specific growth rates. As highlighted in Fig. 2, CHO-K1 2mM and CHO-K1 0mM cell lines had a delayed start of exponential phase compared to cells adapted to growth in 8mM and 4mM glutamine. Moreover, 2mM glutamine-adapted cells had a prolonged growth phase with respect to their parents and the viable cell density was increased. This improvement was reflected in the cumulative viable cell density, particularly in the CHO-K1 2mM cell line (Fig. 3).

Growth rate and integral viable cell density

Effects on transient expression of IgG

Previous studies have shown that glutamine reduction in cell culture media reduced protein production [START_REF] Altamirano | Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine[END_REF]. CHO-K1 8mM, 4mM 2mM and 0mM cells were transfected with a total of 10pmol plasmid encoding IgG heavy chain and light chain genes to verify if any immediate effect on the specific productivity could be observed. The IgG product secreted in culture supernatant was quantified by ELISA at day 2 after transfection. Specific productivity reached 48h after transfection was comparable in all cases (Fig. 7), considering the standard deviation of the method, which was +/-15%.

DISCUSSION

Survival of the best adapted organism to a new environment is generally known as natural selection. In an attempt to transfer the Darwinian Theory to an optimisation procedure for CHO-K1 cells, a simple strategy to improve properties of CHO-K1 was developed. Cells were submitted to an iterative and progressive alteration of milieu conditions by bulk selection of best adapted cells. Previous studies have shown that after decreasing the glutamine concentration in growth media to 0mM, cellular metabolism shifted toward glycolysis leading to a higher glucose consumption rate [START_REF] Jeong | Role of glutamine in hybridoma cell culture: Effects on cell growth, antibody production, and cell metabolism[END_REF]. Because glutamine is an important source for cellular glutamate, its reduction could act as a rate-limiting step in the synthesis of gluthatione (GSH), a tripeptide consisting of glutamate, glycine, and cysteine, that serves to protect cells from oxidative stress [START_REF] Matés | Glutamine and its relationship with intracellular redox status, oxidative stress and cell proliferation/death[END_REF][START_REF] Roth | Regulative potential of glutamine-relation to glutathione metabolism[END_REF]. However, the specific glucose uptake and lactate production rates in the cell lines adapted to lower glutamine concentrations were not significantly changed, nor was the ratio of lactate produced per glucose consumed. The only difference was that in the culture grown without glutamine the cells were able to consume glucose down to a concentration of less than 200mg/l (Fig. 4).

A comparison of ammonia production (Fig. 6) revealed an initially dose dependent reduction in ammonia production with decreasing glutamine concentrations during exponential growth.

On day 8 and 12 the ammonia concentrations were however comparable due to increased uptake of other amino acids in the cells grown at reduced glutamine concentrations. This result contradicts the intention of reducing glutamine to improve the glycosylation properties of produced proteins at later stages of the production process. In previous work on reducing or replacing glutamine [START_REF] Christie | The adaptation of BHK cells to a non-ammoniagenic glutamatebased culture medium[END_REF][START_REF] Bibila | In pursuit of the optimal fed-batch process for monoclonal antibody production[END_REF] the actual ammonia concentration was never tested. Nevertheless, by weening the cells of glutamine, we have achieved an increase in the integral of viable cell days (IVCD) of 20%, this in spite of the prolonged lag during in early batch culture. The IVCD is the main determining factor for total product yield and thus economic process output. The next step in determining the usefulness of this new host cell line, will be to repeat the process with a producer cell line and verify effects on total high level productivity as well as on product quality.

  , including day 0. A volume of 30µl sample mixed with 20µl orthophthalaldehyde (Pierce), was injected on a reversed phase Hypersil ODS column (3µm particle size, 4.0x100mm) thermostated at 25°C, with the following linear gradient of sodium acetate 50mM, pH 5.70 (solvent A) and acetonitrile (solvent B): the concentration of 6% B was kept constant for 2 min., then the concentration of B was increased linearly from 2 to 12 min. to 9% B, from 12 to 18 min. to 19% B, from 18 to 26 min. to 22% B, from 26 to 38 min to 42% B and from 38 to 40 min to 100% . Column equilibrium to original conditions was from 40 to 50 min to 6% B. A flow rate of 1.5ml/min. was used throughout. Peaks were identified by retention time and concentrations determined relative to 3-(2-thienyl)-DLalanine (Fluka), an internal standard added to each sample. Calibration range was from 1mg/l to 25mg/l.

  iterative process of selection for the best adapted cells in reduced-glutamine culture medium.

