Multiple Sclerosis (1998) 4, 243 ± 246

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Myelin basic protein-like material in the urine of multiple sclerosis patients:

relationships to clinical and neuroimaging changes
John N Whitaker
The Department of Neurology and the Center for Neuroimmunology of the University of Alabama at Birmingham and
the Neurology and Research Services of the Birmingham Veterans Medical Center, Birmingham, Alabama 35294, USA

Urinary myelin basic protein-like material (MBPLM) represents material which is cross-reactive with a cryptic epitope in peptide 84 ± 89 of human
myelin basic protein. While normally present at moderate levels in the adult, these levels rise higher in patients who have secondary progressive
multiple sclerosis (MS). The increase in urine MBPLM correlates with the burden of disease detected by T2-weighted cranial magnetic resonance
imaging. There is no correlation between urinary MBPLM and acute disease activity in relapsing-remitting MS. The ®rst major need for improving
the clinical utility of measurements of MBPLM in urine in MS patients is to delineate its exact chemical features so that assays may be improved
and a potential biological role of the MBPLM better understood. The second major task is to apply the group data accumulated and apply them to
individual patients. This could prove to be means to individually direct treatment and determine its effectiveness.
Keywords: multiple sclerosis; urine; myelin basic protein; magnetic resonance imaging; radioimmunoassay

Introduction
Beginning in 1993, the Food and Drug Administration scored clinical examination which are presently
(FDA) of the United States has approved three reagents recommended as primary end points for phase 2 and
for treatment of relapsing-remitting (RR)1 multiple phase 3 trials, respectively.10
sclerosis (MS). These include interferon beta-1b,2,3 A surrogate marker is a nonclinical assessment
interferon beta-1a4 and glatiramer acetate.5 These drug which may predict an ultimate clinical change. The
approvals represent de®nite advances in the care of clinical events in MS which are of greatest interest to
the RR-MS patient; however, all three drugs are relate to a surrogate marker are those of the changes in
expensive (approximately $11 000 US per year), have disease activity, that is, relapses and remissions, and
only a partial effect on relapse reduction, and have the change in status, that is, the transition from RR to
little, if any, effects on the progression of disease. SP disease.1 Whether SP-MS represents the failure of
Furthermore, recent clinical trial experience, espe- remission or other processes that may transpire in
cially for secondary progressive (SP) MS, has been primary progressive (PP) MS remains to be deter-
marked by drug failure or study termination. Thus, the mined. The surrogate marker most commonly used in
trials of deoxyspergualin, sulfasalazine6 and cladribine MS is that of serial cranial MRI which may be
showed no effect on SP-MS. Oral myelin enriched in performed according to a number of techniques.
myelin basic protein (MBP) showed no effect on RR- Segmentation analysis seems particularly attractive
MS in a phase 27 or phase 3 trial, and the phase 3 trial because of its automated and quantitative features.11
of linomide, which had seemed promising based on Other measures such as improved clinical scales,12
two phase 2 trials,8,9 had to be terminated because of electrodiagnostic methods, primarily evoked poten-
unanticipated myocardial toxicity. tials, and measurement of a variety of constituents in
A number of drugs and reagents are candidates for body ¯uids also represent potential avenues to
clinical trials for MS, but the pace of performing explore. The major substances in body ¯uids for
clinical trials is limited because of the long duration of measurement and analysis include myelin constituents
observation needed for determining clinical effective- and related enzymes, glial proteins, markers of general
ness in a pivotal phase 3 trial of new therapy. The neural tissue damage, elements of the immune system
decision to go forward with the expensive testing of a and a number of miscellaneous materials. The subject
phase 3 trial rests on an accurate phase 2 trial which, of the present chapter is to review the current status of
because of the brevity, must rely on nonclinical studies on myelin basic protein-like material (MBPLM)
methods as their primary end points. Although in the urine. Two references13,14 contain previous
expensive and not always paralleling clinical de®cits, reviews by the author on this and related topics.
cranial magnetic resonance imaging (MRI) is the usual
method chosen. All of these circumstances place
emphasis in having more reliable markers for deter-
Features of myelin basic protein
mining treatment effectiveness beyond the changes in Central nervous system (CNS) myelin has at least
quantitated tissue signals on serial cranial MRI or in a seven identi®able protein components existing in
myelin and/or the related oligodendrocyte. These
include proteolipid protein, MBP, 2', 3'-cyclic nucleo-
tide 3' phosphodiesterase, myelin associated glycopro-
Correspondence: JN Whitaker tein, myelin oligodendrocyte glycoprotein, oligo-

