www.elsevier.com/locate/clinchim

Clinica Chimica Acta 342 (2004) 1–12

Review

Peripheral markers of blood–brain barrier damage

Nicola Marchia, Marco Cavagliaa, Vincent Fazioa, Sunil Bhudiab,Kerri Hallenea, Damir Janigroa,*

aDepartment of Neurological Surgery, Cerebrovascular Research Center, NB20, The Cleveland Clinic Foundation,

9500 Euclid Avenue, Cleveland, OH 44195, USAbDepartment of Thoracic and Cardiac Surgery, The Cleveland Clinic, Cleveland, OH 44199, USA

Received 1 September 2003; received in revised form 3 December 2003; accepted 4 December 2003

Abstract

Neurological diseases are often associated with cerebrovascular dysfunction and changes in blood–brain barrier (BBB)

function. This is important for two seemingly conflicting reasons. On the one hand, a leaky BBB may lead to brain disease by

allowing extravasation of cells and molecules normally segregated in the periphery, while on the other hand an intact BBB may

hamper drug delivery to the ailing brain. Under both circumstances, it would be desirable to follow closely over time BBB

‘‘tightness’’. Several lines of evidence have suggested that the astrocytic protein S100h is a potentially useful peripheral marker

of BBB permeability. Other markers of brain-to-blood barriers have been recently discovered by a proteomic approach. These

proteins are virtually absent in normal blood, appear in serum from patients with cerebral lesions, and can be easily detected. We

will present clinical and laboratory evidence supporting the use of these markers as modern neurodiagnostic tools.

D 2004 Elsevier B.V. All rights reserved.

Keywords: S100h; BBB (blood–brain barrier); Blood–CSF barrier; Endothelium; Cerebral ischemia; MRI (magnetic resonance imaging);

Neurological disorders; NSE (neuron-specific enolase); TTR (transthyretin); Neurodiagnostic tools

The discovery that the entry of many substances Not all the blood vessels in the brain constitute a

into the brain is restricted has been generally attribut-

ed to Paul Ehrlich, who observed in the early 1880s

that aniline dyes injected into animals intravenously

colored all the organs except for the brain and spinal

cord [1]. The singular failure of the brain to be stained

at autopsy during jaundice must have been observed

before Ehrlich’s work, but apparently did not steer

inquiry into its mechanisms.

0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.cccn.2003.12.008

* Corresponding author. Tel.: +1-216-445-0561; fax: +1-216-

445-1466.

E-mail address: janigrd@ccf.org (D. Janigro).

blood–brain barrier (BBB): only capillary vessels are

endowed with a full-blown BBB phenotype. Vessels

of increasing diameter have comparably increasing

levels of leakiness and thus superficial vessels of large

diameter are the leakiest while penetrating pial vessels

and descending penetrating vessels tend to have an

intermediate barrier function. Since most animals,

including vertebrates, have some form of barrier

separating their blood circulation from the brain or

the central nervous system, it has been speculated that

profound evolutionary pressure existed to create such

a complex organ [2,13]. At present, however, it is not

clear why such sophistication exists, and we can only

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–122

speculate on the teleological pressure that manifests in

the specialization observed in endothelial cells (EC) of

the brain.

The ontogeny of the mammalian blood–brain bar-

rier may be related to the absolute need for controlled

homeostasis of the central nervous system. It has been

known for many years that the ionic and molecular

composition of the cerebrospinal fluid (CSF) differs

considerably from the composition of the plasma and

remains remarkably constant in spite of intravascular

changes. For example, proteins are few in the CSF, and

ionic gradients for potassium and calcium ions exist

across the blood–brain barrier. Glucose constitutes the

main energy supply to the brain and is avidly con-

sumed by neuronal cells. To ensure a constant supply

of glucose to the brain, even under conditions where

plasma glucose is low, the blood–brain barrier main-

tains nearly a 10-fold gradient for glucose entry into

the brain [3]. Other substances are actively transported

into and out of the brain, including amino acids,

metallic ions, etc.