CONCLUSION

  We presented a new approach to easily and rapidly evolve cells towards improved growth properties using a combination of Assisted Evolution Approach and cell sorting. Cells surviving a progressive stress treatment were sorted out of the improved parent population and typically showed improved robustness, a prolonged life span and enhanced viable cell density. The whole procedure of adapting cells from 8mM glutamine containing medium to 0mM glutamine took less than 10 weeks.

Figure 1 Assisted

 1 Figure 1

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3

Figure 4 (

 4 Figure 4

Figure 5

 5 Figure 5

Figure 6 Final

 6 Figure 6

Figure 7

 7 Figure 7

Wiley-VCH Biotechnology Journal

ACKNOWLEDGMENTS

The authors are grateful to Dominik Jeschek and Gabriele Lhota for amino acid analysis with reverse-phase HPLC.


Markus Schantz

Thomas Erk

Prof. Dr Elke Richling
email: richling@chemie.uni-kl.de

Metabolism of green tea catechins in the human small intestine

Keywords: Chromatography green tea, small intestine, metabolism green tea, small intestine, metabolism, catechins, gallic acid

published or not. The documents may come

Tea is the second most frequently consumed beverage globally, with a consumption of approximately 43.8 L per year [START_REF] Graham | Green tea composition, consumption, and polyphenol chemistry[END_REF][START_REF] Mckay | The role of tea in human health: an update[END_REF]. It is prepared from the dried leaves of Camellia sinensis in non-fermented (green tea), semi-fermented (oolong tea) or fermented (black tea) forms.

Green teas, which account for ~20% of global consumption [START_REF] Ahmad | Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells[END_REF], have well-established beneficial health effects, which are attributed to their polyphenolic contents, particularly contents of a subclass of flavanols the catechins [START_REF] Graham | Green tea composition, consumption, and polyphenol chemistry[END_REF]. Catechins are present in higher quantities in green tea than in other types of tea since they are degraded in the manufacturing processes involved in the production of black and oolong teas [START_REF] Lin | Factors affecting the levels of tea polyphenols and caffeine in tea leaves[END_REF].

In recent decades there has been intense interest in the beneficial health effects of green tea polyphenols. A number of studies have shown them to have antioxidant [START_REF] Shahrzad | Determination of gallic acid and its metabolites in human plasma and urine by high-performance liquid chromatography[END_REF], antibacterial [START_REF] Stapleton | Modulation of beta-lactam resistance in Staphylococcus aureus by catechins and gallates[END_REF] and anti-inflammatory [START_REF] Lambert | Synthesis and biological activity of the tea catechin metabolites, M4 and M6 and their methoxy-derivatives[END_REF] activities when ingested in the human diet. The consumption of green tea has also been associated with reduced risks of cancer [START_REF] Ahmad | Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells[END_REF][START_REF] Lambert | Cancer chemopreventive activity and bioavailability of tea and tea polyphenols[END_REF][START_REF] Katiyar | Tea antioxidants in cancer chemoprevention[END_REF][START_REF] Yang | Possible mechanisms of the cancerpreventive activities of green tea[END_REF][START_REF] Zaveri | Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications[END_REF] and cardiovascular disease [START_REF] Yamada | Tea polyphenols in preventing cardiovascular diseases[END_REF]. Furthermore, flavanols are known to effectively lower plasma low density lipoprotein levels [START_REF] Miura | Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans[END_REF]. These effects have been attributed mostly to the polyphenolic compounds of tea, namely catechins, which represent about 70% of polyphenolic content of green tea [START_REF] Lakenbrink | Flavonoids and other polyphenols in consumer brews of tea and other caffeinated beverages[END_REF]. Structures of five major isolated catechins from different green teas are shown in figure 1, including: (+)-catechin (C), (-)-epicatechin (EC), (-)-epicatechin 3-O-gallate (ECG), (-)-epigallocatechin (EGC), (-)-epigallocatchin 3-O-gallate (EGCG) and gallic acid (GA) [START_REF] Jin | Separation of catechin compounds from different teas[END_REF]. Due to their array of biological properties tea polyphenols, particularly catechins, have attracted the interest of various nutritional researchers [START_REF] Scalbert | Dietary polyphenols and the prevention of diseases[END_REF][START_REF] Yao | Flavonoids in food and their health benefits[END_REF]. To understand the processes mediating the beneficial health effects of green tea catechins in vivo it is essential to acquire information on their bioavailability, absorption and metabolism. Therefore, a number of studies have examined and described various aspects of the biotransformation of tea flavanols 5 by human, mice and rat gut microflora [START_REF] Lambert | Epigallocatechin-3-gallate is absorbed but extensively glucuronidated following oral administration to mice[END_REF]. For example, [START_REF] Das | Studies on Flavonoid Metabolism -Metabolism of (+)-[14c]Catechin in Rat and Guinea Pig[END_REF] detected the formation of two valerolactones after ingestion of radiolabelled compounds in rats and guinea pigs.

