Decrease of serum dipeptidylpeptidase activity in severe sepsis

patients: relationship to procalcitonin

Andreas Bergmann a, Claude Bohuon b,*

aBrahms Diagnostica, Komturstrasse 19-20, D 12099 Berlin, GermanybLaboratoire d’Immunotoxicologie, UER Pharmacie, Rue JB Clement, 92296 Chatenay-Malabry, France

Received 9 July 2001; received in revised form 21 January 2002; accepted 26 January 2002

Abstract

A significant decrease of DPP IV activity has been found in patients with severe sepsis in relationship to the increase of

procalcitonin. These findings might be explained by the high concentration of other substrates for DPP IV present in these

patients. It can be hypothesized that this enzymatic decrease is bound to some changes in immunomodulation. Further studies

will be necessary to elucidate the clinical importance of these findings. D 2002 Published by Elsevier Science B.V.

Keywords: Serum dipeptidylpeptidase; DPP IV; Severe sepsis; Procalcitonin; Immunomodulation

Procalcitonin (PCT), the precursor of calcitonin

(CT), is a polypeptide of 116 amino acids with a

molecular weight of 12,796 kDa. PCT in the plasma of

healthy blood donors is very low (less than 0.15 ng/ml).

On the contrary, in patients with severe bacterial

infections, PCT concentrations are very high (10–

1000 ng/ml), with normal values of CT [1]. Due to a

long half-life in the blood and a great stability after

sampling, PCT is now routinely used for the diagnosis

of bacterial infections and to follow-up the efficiency

of therapy [2].

PCT has 116 amino acids, as it can be deduced

from the DNA sequence of human PCT. But recently,

it has been found (manuscript in preparation ) that the

circulating PCT is principally a shortened form with

114 amino acids, lacking the N-terminal dipeptide

H2N-ALA-PRO-. The 116-amino acid form is absent.

The enzyme most probably responsible for this

truncation is an exopeptidase called dipeptidylpepti-

dase IV or CD26 (DPP IV, E.C.3.4.14.5). DPP IV

catalyses the release of N-terminal dipeptides from

oligo- and polypeptides preferentially with proline in

the penultimate position [3]. DPP IV has been shown

to degrade also PCT 1–116 to form PCT 3–116 [4]. It

is present in the cell membrane of many tissues,

especially on T lymphocytes. Also, a soluble circulat-

ing form with the ability to bind adenosine desami-

nases has been described [5].

This presence in the plasma of DPP IV is probably

due to the shedding of T cell membranes. A recent

paper confirms this origin [6].

Therefore, it seemed interesting to study DPP IV

activity in the plasma of septic patients simultane-

ously with PCT measurements.

0009-8981/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S0009 -8981 (02 )00042 -6

* Corresponding author.

www.elsevier.com/locate/clinchim

Clinica Chimica Acta 321 (2002) 123–126

1. Materials and methods

Thirty blood samples were collected from septic

patients with various ranges of severity: 10 with

moderate sepsis, 20 with severe sepsis. Furthermore,

the blood of 20 health volunteers were collected. After

clotting, the blood was centrifuged and the serum used

for PCT and DPP IV assays.

PCT assay was made using a specific luminometric

assay (Brahms, Berlin). DPP IV was measured by a

fluorometric assay of the released 4-methoxy-2 naph-

thylamine and the substrate was Lys-Pro-4-methoxy-2

Table 1

Enzyme activity of DAP IV in serum of sepsis patients vs. controls

Sepsis Controls

Sample

no.

PCT

(ng/ml)

Enzyme activity

(nmol/min/ml)

Sample

no.

PCT

(ng/ml)

Enzyme activity

(nmol/min/ml)

147 95.6 0.28 1 < 0.15 0.59

148 231 0.18 2 < 0.15 0.86

149 127 0.36 3 < 0.15 0.68

150 110 0.29 4 < 0.15 0.71

151 111 0.30 5 < 0.15 0.50

152 98.3 0.30 6 < 0.15 0.71

1244 77.6 0.45 7 < 0.15 0.90

1245 145 0.61 8 < 0.15 0.94

1246 223 0.53 9 < 0.15 0.86

1250 83.4 0.53 10 < 0.15 0.70

41 77.9 0.24 11 < 0.15 0.88

42 145 0.15 12 < 0.15 0.65

43 245 0.23 13 < 0.15 0.76

44 289 0.28 14 < 0.15 1.10

45 231 0.26 15 < 0.15 0.83

46 206 0.20 16 < 0.15 0.79

47 160 0.18 17 < 0.15 1.09

48 250 0.13 18 < 0.15 0.76

49 306 0.09 19 < 0.15 0.68

50 385 0.10 20 < 0.15 0.48

1 14.6 0.34

2 25 0.40

3 6.3 0.76

4 31 0.31

6 3 0.26

7 27 0.58

8 7.5 0.35

9 3.5 0.31

10 5 0.24

12 3.8 0.48

Mean 0.32 Mean 0.77

1 s 0.16 1 s 0.17

Mean�1 s 0.16 Mean� 1 s 0.61

Mean +1 s 0.48 Mean + 1 s 0.94

PCT< 0.15

(n= 20)

PCT 1–99

(n= 15)

PCT > 100

(n= 15)

Enzyme m 0.77 * 0.39 0.259

Activity DS 0.6 0.15 0.15

p< 0.001, Student’s Test p< 0.05, Student’s Test

* p< 0.001, Student’s Test.