Homeostatic control of the brain parenchyma by

vascular endothelial cells is also achieved by a func-

tion that is seemingly opposed to their ‘‘barrier’’

function. For example, the abluminal membrane of

the endothelium facing the glia end feet is endowed

with a high concentration of sodium/potassium trans-

porter molecules that permit absorption of excess

potassium from the brain into the blood [4–8]. Failure

of such a mechanism could lead to an abnormal

accumulation of potassium into the brain leading to

neurological disorders such as seizures. Control of

parenchymal pH may also depend on the integrity of

brain endothelial cells.

1. Anatomy and physiology of the normal blood–

brain barrier

The BBB is primarily composed of microvascular

EC linked by tight junctions that largely prevent

molecular communication between the plasma and

the brain or central nervous system [9–12]. Only

capillary vessels have complete BBB properties. As

vessel diameter increases, leakiness also increases:

superficial vessels of large diameter are the leakiest,

and penetrating pial vessels and descending penetrat-

ing vessels have intermediate degrees of barrier func-

tion. Surprisingly, in spite of the large body of

evidence on the neurophysiological properties of the

central nervous system, comparatively little is known

about the physiology and pathophysiology of micro-

vascular EC.

The long axis of these cells is perpendicular to

blood flow, and they are exquisitely sensitive to shear

stress. Recent evidence has shown that shear stress

promotes the expression of numerous genes involved

in various aspects of endothelial cell function [12,14–

16]. It is unknown whether flow-induced changes in

EC affect neighboring glia, and whether these changes

affect expression of astrocytic proteins that may be

detected in plasma. Some of the unique properties of

the BBB are induced by the perivascular glia. This is

known from two lines of evidence. First, it is evident

that shear stress alone cannot be responsible for BBB

properties in EC, because systemic microvascular EC

are exposed to comparable levels of shear stress.

Second, glia cells have been shown to induce

blood–brain barrier properties in vessels of systemic

origin [17,18]. Thus, the blood–brain barrier is com-

monly understood to be constituted of both endothe-

lial cells and glial end feet. Perivascular pericytes and

microglia may also be considered active components

of the blood–brain barrier.

The microvascular endothelium shares a common

basement membrane with astrocytes and pericytes.

Beyond the basement membrane in the parenchymal

vessels of the brain lies a close investment of end feet

from neuroglial cells, predominantly astrocytes.

Astrocytes and their processes invest more than 90%

of endothelial capillaries, and their end feet are

projected tightly around the endothelial cells. There-

fore, the glial end feet are a natural candidate for

mediating communication between neurons and cap-

illaries. Astrocytic proteins are synthesized and re-

leased next to capillaries, but it is thought that they

extravasate into the plasma only when the BBB is

breached.

2. Protein permeability at the BBB

Brain-derived proteins may be useful markers of

BBB integrity because they have several possible

mechanisms of passage across the BBB. Under

physiologic conditions, the production of CSF from

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–12 3

plasma involves efficient removal of plasma pro-

teins by filtering mechanisms in the choroid plexus.

The resulting CSF contains extremely small quan-

tities of proteins [19]. However, many neurological

disorders are associated with elevated CSF pro-

tein levels. Furthermore, CSF-specific proteins exist

[20].

Fig. 1. Gradients and topography of peripheral markers of BBB/brain dam

immunopositive astrocytes and NSE immunoreactivity in human neocorte

highly concentrated in proximity to blood vessels (indicated by small arro

indicate 20 Am. n = neuron, a = astrocyte.