Valerolactones have also been identified as metabolites of catechins in human urine and plasma after oral ingestion of cups of green tea (1.2 g green tea powder in 300 ml hot water) [START_REF] Li | Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry[END_REF][START_REF] Li | Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion[END_REF]. In addition, [START_REF] Meselhy | Biotransformation of (-)-epicatechin 3-Ogallate by human intestinal bacteria[END_REF] observed these valerolactone metabolites after in vitro incubation of EGCG, ECG, EC and EGC for 24 h with a human fecal suspension. Other studies have demonstrated glucuronidation, sulfation and methylation of catechins in the liver, kidney, brain and gastrointestinal tract of rats and mice [START_REF] Lambert | Epigallocatechin-3-gallate is absorbed but extensively glucuronidated following oral administration to mice[END_REF][START_REF] Donovan | Catechin is metabolized by both the small intestine and liver of rats[END_REF][START_REF] Kuhnle | Epicatechin and catechin are O-methylated and glucuronidated in the small intestine[END_REF][START_REF] Tsang | The absorption, metabolism and excretion of flavan-3-ols and procyanidins following the ingestion of a grape seed extract by rats[END_REF]. Further, in a recently published study, more than 20 metabolites --glucuronides, methylated and ring-fission products --have been detected in human urine after ingestion of a green tea solution using HPLC-ESI-MS/MS [START_REF] Sang | Human urinary metabolite profile of tea polyphenols analyzed by liquid chromatography/electrospray ionization tandem mass spectrometry with data-dependent acquisition[END_REF]. In addition, [START_REF] Auger | Bioavailability of polyphenon E flavan-3ols in humans with an ileostomy[END_REF] have investigated food matrix effects on the absorption and metabolism of catechins using samples from five ileostomy volunteers who had consumed a green tea extract. The most abundant metabolites found in the urine of these subjects were sulfates of catechin, epicatechin and their methylated forms. After ingestion of 200 mg green tea extract 43% of the ingested flavan-3-ols were recovered in ileal fluid. In another ex vivo study, tea catechins have been shown to be unstable in intestinal juice and buffered systems with pH >7.4, notably the concentration of catechins incubated in Krebs-Ringer buffer decreased by 80% within an hour [START_REF] Zhu | Stability of the flavan-3-ols epicatechin and catechin and related dimeric procyanidins derived from cocoa[END_REF][START_REF] Zhu | Stability of Green Tea Catechins[END_REF].

The specific objective of the study presented here was to examine in detail the microbial metabolism and chemical stability of green tea-derived catechins (shown in figure 1) in the small intestine under experimental conditions. More specifically, tea flavan-3-ols were incubated with fresh ileostomy fluid (obtained from volunteers with terminal ileostomies) and the metabolites formed were identified and quantified using HPLC-DAD and HPLC-ESI-MS/MS. In addition, the microbiota and enzymatic activities of the ileostomy effluents were characterized.

Subjects

Two females between 45 and 60 years old, both with a terminal ileostomy, donated ileostomy effluents voluntarily for this study. Both probands suffered from Crohn's disease, without any involvement of the ileum and had undergone colectomy (with no surgical ileal resection) several years prior to the study, but were otherwise healthy.

A leaflet was given to both probands, asking them to avoid consuming food such as tea containing EC, EGC, EGCG, ECG, C and GA for 48 h before their ileostomy bag was removed. No drugs such as antibiotics were administered. The study protocol was approved by the Ethics Committee of the Mainz Medical Association (Landesärztekammer, no. 837.242.07 (5777)) in 2007.

