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
(FDA) of the United States has approved three reagents
for treatment of relapsing-remitting (RR)1 multiple
sclerosis (MS). These include interferon beta-1b,2,3
interferon beta-1a4 and glatiramer acetate.5 These drug
approvals represent de®nite advances in the care of
the RR-MS patient; however, all three drugs are
expensive (approximately $11 000 US per year), have
only a partial effect on relapse reduction, and have
little, if any, effects on the progression of disease.
Furthermore, recent clinical trial experience, espe-
cially for secondary progressive (SP) MS, has been
marked by drug failure or study termination. Thus, the
trials of deoxyspergualin, sulfasalazine6 and cladribine
showed no effect on SP-MS. Oral myelin enriched in
myelin basic protein (MBP) showed no effect on RR-
MS in a phase 27 or phase 3 trial, and the phase 3 trial
of linomide, which had seemed promising based on
two phase 2 trials,8,9 had to be terminated because of
unanticipated myocardial toxicity.
A number of drugs and reagents are candidates for
clinical trials for MS, but the pace of performing
clinical trials is limited because of the long duration of
observation needed for determining clinical effective-
ness in a pivotal phase 3 trial of new therapy. The
decision to go forward with the expensive testing of a
phase 3 trial rests on an accurate phase 2 trial which,
because of the brevity, must rely on nonclinical
methods as their primary end points. Although
expensive and not always paralleling clinical de®cits,
cranial magnetic resonance imaging (MRI) is the usual
method chosen. All of these circumstances place
emphasis in having more reliable markers for deter-
mining treatment effectiveness beyond the changes in
quantitated tissue signals on serial cranial MRI or in a
Correspondence: JN Whitaker
scored clinical examination which are presently
recommended as primary end points for phase 2 and
phase 3 trials, respectively.10
A surrogate marker is a nonclinical assessment
which may predict an ultimate clinical change. The
clinical events in MS which are of greatest interest to
relate to a surrogate marker are those of the changes in
disease activity, that is, relapses and remissions, and
the change in status, that is, the transition from RR to
SP disease.1 Whether SP-MS represents the failure of
remission or other processes that may transpire in
primary progressive (PP) MS remains to be deter-
mined. The surrogate marker most commonly used in
MS is that of serial cranial MRI which may be
performed according to a number of techniques.
Segmentation analysis seems particularly attractive
because of its automated and quantitative features.11
Other measures such as improved clinical scales,12
electrodiagnostic methods, primarily evoked poten-
tials, and measurement of a variety of constituents in
body ¯uids also represent potential avenues to
explore. The major substances in body ¯uids for
measurement and analysis include myelin constituents
and related enzymes, glial proteins, markers of general
neural tissue damage, elements of the immune system
and a number of miscellaneous materials. The subject
of the present chapter is to review the current status of
studies on myelin basic protein-like material (MBPLM)
in the urine. Two references13,14 contain previous
reviews by the author on this and related topics.
Features of myelin basic protein
Central nervous system (CNS) myelin has at least
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-
tein, myelin oligodendrocyte glycoprotein, oligo-
 Urine MBPLM in MS
JN Whitaker
244
dendrocyte myelin glycoprotein and myelin oligoden-
drocyte speci®c protein.15 While the quantitation of all
of these in body ¯uids may have potential as surrogate
markers, MBPLM has been the most intensively
studied and offers the greatest clinical use at present.
As recently reviewed,14 MBP accounts for 30% of
CNS myelin proteins,16 is encoded by a single gene of
seven exons located on chromosome 1817 and is
normally expressed only by oligodendrocytes and
Schwann cells.18,19 Alternate splicing of the MBP
transcript gives rise to at least four isoforms of human
MBP with the major ones having molecular weights of
21.5 kd (all seven exons) and 18.5 kd (exons 1 and 3 ±
7).19 The 18.5 kd isoform containing 170 amino acid
residues dominates in adult human CNS myelin. In
addition to multiple isoforms, MBP may have a
number of post-translational modi®cations,20 such as
amino-terminus acylation,21 selected phosphorylation
of serine and threonine,22,23 deamidation of glutamine,
loss of C-terminal arginine23 ± 25 and methylation of the
arginine at residue 10726 that result in charged isomers.
Of particular interest is the heavily citrullinated
isomer, designated as MBP-C8, which, created by the
citrullination of six of the 19 arginine residues present
in MBP, results in a less basic MBP.27 MBP-C8 is
formed early in myelinogenesis,28 is relatively well
preserved in MS brain tissue and is immunogenic for
humoral29 and cellular30 respones.
MBP has multiple independent epitopes throughout
its 170 residues,31,32 and a multideterminant model is
likely.32 Certain regions of MBP appear to have special
relevance to MS. MBP peptide 80 ± 100 contains the
encephalitogenic epitopes for the SJL mouse33,34 and
Lewis rat,35 the dominant epitope for antibody to MBP
in brain and cerebrospinal ¯uid (CSF),36 the dominant
epitope for T cell reactivity in MS37 and the regions
cross-reacting most closely with MBPLM in CSF38 and
urine.39,40 There is evidence for intramolecular folding
and b-sheet structure in this region.41 The humoral
response to MBP appears to re¯ect restricted V-region
use leading to the existence of cross-reactive idio-
topes.42,43
MBPLM in body ¯uids
In studies previously reviewed14 the general features of
MBPLM in CSF and urine have been described.