Proteins in CSF can be detected by directly sam-

pling CSF, which requires invasive techniques such as

lumbar puncture or intraoperative sampling from

ventricles or subarachnoid space. BBB integrity can

also be assessed by contrast-enhanced computed to-

mography or MRI (see above). An obvious limitation

of intrathecal detection methods is that they are

age. See text for details. The micrographs show GFAP and S100hx (temporal lobe). Note that GFAP and S100h immunoreactivity is

ws). A large NSE+ neuron neighboring a vessel is also shown. Bars

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–124

invasive and that the sample itself may be contami-

nated by the procedure. Accurate non-invasive tech-

niques would clearly be preferable, particularly to

analyze multiple longitudinal samples. A small group

of proteins are found exclusively or almost exclusive-

ly in the cerebrospinal fluid. Any disruption in BBB

integrity may allow protein leakage in both directions

(see Fig. 1). Thus, testing serum levels of CSF

proteins represents a non-invasive means for evaluat-

ing BBB integrity that may be of diagnostic value

[21]. Fig. 2 shows the predicted behavior of proteins

in conditions of intact vs. impaired BBB function.

Scenarios A and B demonstrate the observed elevation

in S100h serum levels as the result of BBB opening

following and prior to neuronal damage (respective-

Fig. 2. Possible mechanisms of release of putative markers of neuronal func

dots) may be released after neuronal injury; (B) before injury across a leak

the absence of neuronal damage. Hypothesis A predicts that a marker tha

plasma compartment only after brain injury. In contrast, if BBB dysfunctio

under normal conditions, appearance in plasma will be temporally relate

release due to increased synthesis or release in the CSF. However, if the

availability in the CNS from neuronal damage, the appearance in plasma

ly). Scenario C exemplifies the use of a chemical

means (mannitol infusion) that has been used to

produce a transient opening of the BBB without

producing accompanying neuronal damage.

3. Blood–brain barrier in neurological disorders

Substantial progress has been made in the under-

standing of the pathophysiology and mechanisms

involved in the attenuation of BBB permeability. In

many diseases that affect the brain, the cerebral

endothelium plays an active part in the disease process

with the BBB becoming disrupted, or modified, in

such a way that there is a dramatic increase in vascular

tion or blood–brain barrier damage across the BBB. (A) Marker (red

y blood–brain barrier, with additional release after injury; or (C) in

t is released or synthesized in response to injury will appear in the

n precedes injury and if the marker is present in CSF but not plasma

d to opening of the blood–brain barrier, with a possible additional

BBB opening is not followed by significant increase in marker’s

will be brief in duration.

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–12 5

permeability. Theoretically, several ways exist in

which various molecules can pass the endothelium.

These include intercellular routes, vesicular transport

or direct transcellular penetration through damaged

endothelium. BBB dysfunction may be a cause or

consequence of a particular disease process. Cerebral

vessels from pathological brain tissue often resemble

peripheral endothelium, with significant increases in

vesicular transport and abnormal tight junctions. Dis-

eases in which increased BBB permeability have been

reported include neoplasia, ischemia, hypertension,

dementia, epilepsy, infection, multiple sclerosis and

trauma. The effect of a disease on BBB function will

secondarily affect the cerebral blood flow and vascu-

lar tone in the brain, which further influences transport

across the BBB. Besides the effects of increased

vascular permeability on the brain parenchyma, a

question of great significance is whether, in certain

neuropathological conditions, the BBB disturbance

constitutes the main pathogenic factor itself, which

then triggers a sequence of events molding the final

pathological state.

While loss of blood brain barrier (BBB) function is

an etiologic component of many neurological diseases

an intact BBB may restrict the delivery of certain

therapeutic substances to the brain. Thus, measuring

BBB function may be important to diagnose disease

progression and monitor time-dependent changes in

BBB integrity when chemotherapic penetration may

be enhanced. At present, only invasive and expensive

techniques such as contrast-enhanced magnetic reso-

nance imaging, CT-scan and lumbar puncture are

available to clinically assess BBB integrity. An alter-

native approach has been proposed, consisting of

detection of changes in blood composition that indi-

cate BBB disruption [21].

Current BBB assessment by imaging or cerebro-

spinal fluid sampling is based on direct or indirect

determination of protein permeability across the

BBB. CNS proteins are normally asymmetrically

distributed, with generally much higher concentration

in plasma than in CSF. Thus, the appearance of

plasma proteins in CSF is a hallmark of numerous

CNS disorders with presumed or overt BBB disrup-

tion. Only a few proteins are synthesized exclusively

by, or are present in higher concentrations in CSF or

interstitial compartment compared to the blood.