Chemicals

All chemicals and solvents were of analytical grade. The flavanols (+)-catechin (C), (-)epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epigallocatechin 3-O-gallate (EGCG) and (-)-epicatechin 3-O-gallate (ECG) were purchased from Extrasynthése (Lyon, France), while gallic acid (GA) and p-nitrophenol 3-O-α-L-rhamnopyranoside (p-nprha) were ordered from Sigma Aldrich (Munich, Germany). The reference substance 1,2,3-trihydroxybenzol (pyrogallol) and internal standard (IS) 3,4,5-trimethoxycinnamic acid were also purchased from Sigma Aldrich (Munich, Germany), while 3',4',5'-trihydroxyphenyl-γ-valerolactone and 3',4',-dihydroxyphenyl-γ-valerolactone were kindly provided by Prof. Rice (State University of New Jersey, USA). The two healthy volunteers (A and B) removed their fresh ileostomy bags, which were immediately closed and placed in an anaerobic box containing Anaerocult ® (Merck, Darmstadt, Germany) to provide an anaerobic atmosphere. The samples were transported to the laboratory at +4°C and the anaerobic box was placed in an anaerobic chamber flushed with an 80:20 N 2 :CO 2 Aligal gas mixture (Air Liquide, Kaiserslautern, Germany). The ileal fluids from volunteers A and B were each diluted with an equal volume of anaerobic carbonate-phosphate buffer, following [START_REF] Lebet | Measurement of fermentation products and substrate disappearance during incubation of dietary fibre sources with human faecal flora[END_REF], and filtered through a strainer to remove particles. The resulting filtrates were designated ileal preparations A and B, respectively, and used for the incubation experiments (see below).

Preparation of ileal fluids

Incubation conditions

Portions (10 µl) of 25 mM stock solutions of the test flavan-3-ols, GA and p-nitrophenol 3-Oα-L-rhamnopyranoside in dimethylsulfoxide (DMSO) were placed in separate, small incubation tubes (Sarstedt, Nuembrecht, Germany) together with 2.5 ml portions of ileal samples A and B. The tubes were closed airtight, shaken and incubated for 0, ½, 1, 2, 4, 6, 8 and 24 h at 37 °C. The microbial reactions were then stopped by placing the tubes in liquid nitrogen. As controls, samples with carrier (10 µl DMSO) and human inoculum but no test substance, and others with test substances (C, EC, ECG, EGC, EGCG and G) but no inoculum (stability controls) were also prepared and treated in the same manner. In addition, the stability of each substance in carbonate-phosphate buffer without inoculum under anaerobic conditions was evaluated following the above protocol. Each incubation experiment was performed in triplicate. Frozen samples were lyophilized and extracted twice in 1 ml portions of a 70:30 (v/v) methanol:water mixture containing 1% acetic acid, with 10-min treatments in an ultrasonic bath. The two extracts from each sample were then pooled, centrifuged for 10 min at 5.000 rpm and filtered using 0.45 µm membrane filters (Buddeberg GmbH, Mannheim, Germany).

Sample preparation

Finally, the IS 3,4,5-trimethoxycinnamic acid was added and 200 µl portions of each pooled extract were analyzed by HPLC-DAD and HPLC-ESI-MS/MS (see below for chromatographic conditions). The recovery rates of the catechins ranged from 39 to 99%.

HPLC-DAD data analysis

All samples were analysed using a Hewlett-Packard 1090 HPLC station equipped with a photodiode array detector (DAD) (Waldbronn, Germany) and a 4.6 x 250 mm, 5 µm particle size Symmetry C 18 column (Waters, Eschborn, Germany). The column oven temperature was adjusted to 40°C, and the mobile phase consisted of aqueous 0.1% (v/v) formic acid (A) and acetonitrile (B), in a 40-min linear gradient of 1-50% (B) at a flow rate of 1 ml/min. In each case the injection volume was 20 µl. Eluting substances were identified by comparing their retention times and UV spectra with those of authentic references, and the acquired data were evaluated with Hewlett-Packard Chemstation ® Software. To quantify the compounds calibration curves (at appropriate wavelengths according to the absorption maxima of the compounds) were generated. Linear responses were obtained for all the compounds at concentrations up to 1 g/L, and their limits of quantification (LOQ) and determination (LOD), defined as signal to noise ratios of 3:1 and 10:1, ranged from 0.6 to 4.0 mg/L and 0.1 to 0.6 mg/L, respectively. To establish degradation rates for each substance, in µmol/h, a tangent was drawn through the inflection points of the degradation curves obtained from analyses of samples incubated for various times, as described above, and a linear equation was fitted to it [START_REF] Knaup | Human intestinal hydrolysis of phenol glycosides -a study with quercetin and p-nitrophenol glycosides using ileostomy fluid[END_REF].