A. Bergmann, C. Bohuon / Clinica Chimica Acta 321 (2002) 123–126124

naphthylamine. The activity was expressed in nano-

mole per minute per milliliter of serum [7].

2. Results

The patients were divided into two groups accord-

ing the levels of PCT, the first group ranging from 1 to

100 ng/ml; the second group ranging from 100 to 385

ng/ml including very severe sepsis patients.

It is interesting to note that PCT in the patients

ranged from 30 to about 400 times the normal values

which are always less than 0.15 ng/ml.

Concerning DPP IV, it was clear that all the

patients had a large decrease of the activity in com-

parison to the normal individuals. The means were

0.39 and 0.26 nmol/min/ml, respectively, for groups 1

and 2. For healthy blood donors, the mean was 0.77

nmol/min/ml.

Table 1 summarizes these data.

3. Discussion

Clearly, there is a definite decrease of DPP IV

activity in septic patients in comparison to normal

people.

Generally, more DPP IV activity is low, more PCT

concentrations are high. However, there is not always a

perfect relationship for each patient between PCT and

DPP IV. Nevertheless, it can be observed that for two

patients with very high levels of PCT (respectively, 306

and 385 ng/ml), the activities of DPP IV are very low:

0.09 and 0.10 nmol/min/ml. The cause of this decrease

of DPP IV could be due to the high levels of many

potential substrates circulating in the blood of septic

patients. Many cytokines, e.g., TNFh, IL1h, IL2, IL6,IL8, IL10, IL13, but also some growth factors as

GMCSF, GSF and chemokines (such as Rantes and

IP10) also have an N-terminal dipeptide H2N-X-Pro

which might be cut by DPP IV. However, it must be

specified that it is only the circulating DPP IV activity

which has been studied. Possibly, DPP IV present also

as a membrane protein is decreased simultaneously

with the soluble enzyme. A decrease of membrane DPP

IV has been observed during HIV infection and related

to a defective immunological memory [8]. In fact, few

studies have been published on human soluble DPP IV.

Studies have found a reduced expression of DPP

IV in T lymphocytes of cancer patients. In the study of

Vermaten, a clear relationship between DPP IV activ-

ity in serum and in CD 26 T lymphocyte plasma

membrane was observed [9].

In our preliminary study, the number of peripheral

CD 26 T lymphocytes was not analyzed, but it may be

hypothesized that membrane DPP IV activity was

lower in the patients than in healthy subjects. It must

be also pointed out that the origin of DPP IV in serum

remains partly unknown and other cells may release

the enzyme by shedding. Hino et al. [10], studying

rats with hepatitis induced by carbon tetrachloride,

noted an increase of serum DPP IV in correlation to

the decrease of liver DPP IV.

Although further investigation is necessary, it can

be suggested that in the septic patients, the origin of

the plasma DPP IV decrease is principally related to

the levels of DPP IV in lymphocytes. But also the

large overproduction of many substrates (cytokines,

chemokines, growth factors) during acute sepsis might

explain this decrease. In this case, it would be inter-

esting to study serum DPP IV after injection of

pharmacological amounts of GMCSF, EPO or IL2

for a therapeutic purpose.

The role of this DPP IV decrease may also be

questioned. It is now well known that the inhibition of

DPP IV has a therapeutic potential. Particularly,

synthetic DPP IV inhibitors have a powerful antiin-

flammatory capability [11,12]. Also, it has been found

that DPP IV plays a role in the inactivation of

endomorphin in vivo in mice [13]. Therefore, the

inhibition of DPP IV might be indirectly an adapted

response to maintain some central analgesic activity.

Finally, many peptides including PCT are short-

ened by DPP IV. The influence of protein chain length

has been discussed recently by Vanhoff [14]. Accord-

ing these authors chromogranin with 431 AA and N-

terminal with H2N-Leu-Pro is not attacked by DPP IV,

but a subproduct vasostatin I with 76 AA and the

same N-terminal H2N-Leu-Pro is a substrate. PCT, a

peptide with 116 AA with N-terminal H2N-Ala-Pro,

has been recently found to be a good substrate. The

principal circulating form of PCT has only 114 AA

[15].

In fact, many large peptides are potential sub-

strates, but few of them have been studied for this

attack by DPP IV. In vivo, the circulating forms of

A. Bergmann, C. Bohuon / Clinica Chimica Acta 321 (2002) 123–126 125

some very essential large peptides as GCSF, GMCSF,

IL2, TPO have not been studied as much as we know.

The usefulness of this structure H2N-X-Pro-X is

understood in some cases, perhaps as a protection

against specific N-terminal degradation. Furthermore,

this importance of the proline signal for the immuno-

modulating peptides may lead to a broader under-

standing of the immune response.

Acknowledgements

We thank E. Seidel-Muller for excellent technical

support.

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