These CSF markers may appear or increase their

plasma concentration after passage across a failed

BBB. Therefore, measuring levels of CSF proteins in

plasma may be a reliable way to monitor blood to

CNS barrier integrity without the use of invasive

methods.

4. Neuronal damage vs. glial/BBB damage

Most research into brain damage has focused on

neuronal damage, because this is the cause of most

deficits from neurological disease. In fact, ‘‘brain

damage’’ has often been used as a synonym for

neuronal damage or death. This research has shown

that neuronal sensitivity to insult is region-and dis-

ease-specific. As example, ischemic insult selectively

affects the CA1 region of the hippocampus, leaving

the neighboring dentate gyrus and CA3 practically

intact [22]. In contrast, damage resulting from epilep-

tic seizures is more prominent in CA3 hippocampal

subfield [23]. In addition to these patterns of speci-

ficity, it has also been observed that neuronal cell

death does not occur concomitantly with the insult,

but rather after a delay.

In acute insults such as ischemia [24,25], the delay

provides a potential therapeutic window for neuro-

protective intervention. In chronic and progressive

neurological diseases, such as multiple sclerosis, the

delay may be even longer. Ideal markers of BBB

permeability and of neuronal damage share several

characteristics (Table 1): both should be virtually

undetectable in normal subjects and should show

distinct alterations in response to insults that are

correlated with the severity of the damage.

Distinguishing between BBB defects and neuronal

damage has enormous clinical relevance. For exam-

ple, in acute CNS disturbances such as ischemic

stroke, the delay between insult and irreversible

neuronal cell death offers a window of therapeutic

opportunity. If BBB openings develop early after the

initial arterial occlusion [26–32], clinicians would

have a unique opportunity to administer drugs that

are normally BBB-impermeant (e.g., nerve growth

factors) before neurons were damaged. The duration

of these openings may be unpredictable, so a periph-

eral, non-invasive, easily repeatable test would be

extremely useful. In chronic neurological diseases,

such as multiple sclerosis, BBB openings may have

Table 1

Properties of selected proteins and their use as potential markers

BBB integrity

S100h NSE GFAP

(1) Low or

undetectable

levels in plasma

in normal subjects

yes no no

(2) Normally

present in CSF

yes yes yes

(3) [Plasma] < [CSF] yes no no

(4) Increase [CSF] in

response to insult

yes yes yes

(5) Normally excluded

by BBB

yes yes yes

(6) Expected flux

after BBBD

blood-to-

brain

brain-to-

blood

brain-to-

blood

(1) Stroke 28 (1997) 1956–1960; (2) Clin. Chem. 43 (1997) 541–

543; (3) Clin. Chem. 46, (2000) 993–996; (4) Clin. Chem. 44

(1998) 1056–1058; (5) Stroke 31 (2000) 2670–2677; (6) Perfusion

12 (1997) 171–177; (7) Glia 22 (1997) 1–11.

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–126

both therapeutic and etiologic significance. Severity

of symptoms has been suggested to correlate with

BBB function in these conditions, and promising

therapies using brain-derived proteins have failed

largely because the compounds are poorly transported

across the BBB. When a patient experiences both

blood–brain barrier opening and neuronal damage,

plasma levels of both markers would be expected to

exceed normal levels [54]. Also, as we will discuss

below, S100 protein levels appear to be directly corre-

lated with BBB integrity rather than with neuronal

damage, while another neuronal protein, monomeric-

transthyretin (TTR) represents a potential marker of

opening to the blood–CSF barrier [48].

Because of this focus on neuronal damage, much

of the previous research on biochemical markers has

focused on markers that measure neuronal damage.