HPLC-ESI-MS/MS data analysis

To identify flavan-3-ols and their corresponding metabolites a HPLC-ESI-MS/MS system with a SCIEX API 3200 MS/MS spectrometer equipped with an ESI interface (Applied Biosystems, Darmstadt, Germany), and a Jasco PU-980 pump (Groß-Umstadt, Germany) was used. The chromatographic system, including the column, mobile phase gradient, flow rate and injection volume, were all the same as in the HPLC-DAD analyses described above. MS analyses of eluting substances were carried out in negative ion, full-scan (100 to 900 atomic mass units) mode with a total scan duration of 1.0 s. Following optimization of the ionization, by infusing (-)-epicatechin with a syringe pump, the capillary temperature was set at 450 °C and the spray capillary voltage at -4.5 kV. Nitrogen was used as curtain gas (25 psi), nebulizer gas (GS1, 50 psi) and turbo gas (GS2, 40 psi). The declustering potential was -40 V and the entrance potential -4.5 V. The collision cell was operated with nitrogen, and a collision energy of -30 V was used. Both quadrupoles were set at unit resolution, and data were acquired and evaluated by Analyst Software 1.4.2 (Applied Biosystems, Darmstadt, Germany). The quasimolecular ions and product ion spectra obtained were compared to those of previously analyzed standards.

Api ZYM Tests

Api ZYM Test strips (Biomérieux, Lyon, France) were inoculated and incubated according to the manufacturer's instructions. Briefly, 2 ml of fresh ileal fluid from each proband was mixed with 4 ml distilled water, centrifuged at 2.000 rpm for 2 min, then each microcup of a test strip was inoculated with 65 µl of the resulting supernatant. The test strips were closed and incubated for 4 h under anaerobic conditions at 37 °C. After the incubation one drop of each of the two API reagents A and B were added to each microcup. Within 5 min colour

Microorganism Diagnostics

In order to characterise the microbiota in the ileal fluids we sent cooled (+4°C) ileostomy samples from each volunteer to an accredited laboratory (L+S AG, Bad Bocklet, Germany) to determine the abundance of specific classes of microorganisms in them using standard aerobic and anaerobic cultivation techniques.

Results

In order to imitate the anaerobic metabolism of the flavan-3-ols (C, EC, ECG, EGC, EGCG) and GA in the upper gastrointestinal tract, an ex vivo model simulating passage through the small intestine was used, in which the studied compounds were incubated for up to 24 h under anaerobic conditions with inocula from the ileostomists. Samples were drawn at 30 min, 1, 2, 4, 6, 8, and 24 h then extracted and analyzed by HPLC-DAD to quantify the parent compounds and metabolites, and by HPLC-ESI-MS/MS to confirm their identities.

Stability of the compounds in the carbonate-phosphate buffer

The stability of each studied compound in the absence of ileostomy fluid was evaluated by incubating them in anaerobic carbonate-phosphate buffer solution for 24 h and analyzing the solution at various time points. No degradation of C, EC, ECG, EGC, EGCG or GA was observed within the first 8 h of these incubations (data not shown). To determine the flavanol recovery rates provided by the sample preparation procedure, lyophilized inocula from probands A and B were spiked with 25 mM of the studied compounds, subjected to the procedure and then analyzed in the same manner as the samples.

Recoveries of the flavanols from lyophilized inoculum

The recoveries of the incubated substances are shown in table 1.

Degradation of green tea catechins in human ileostomy fluid

Degradation rates of the green tea catechins (summarised in table 2) were consistently lower when incubated with the ileostomy preparations from proband A than with those from proband B. The fates of the specific catechins are described in more detail in the following sections.

ECG. The kinetics of the degradation of ECG and the appearance of metabolites observed during incubations of the substance with fresh human ileostomy preparations for 24 h are shown in figure 2. During the first two hours ECG was almost completely degraded to GA and EC, at 0.123 µmol/h, in the presence of the ileal preparation from proband B. After reaching peak concentrations at two hours the level of GA then decreased, while the level of EC remained roughly constant. In the presence of ileal preparation A the degradation rate of ECG was much slower (0.024 µmol/h), and a substantial proportion (33%) still remained after the 24 h incubation. Nevertheless, GA and EC were still detected within 2 h, and their levels rose for ca. The kinetics of the degradation of EGCG and the appearance of metabolites observed during incubations of the substance with fresh human ileostomy preparations for 24 h are shown in figure 4. The degradation patterns were very similar to those observed for EGC, in that the substance was degraded more rapidly in contact with ileal preparation B than with ileal preparation A, but in both cases two metabolites were detected within 2 h. Comparison of their UV spectra, retention times and mass spectra in negative ionisation mode with those of authentic standards confirmed these metabolites to be GA and EGC (quasimolecular ions m/z 169 and m/z 305, respectively. The structures of the degradation products are shown in figure 1).