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 Urine MBPLM in MS
JN Whitaker
244
dendrocyte myelin glycoprotein and myelin oligoden- Moreover, the MBP epitope which cross-reacts with
drocyte speci®c protein.15 While the quantitation of all urinary MBPLM is cryptic, i.e., antibodies to MBP
of these in body ¯uids may have potential as surrogate which recognize this epitope react with certain MBP
markers, MBPLM has been the most intensively peptides but not the intact MBP molecule. Urine
studied and offers the greatest clinical use at present. contains low amounts of MBPLM shortly after birth
As recently reviewed,14 MBP accounts for 30% of that rise to adult levels at 1 ± 2 years of age and then to
CNS myelin proteins,16 is encoded by a single gene of above normal adult levels during childhood.45 These
seven exons located on chromosome 1817 and is levels return to adult levels at about the age of 10
normally expressed only by oligodendrocytes and years. During the differing phases in disease activity
Schwann cells.18,19 Alternate splicing of the MBP typical of RR-MS, urine MBPLM does not increase but
transcript gives rise to at least four isoforms of human does so when RR-MS patients change status from the
MBP with the major ones having molecular weights of RR to the SP pattern of disease.40 Urinary MBPLM is
21.5 kd (all seven exons) and 18.5 kd (exons 1 and 3 ± higher in MS patients than in controls.46
7).19 The 18.5 kd isoform containing 170 amino acid Perhaps instructive for the detection of peptides,
residues dominates in adult human CNS myelin. In whether free or bound, of other proteins in body ¯uids,
addition to multiple isoforms, MBP may have a several major laboratory steps were required for the
number of post-translational modi®cations,20 such as RIA for urine MBPLM to work. First, it was necessary
amino-terminus acylation,21 selected phosphorylation to understand the epitopes of MBP, especially that it
of serine and threonine,22,23 deamidation of glutamine, could have cryptic determinants that were not
loss of C-terminal arginine23 ± 25 and methylation of the expressed in the intact molecule.47 Second, the correct
arginine at residue 10726 that result in charged isomers. sequence of MBP in the span of residues in 80 ± 89 had
Of particular interest is the heavily citrullinated to be corrected. This included the correct assignment
isomer, designated as MBP-C8, which, created by the at residues 83 ± 84 of glutamine-aspartic acid rather
citrullination of six of the 19 arginine residues present than glutamic acid-asparagine.39,48 Third, the RIA
in MBP, results in a less basic MBP.27 MBP-C8 is components, especially the choice of standards, had
formed early in myelinogenesis,28 is relatively well to be optimized in order to enhance assay validity
preserved in MS brain tissue and is immunogenic for when the exact chemical identity of MBPLM is not yet
humoral29 and cellular30 respones. known.
MBP has multiple independent epitopes throughout The relationship of MS events to urinary MBPLM
its 170 residues,31,32 and a multideterminant model is has been more complicated to delineate than that of
likely.32 Certain regions of MBP appear to have special CSF MBPLM. It is clear that the urinary MBPLM
relevance to MS. MBP peptide 80 ± 100 contains the does not correlate with acute disease activity in RR-
encephalitogenic epitopes for the SJL mouse33,34 and MS indicated either by clinical changes, cranial MRI
Lewis rat,35 the dominant epitope for antibody to MBP with gadolinium-enhancing lesions or an increase in
in brain and cerebrospinal ¯uid (CSF),36 the dominant MBPLM in CSF.46 However, based primarily on the
epitope for T cell reactivity in MS37 and the regions urine studies performed in conjunction with the
cross-reacting most closely with MBPLM in CSF38 and multicenter trial of interferon beta-1b,2,3 there was a
urine.39,40 There is evidence for intramolecular folding relationship of urinary MBPLM with the disability as
and b-sheet structure in this region.41 The humoral measured by the Kurtzke Expanded Disability Status
response to MBP appears to re¯ect restricted V-region Scale,49 and the transition from RR-MS to SP-MS.40 In
use leading to the existence of cross-reactive idio- addition to these clinical changes of disease status,
topes.42,43 there were also changes to show that urinary
MBPLM levels correlated in a statistically signi®cant
fashion with the burden of disease on T2-weighted
MBPLM in body ¯uids images on annual cranial MRI for both lesion number
In studies previously reviewed14 the general features of and lesion volume.40 There was an inverse correla-
MBPLM in CSF and urine have been described. tion between urinary MBPLM and new lesions as
Although potentially detectable in blood, there is no detected by cranial MRI. Urine measurements of
validated assay for MBPLM in that body ¯uid. In CSF MBPLM must be expressed in regard to renal
the size of MBPLM is greater than 30 000, presumably function. Although not ideal, at present the best
re¯ecting its attachment to another protein of un- approach is to relate the urinary MBPLM to
known identity, and, based on the binding features of creatinine.46,50 In studies of 24 h urine collections
the antibody to MBP which detects it, cross-reacts with which lead to a more accurate measure of the total
an epitope residing in residues 80 ± 89 exposed, i.e., MBPLM in urine, patients with SP disease also had
noncryptic, on the surface of the MBP molecule.44 higher urinary MBPLM values.40
Using the double antibody radioimmunoassay (RIA) In addition to their immunochemical differences,51
established in our laboratory, MBPLM is not found there are apparent differences in mechanism for the
normally in CSF but increases immediately after acute increase in MBPLM in CSF and urine. Currently, it is
CNS myelin injury in an etiologically nonspeci®c only possible to speculate about the difference in
manner. In contrast, urinary MBPLM has a molecular mechanisms as follows. Normally MBPLM appears in
weight of 51000 daltons, is not attached to any other urine, presumably originating from CNS myelin or
molecule and most closely simulates an epitope oligodendrocytes as part of the normal turnover of
residing within residues 83 ± 89 or 84 ± 89 of MBP. MBP. As remissions fail, remyelination does not occur,