However, most neurologic diseases are accompanied

by increased BBB permeability, and thus the markers

thought to indicate neuronal damage may in fact

indicate BBB defects. Marker proteins under investi-

gation have included neuron-specific enolase (NSE),

glial fibrillary acidic protein (GFAP), NSE and S100h(see Fig. 1). S100h seems particularly promising. In

normal subjects, NSE is more concentrated in plasma

and S100h is primarily present in central nervous

system fluids [33]. Thus, opening the blood–brain

barrier in the absence of neuronal damage would be

expected to markedly increase plasma S100h levels

while leaving NSE levels unchanged.

5. Markers of blood–brain barrier integrity:

S100B

S100B is primarily synthesized in the brain by the

end feet process of the astrocytes and is quickly

released from the brain in the blood when the BBB

is disrupted [21,34–37]. S100B has also been found

in other tissues but at lower concentrations [38–40].

While S100B appearance in plasma correlated well

with BBB openings, S100B has been shown to in-

crease in plasma, CSF or both as a consequence of

other pathologies not limited to the CNS. S100B may

also detect brain damage, or indicate advanced me-

tastasis in melanoma patients [41–45].

The fact that S100B can increase in serum inde-

pendent of brain (or neuronal) damage was demon-

strated indirectly by a team of scientists who studied

the affects of boxing as well as other high cardiovas-

cular output activities on the levels of S100B in serum.

Interestingly, this study found that a significant in-

crease in S100B was observed in serum of subjects

undertaking activities that involved repetitive, jarring

movement or contact to the head (such as boxing,

sparring, running and jogging), but essentially no

increase was observed in persons exerting themselves

through exercise on a stationary bicycle. Clearly, these

activities did neither cause nor promoted brain dam-

age but the rise in S100B protein in running activities

may be due to astroglial activation, astroglial destruc-

tion or blood–brain barrier disruption or a combina-

tion of the three. This finding also indicated that the

source of S-100B was not influenced greatly from

excretion of the protein from extra-cranial tissue.

Another study demonstrated dissociation between

serum S100B and brain damage. S100B was measured

in sera from patients diagnosed with major depression

[46]. This study discovered a positive correlation of

the severity of depression with S100B levels; patients

given antidepressive therapy (prescription anti-depres-

sants) experienced significantly decreased levels of

serum S100B.

A direct demonstration proving that S100B may

increase in serum in spite of an obvious lack of

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–12 7

additional brain damage was provided by studies

where S100B was measured at short intervals

(minutes) in patients undergoing osmotic opening of

the blood–brain barrier [21,34,47,48]. These authors

demonstrated that S100B elevation correlated tempo-

rally with BBB disruption; these findings were also

confirmed radiologically by Kanner et al. [47]. To test

the connection between S100B and blood–brain bar-

rier integrity, NSE and S100B were measured in

serum of patients with primary central nervous lym-

phoma who underwent blood–brain barrier disruption

by intra-arterial mannitol infusion before receiving

methotrexate infusion. Mean serum levels of S100B

increased significantly after mannitol infusion and

again after methotrexate infusion, and they remained

elevated through recovery. However, NSE serum

levels remained constant throughout the procedure

and during recovery. Previous investigators have

shown that blood–brain barrier disruption with intra-

arterial methotrexate does not lead to brain damage

[49–51]. To rule out the possibility that the increased

plasma S100B levels were caused by the methotrex-

ate, the authors measured S100B and NSE in the

blood of three patients who were given methotrexate

Fig. 3. Different distribution between S-100h and TTR in the brain. In the

the BBB, whereas TTR is synthesized by the choroid plexuses and is foun

the different roles of these markers.

without blood–brain barrier disruption. Under these

conditions (no BBB disruption) the levels of both

markers remained within normal ranges. It was con-

cluded that the increase in S100B immediately after

the blood–brain barrier disruption was almost cer-

tainly too soon to be the result of synthesis and

release from ‘‘reactive’’ glia and that S100B may be

an early marker of blood–brain barrier disruption that

is not necessarily related to either neuronal or glial

brain damage. These findings of course do not chal-

lenge the traditional understanding that NSE is related

to neuronal damage.