In the presence of ileal preparations from both probands the level of the degradation product EGC rapidly rose in the first 2 h. However, with preparation A it subsequently declined, while in the presence of preparation B it remained roughly constant for the following 22 h. In addition, GA levels peaked after 4 h then gradually declined during the following 20 h in the presence of preparation B, while they rose to a much lower maximum after 2 h with ileal preparation A then remained roughly constant. After 8 h incubation traces of the degradation products of EGC pyrogallol and 3',4',5'-trihydroxyphenyl-γ-valerolactone were detectable by HPLC-ESI-MS/MS (data not shown). With preparation B the concentration declined much more rapidly in the first 2 h (at 0.051 µmol/h), but the level remained almost stable thereafter. The incubations with EC yielded similar patterns. In addition, 3',4',5'-trihydroxyphenyl-γ-valerolactone and 3',4',dihydroxyphenyl-γ-valerolactone were detected as metabolites of both EC and C. The identities of these metabolites were confirmed by HPLC-ESI-MS analysis (quasimolecular ions m/z 223 and 207, respectively, with identical product ion profiles to those of authentic standards). C was also detected as an EC metabolite. Due to the low concentrations of the metabolites (less than 1% of the initial concentration of the parent compounds) they could not be quantified.

GA.

GA was degraded at similar rates by the preparations from probands A and B (0.022 and 0.051 µmol/h, respectively), continuously during the 24 h incubations (figure 7), and at the end of the incubations approximately 12% of the initial concentrations were still detectable in both cases. However, we would specify the time periods for which the rates apply, because it would be impossible for the final concentrations to be so similar if the rates diverged so much

Glycosidase activity of the ileal microflora

The glycosidase activity of the microflora in the ileostomy fluids was assayed by incubating p-nitrophenol 3-O-α-L-rhamnopyranoside (p-nprha) under anaerobic conditions with the ileostomy fluids, as described above, and monitoring its degradation. The results showed that the ileostomy fluids differed significantly in glycosidase activity; complete liberation of pnitrophenol from p-nprha took 0.5 h and 1 h in the presence of the preparations from probands A and B, respectively.

Analyses of the ileal fluids

Since the pH is also an important factor for the stability of catechins in the ileostomy fluid, it was determined before all experiments and was found to be consistently between 6.08 and 6.24. In addition, characterisation by a commercial laboratory (L+S AG, Bad Bocklet, Germany) showed there were significant differences in the microbial flora of the ileal fluids of the two probands in terms of concentrations of fungi and bacteria (anaerobic and aerobic) (see table 3). However, both preparations were able to cleave 2-naphthyl caprylate, according to the Api Zym tests. Heating of fresh ileostomy fluid to inactivate the intestinal microflora significantly reduced the degradation of the compounds under study. In the presented work we used an ex vivo ileostomy model to study the microbial metabolism of green tea catechins and gallic acid in the small intestine. The catechins and their metabolites were quantified by HPLC-DAD and identified by HPLC-ESI-MS/MS. All of the studied tea catechins (C, EC, ECG, EGC, EGCG) and GA were metabolised during incubation with human ileal fluid, and cleavage of the esters ECG and EGCG into the corresponding aglycones and GA was observed. Furthermore, pyrogallol was identified as a degradation product of GA, and two metabolites of C, EC and EGC were identified by HPLC-ESI-MS/MS (3',4',5'-trihydroxyphenyl-γ-valerolactone and 3',4',-dihydroxyphenyl-γvalerolactone). There is no evidence of valerolactone metabolites in black tea after the fermentation process. The fermentation is performed by a polyphenol oxidase catalysed oxidation of fresh tea leafs. During this process, polyphenols such as catechins are oxidized which is associated with an increase in monoterpene alcohol [START_REF] Wang | Tea flavonoids: their functions, utilisation and analysis[END_REF]. Due to this fermentation process 60 to 80% of green tea catechins are transformed into theaflavins, thearubigins and other epimerization products as reported by literature [START_REF] Wiseman | Tea flavonoids: Bioavailability in vivo and effects on cell signaling pathways in vitro[END_REF][START_REF] Wang | Polyphenolic Chemistry of Tea and Coffee: A Century of Progress[END_REF].