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 Urine MBPLM in MS
JN Whitaker
245
axonal damage mounts and progression is detected. 2 The IFNB Multiple Sclerosis Study Group. (1993)
The rise in MBPLM in SP-MS may indicate the Interferon beta-1b is effective in relapsing-remitting
synthesis of MBP without its incorporation into the multiple sclerosis. I. Clinical results of a multicenter,
CNS myelin sheath and the subsequent release and randomized, double-blind, placebo-controlled trial. Neu-
rology 43: 655 ± 661.
degradation of MBP. The rise in the MBPLM in
3 Paty DW, Li DKB, UBC MS/MRI Study Group, IFNB
children at the end of active myelinogenesis presum- Multiple Sclerosis Study Group. (1993) Interferon beta-
ably correlates with the curtailment of MBP incorpora- 1b is effective in relapsing-remitting multiple sclerosis.
tion into myelin as myelinogenesis is completed.45 II. MRI analysis results of a multicenter, randomized,
MBPLM in CSF most likely comes from damage to double-blind, placebo-controlled trial. Neurology 43:
oligodendrocytes and CNS myelin which leads to the 662 ± 667.
release into CSF of MBP linked, as mentioned above, 4 Jacobs LD et al. (1996) Intramuscular interferon beta-1
to another protein. alpha for disease progression in relapsing multiple
Along with these correlative studies of the annual sclerosis. Ann Neurol 39: 285 ± 294.
changes in cranial MRI and urine MBPLM from the 5 Johnson KP et al. (1995) Copolymer 1 reduces relapse rate
and improves disability in relapsing-remitting multiple
interferon beta-1b trial,46 there were also serial
sclerosis: Results of a phase III multicenter, double-blind,
measures from the cohort of patients at the University placebo-controlled trial. Neurology 45: 1268 ± 1276.
of British Columbia where urine was collected and 6 Noseworthy JH, O'Brien PC, Mayo Clinic-Canadian
cranial MRIs performed every six weeks. In this group Cooperative MS Study Group. (1997) The Mayo Clinic-
of patients it was possible to examine the relationship Canadian Cooperative study of sulfasalazine (Salazopyr-
of the timing of the increase of urinary MBPLM to the in EN) in active multiple sclerosis: preliminary report.
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disease. Based on this group of frequently studied 7 Weiner HL et al. (1993) Double-blind pilot trial of oral
patients, the transition clinically from RR to SP MS tolerization with myelin antigens in multiple sclerosis.
occurred some time after the rise in the urinary Science 259: 1321 ± 1324.
8 Andersen O et al. (1996) Linomide reduces the rate of
MBPLM level.40 There is a suggestion that the RR
active lesions in relapsing-remitting multiple sclerosis.
patients who were to become SP may have been Neurology 47: 895 ± 900.
different from the onset of the study in that a 9 Karussis DM et al. (1996) Treatment of secondary
difference between RR and SP MS patients in urinary progressive multiple sclerosis with the immunomodu-
MBPLM is signi®cant in the ®rst urine specimen lator linomide: A double-blind, placebo-controlled pilot
collected after six weeks in the trial. Neither the study with monthly magnetic resonance imaging evalua-
interferon beta-1b nor any subsequent trial has had tion. Neurology 47: 341 ± 346.
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11 Bedell BJ, Narayana PA, Wolinsky JS. (1997) A dual
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