In addition to S100B, another putative marker of

blood–brain barrier function was unveiled in these

studies. The CSF protein, monomeric-TTR was also

temporally linked to S100B movement from brain to

serum [48]. However, since the authors observed a

slight, but significant delay (within minutes) between

the peak serum elevation of S100B (immediately

following chemical opening of the BBB) and TTR

(occurring shortly thereafter following the administra-

tion of chemotherapy), it was concluded that TTR was

a marker of blood-to-CSF rather than a BBB marker

(see Fig. 3).

brain, S-100h is primarily synthesized by the astrocytes surrounded

d in the ventricular CSF. This topographic segregation may explain

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–128

6. Mathematical modeling of serum S100B levels

after brain damage or BBB leakage

While these results clearly demonstrated a relation-

ship of serum S100B with BBB function, previous

findings by others demonstrated a positive correlation

with brain damage [43,44,52,53]. How could these

seemingly contrasting two findings be explained? If

one utilizes values from the literature related to S100B

Fig. 4. S100h levels after BBB disruption: (A, B) schematic representatio

details). Sigmoidal (Boltzmann) fit of S100hserum levels measured after h

leakage produced by opening of the BBB of one hemisphere. The as

representation of Eq. (2). The initial values of S100hserum and S100hCSF w

hemispheric BBB disruption depend on both CSF and blood volumes. Sim

the contribution of neuronal damage to plasma levels (box 3, C) under con

Figure was reproduced and modified with permission from Ref. [34].

levels in tissue as well as the measured dimensions of

various body cavities, a kinetic model for S100B

release in serum can be deduced [34,38]. The predic-

tive value for these parameters were independently

confirmed by mathematical modeling of the range of

steady-state concentrations that S100B serum would

reach when the BBB is greatest (schematically repre-

sented in Fig. 4A) or when brain damage occurs

concomitantly. This model was used to assess the

n and mathematical modeling of S100h kinetics (see Ref. [10] for

emispheric BBBD. The average of 36 openings is shown reflecting

ymptotic value determined was 0.176 ng/ml. (C) Tri-dimensional

ere 0.05 and 2 ng/ml, respectively. Note that S100hs-s obtained after

ilar plots were constructed at different S100hCSF levels to estimate

ditions of breached BBB. (D) shows the results of these calculations.

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–12 9

dependency of S100B steady state on serum and CSF

volumes as well as CSF levels of the protein. The

initial values used were derived from the literature

(e.g., S100B CSF= 2 ng/ml and S100B serum = 0.05

ng/ml). Data were fitted according to the following

equation:

S100hs�s ¼ ½ðS100hCSF*1=2CSFvolÞ þ ðS100hser

*SerumvolÞ�=½1=2CSFvol þ Serumvol�ð1Þ

where S100hs-s is the steady-state serum concentration

after hemispheric opening of the barrier; S100hser/CSF

are the reference concentrations of S100h in serum and

CSF expressed in ng/ml; CSFvol and serumvol are

volumes of these compartments expressed in liters.

The resulting three-dimensional plot is shown in Fig.

4C to demonstrate the dependence of S100hs-s on CSF

and blood volume. As expected, the peak levels of

S100hs-s are achieved when CSF volume is greatest

and serum lowest. As predicted by our direct experi-

mental observation and fitting, these values were again

close to 0.18 ng/ml (green arrow), which closely

parallels the amount leaked from the CNS after hemi-

spheric BBB disruption. These data and fits were

based on CSF S100h levels typical of uninjured brain.