Discussion

Despite our ex vivo findings of catechin degrading activities and observations of cleavage of the gallate moieties of EGCG and ECG in ileal fluids, in a study where five volunteer ileostomists ingested 300 ml portions of a solution of a green tea extract (Polyphenon E) [START_REF] Auger | Bioavailability of polyphenon E flavan-3ols in humans with an ileostomy[END_REF] found that some catechins, mainly EGCG, reach the colon of healthy humans. In addition, cleavage of the ester bonds of galloylated catechins by intestinal microbiota has been observed in incubations with the caecum of freshly slaughtered pigs [START_REF] Van't Slot | Degradation and Metabolism of Catechin, Epigallocatechin-3-gallate (EGCG), and Related Compounds by the Intestinal Microbiota in the Pig Cecum Model[END_REF]. However, [START_REF] Stalmach | Absorption, metabolism, and excretion of green tea flavan-3-ols in humans with an ileostomy[END_REF] found that 31% of ingested green tea flavanols were absorbed in the small intestine and preceding parts of the gut in the 24 h following ingestion. Furthermore, a microbial esterase activity has been detected in human saliva [START_REF] Yang | Human salivary tea catechin levels and catechin esterase activities: Implication in human cancer prevention studies[END_REF] and observed in our assays by cleavage of the substrate 2-naphthyl caprylate in the Api ZYM tests. Therefore, microbial esterase activity was presumably responsible for the cleavage of ECG and EGCG during our incubations, which were intended to simulate passage through the small intestine. Overall, the findings of However, extensive bacterial degradation of catechins may also occur in the colon. Meselhy and coworkers [START_REF] Meselhy | Biotransformation of (-)-epicatechin 3-Ogallate by human intestinal bacteria[END_REF] incubated tea catechins with a human fecal suspension, and most of the incubated catechins (EGCG, ECG, EC, EGC) were metabolized to 3',4',5'-trihydroxyphenylγ-valerolactone, 3',4'-dihydroxyphenyl-γ-valerolactone, 3'-hydroxyphenyl-γ-valerolactone and other metabolites. Two of these substances (3',4',5'-trihydroxyphenyl-γ-valerolactone and 3',4'-dihydroxyphenyl-γ-valerolactone) have also been detected in our studies and by other groups in human urine and blood after tea ingestion [START_REF] Li | Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry[END_REF][START_REF] Li | Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion[END_REF][START_REF] Lee | Pharmacokinetics of tea catechins after ingestion of green tea and (-)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability[END_REF]. These metabolites have also been observed (presumably due to microbial metabolism) following oral administration of EC, EGC and GA in human plasma and urine, as well as rat and mouse tissues, by [START_REF] Meng | Identification and characterization of methylated and ring-fission metabolites of tea catechins formed in humans, mice, and rats[END_REF].

The formation of GA and further degradation to pyrogallol in incubations of EGCG and ECG with human fecal suspension have also been described [START_REF] Meselhy | Biotransformation of (-)-epicatechin 3-Ogallate by human intestinal bacteria[END_REF]. However, our findings indicate that some formation of GA and further metabolism to pyrogallol (at least) occurs by the end of the small intestine since low amounts of pyrogallol were detected following our incubations with ileal fluids. These divergences from previous findings may be due to the rapid autoxidation of pyrogallol in the presence of oxygen during sample preparation [START_REF] Marklund | Involvement of Superoxide Anion Radical in Autoxidation of Pyrogallol and a Convenient Assay for Superoxide-Dismutase[END_REF]. All metabolites we identified are shown in figure 8.

Due to the substantial differences in the degradation rates observed in the incubations with ileal preparations of probands A and B, the microbiotic flora of the ileal fluids were examined (table 3), and substantial differences in their fungal and bacterial (anaerobic and aerobic) populations were observed. Furthermore, glycosidase activity assays showed that the p-nprha degradation rate was much higher in the inoculum from proband B than in the inoculum from proband A, providing further indications of microbiological differences between them. The diversity of microbial populations in the human gastrointestinal tract has been previously reported, and various groups have shown that diet and lifestyle can substantially affect human microbiota, so there may be high intra-and inter-individual variations in intestinal bacteria communities [START_REF] Jacobs | H-1 NMR metabolite profiling of feces as a tool to assess the impact of nutrition on the human microbiome[END_REF][START_REF] Eckburg | Diversity of the human intestinal microbial flora[END_REF][START_REF] Muyzer | Profiling of Complex Microbial-Populations by Denaturing Gradient Gel-Electrophoresis Analysis of Polymerase Chain Reaction-Amplified Genes-Coding for 16s Ribosomal-Rna[END_REF]. However, in contrast to previous authors, who found a catechin metabolism in the colon, we observed (using our well-established ex vivo model) indications that significant metabolism of green tea catechins may also occur in the upper part of the GIT namely in the ileum. Further studies are needed to elucidate the metabolism and bioavailability of catechins more comprehensively, in order to correlate their metabolism to the disease-preventive effects of green tea. Values are means of threefold determinations ± SD.

  the colour intensity could be evaluated using an Api ZYM colour reaction chart.

  4 h then remained roughly constant. Comparison of retention times and the UV spectra of the products with those of authentic references confirmed their identities. The expected quasimolecular ions of m/z 169 for GA and m/z 289 for EC were observed by HPLC-ESI-MS in negative ion mode, and identical profiles of daughter ions to those obtained from commercially available references (characteristic fragments with m/z ratios of 69, 79, 97, 107 and 125 for GA, and m/z ratios of 109, 125, 137, 151 and 203 for EC). After a further 6 h incubation traces of the metabolites 4',5'-trihydroxyphenyl-γ-valerolactone (m/z 223), 3',4',-dihydroxyphenyl-γ-valerolactone (m/z 207), and pyrogallol (m/z 125) were detected by HPLC-ESI-MS/MS, and confirmed by comparison of retention time and spectra of authentic references.

Figure 3

 3 Figure3shows two HPLC chromatograms obtained from samples of the incubations of ECG

13 EGC.

 13 figure 6, this substance was very slowly degraded (at 0.27 µmol/h) in the presence of the ileal

  incubations. The metabolite pyrogallol was identified in the samples by comparing its retention time and UV spectra with those of an authentic reference, and verified by HPLC-ESI-MS/MS in negative ionisation mode, in which its retention time and product ion spectrum (dominant fragments with m/z ratios of 69, 79, 97 and 125) was identical to that of the standard.

  previous studies indicate that some catechin metabolism (at least) occurs during passage through this part of the gut.

Fig. 1 :

 1 Figure legends

Fig. 2 :

 2 Fig. 2: Changes in concentrations [µmol] of (-)-epicatechin 3-O-gallate (ECG) and its

Fig. 3 :

 3 Fig. 3: HPLC chromatograms recorded at 280 nm of samples obtained from anaerobic

Fig. 4 :

 4 Fig. 4: Changes in concentrations [µmol] of (-)-epigallocatechin 3-O-gallate and its

Fig. 5 :

 5 Fig. 5: Changes in concentrations [µmol] of (-)-epigallocatechin (EGC) during incubations

22 Fig. 6 :

 226 Fig. 6: Changes in concentrations [µmol] of (+)-catechin (C) during incubations with

Fig. 7 :

 7 Fig. 7: Changes in concentration [µmol] of gallic acid (GA) during incubations with

Fig. 8 :Tab. 1

 81 Fig.8: Chemical structures of metabolites detected from the incubations of (+)-catechin, (-)-

  -epicatechin 3-O-gallate (ECG) (-)-epigallocatechin 3-O-gallate (EGCG) (-)-epigallocatechin (EGC) gallic acid (GA)

  Microbiota analysis. Significant differences between the microbiota of the two probands are marked bold. cfu = colony forming units. LOD = 10 [cfu/g]

	Page 23 of 33			Biotechnology Journal
	Tab. 2 Degradation rates [µmol/h] of green tea catechins under study incubated with preparations of human ileostomy fluids from both probands Tab. 3 proband A proband B
		catechins	Degradation rate	Degradation rate
				[cfu/g]	[cfu/g]
		).	proband A [µmol/h]	proband B [µmol/h]
	aerobian
	C	compound	recovery [%]	0.027	0.051
	-E.coli		7*10 7	2*10 8
	C EC	82 ± 13	0.035	0.089
	-E.coli-variants		< 10	< 10
	EC ECG ECG EGC EGCG EGC EGCG GA -Proteus sp. -Enterococcus sp. -other aerob GA anaerobian -Bacteroides sp.	0.024 r 36 ± 2 74 ± 8 72 ± 9 F o 0.026 0.015 0.022 < 10 F 5*10 6 o < 10 r 82 ± 5 P P 94 ± 5 e e 3*10 3	0.123 0.033 0.113 0.051 1*10 8 < 10 < 10 1*10 6
				e r e r
				R R
				e e
				v i e v i e
					w w
				25
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Acknowledgments

We thank the volunteers for their participation, Prof. J.E. Rice (State University of New Jersey, USA) for providing the valerolactone standards, and Dr. G. Krammer/ Symrise GmbH & Co. KG (Holzminden, Germany) for their support with HPLC-DAD equipment.

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