To estimate the steady-state values of S100h serum at

different S100h CSF and under condition of bi-hemi-

spheric BBB damage, we used the following equation

(Fig. 4D):

S100hs�s ¼ ½ðCSFvol*S100hCSFÞ þðserumvol*0:05Þ�=½CSFvol þ serumvol� ð2Þ

Values for CSF and serum volume were arbitrarily set

at 0.15 and 1.5 l, respectively, to reflect the average

volumes for serum and cerebrospinal fluid. The red

region in Fig. 4D represents S100hs-s within a range

that includes normal values and levels that may be

achieved by breaching the BBB in absence of damage

(our findings). The data point 2 refers to data from

Buttner et al. [55], where experimentally measured

CSF values of 6 Ag/l corresponded to serum levels of

around 0.6 Ag/l. Note that these values were identicalto those predicted by our model. The boxed blue region

(3) represents S100hCSF levels measured in patients

affected by a variety of neurological diseases

[38,43,44,52,54–60]. These levels were compared in

the same study with S100serum. Again, a perfect

correlation of CSF/serum ratios with data obtained

from our model was found.

Taken together, these experimental results and

mathematical modeling demonstrate that the maximal

levels of S100hs-s achievable after BBB failure are

around 0.34 ng/ml (Fig. 4C). Thus, levels of S100hs-s

exceeding this value may be due to other factors, such

as non-CNS release [57], synthesis ex novo due to

damage, or other mechanisms.

7. Conclusions

S100h, neuron-specific enolase and other putative

markers of brain damage have been shown to corre-

late with outcome in a variety of neurological dis-

orders [57–59,61,62]. The interpretation of the

clinical significance of the appearance of S100h in

serum of neurological patients is complicated by the

fact that the cerebral circulation, unlike the coronary

vascular network, is characterized by tight junctions

between endothelial cells. The presence of tight junc-

tions is the molecular basis of the so-called blood–

brain barrier, a specialized endothelial structure effec-

tively shielding the brain from systemic influences.

The presence of this endothelial barrier minimizes the

extravasation of a variety of molecules including CSF

(or serum) S100h. Thus, detection of passage of

albumin from serum to brain is the preferred clinical

method to evaluate BBB intactness by either direct

measurements (lumbar puncture) or contrast-enhanced

CT-MRI where albumin is chemically linked to radio-

opaque ions (e.g., gadolinium). The opposite ap-

proach, detection of S100h protein in serum, is also

possible in virtue of the fact that this protein is almost

exclusively present in brain astrocytes.

The fact that S100hserum may be used as marker of

BBB integrity is not necessarily in disagreement with

the notion that S100h is a marker of brain damage,

since both phenomena (BBB failure and brain dam-

age) are temporally and topographically associated.

A possible explanation of the dual message that

levels of S100hserum may convey was derived mathe-

matically [34]. According to these authors, low levels

of S100h are normally present at the blood-to-brain

interface and in the CSF while disruption of the BBB

N. Marchi et al. / Clinica Chimica Acta 342 (2004) 1–1210

will result in sudden appearance of cerebral S100h in

serum. It was possible to estimate the steady-state

levels of S100h that are when (1) The BBB is com-

pletely leaky; (2) levels of S100hCSF do not increase

over time due to neuroglial damage; and (3) CSF and

serum concentrations are constant. Furthermore, simi-

lar analysis was performed for S100hCSF levels typical

of a broad range of cerebral dysfunction. Serum levels

of S100h exceeding the ‘‘BBB ceiling’’ may implicate

brain damage or release from non-CNS sources.

In conclusion, interpretation of recent results and

existing literature compelled us to reinterpret the sig-

nificance of S100h as marker of brain damage. Exper-

imental, clinical and theoretical data show that: (1)

S100h is a marker of both BBB and neuronal damage;

(2) threshold serum values indicating brain damage can

be estimated; (3) conditions exist when S100hserum is

low in spite of massive brain damage; and (4) detection

of slightly elevated levels of S100hserum may be an

early sign of future neuronal damage, triggered or

accompanied by blood–brain barrier failure.

Acknowledgements

The authors would like to thank Anne-Charlotte

Aronsson for her helpful comments and support. This

work was supported by NIH-2RO1 HL51614, NIH-

RO1 NS 43284 and NIH-RO1 NS38195 to Damir

Janigro.

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