Although potentially detectable in blood, there is no
validated assay for MBPLM in that body ¯uid. In CSF
the size of MBPLM is greater than 30 000, presumably
re¯ecting its attachment to another protein of un-
known identity, and, based on the binding features of
the antibody to MBP which detects it, cross-reacts with
an epitope residing in residues 80 ± 89 exposed, i.e.,
noncryptic, on the surface of the MBP molecule.44
Using the double antibody radioimmunoassay (RIA)
established in our laboratory, MBPLM is not found
normally in CSF but increases immediately after acute
CNS myelin injury in an etiologically nonspeci®c
manner. In contrast, urinary MBPLM has a molecular
weight of 51000 daltons, is not attached to any other
molecule and most closely simulates an epitope
residing within residues 83 ± 89 or 84 ± 89 of MBP.
Moreover, the MBP epitope which cross-reacts with
urinary MBPLM is cryptic, i.e., antibodies to MBP
which recognize this epitope react with certain MBP
peptides but not the intact MBP molecule. Urine
contains low amounts of MBPLM shortly after birth
that rise to adult levels at 1 ± 2 years of age and then to
above normal adult levels during childhood.45 These
levels return to adult levels at about the age of 10
years. During the differing phases in disease activity
typical of RR-MS, urine MBPLM does not increase but
does so when RR-MS patients change status from the
RR to the SP pattern of disease.40 Urinary MBPLM is
higher in MS patients than in controls.46
Perhaps instructive for the detection of peptides,
whether free or bound, of other proteins in body ¯uids,
several major laboratory steps were required for the
RIA for urine MBPLM to work. First, it was necessary
to understand the epitopes of MBP, especially that it
could have cryptic determinants that were not
expressed in the intact molecule.47 Second, the correct
sequence of MBP in the span of residues in 80 ± 89 had
to be corrected. This included the correct assignment
at residues 83 ± 84 of glutamine-aspartic acid rather
than glutamic acid-asparagine.39,48 Third, the RIA
components, especially the choice of standards, had
to be optimized in order to enhance assay validity
when the exact chemical identity of MBPLM is not yet
known.
The relationship of MS events to urinary MBPLM
has been more complicated to delineate than that of
CSF MBPLM. It is clear that the urinary MBPLM
does not correlate with acute disease activity in RR-
MS indicated either by clinical changes, cranial MRI
with gadolinium-enhancing lesions or an increase in
MBPLM in CSF.46 However, based primarily on the
urine studies performed in conjunction with the
multicenter trial of interferon beta-1b,2,3 there was a
relationship of urinary MBPLM with the disability as
measured by the Kurtzke Expanded Disability Status
Scale,49 and the transition from RR-MS to SP-MS.40 In
addition to these clinical changes of disease status,
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
images on annual cranial MRI for both lesion number
and lesion volume.40 There was an inverse correla-
tion between urinary MBPLM and new lesions as
detected by cranial MRI. Urine measurements of
MBPLM must be expressed in regard to renal
function. Although not ideal, at present the best
approach is to relate the urinary MBPLM to
creatinine.46,50 In studies of 24 h urine collections
which lead to a more accurate measure of the total
MBPLM in urine, patients with SP disease also had
higher urinary MBPLM values.40
In addition to their immunochemical differences,51
there are apparent differences in mechanism for the
increase in MBPLM in CSF and urine. Currently, it is
only possible to speculate about the difference in
mechanisms as follows. Normally MBPLM appears in
urine, presumably originating from CNS myelin or
oligodendrocytes as part of the normal turnover of
MBP. As remissions fail, remyelination does not occur,

axonal damage mounts and progression is detected.
The rise in MBPLM in SP-MS may indicate the
synthesis of MBP without its incorporation into the
CNS myelin sheath and the subsequent release and
degradation of MBP. The rise in the MBPLM in
children at the end of active myelinogenesis presum-
ably correlates with the curtailment of MBP incorpora-
tion into myelin as myelinogenesis is completed.45
MBPLM in CSF most likely comes from damage to
oligodendrocytes and CNS myelin which leads to the
release into CSF of MBP linked, as mentioned above,
to another protein.
Along with these correlative studies of the annual
changes in cranial MRI and urine MBPLM from the
interferon beta-1b trial,46 there were also serial
measures from the cohort of patients at the University
of British Columbia where urine was collected and
cranial MRIs performed every six weeks. In this group
of patients it was possible to examine the relationship
of the timing of the increase of urinary MBPLM to the
clinical change in disease status from RR to SP
disease. Based on this group of frequently studied
patients, the transition clinically from RR to SP MS
occurred some time after the rise in the urinary
MBPLM level.40 There is a suggestion that the RR
patients who were to become SP may have been
different from the onset of the study in that a
difference between RR and SP MS patients in urinary
MBPLM is signi®cant in the ®rst urine specimen
collected after six weeks in the trial. Neither the
interferon beta-1b nor any subsequent trial has had
patients strati®ed at randomization or entry by
measures of urinary MBPLM. It is possible that such
a distinction might allow for more appropriate group-
ing of patients for determining effectiveness of
treatment.
Many questions remain about surrogate markers in
general. Two major goals relate to further research on
urinary MBPLM. First, it is necessary to de®ne the
exact chemical identity of MBPLM in urine and CSF.
This would permit assays to be improved, furnish
guidance to try again to detect and quantitate MBPLM
in blood, and help clarify the relationships and
potential roles of MBPLM in body ¯uids. Second, it
must be determined if and how the group data relating
changes in urinary MBPLM to disease progression and
MRI change can be applied to individual patients.
Once accomplished, urinary MBPLM could be utilized
in individual patients to direct treatment and gauge
effectiveness.
Acknowledgements
The author wishes to thank Mr R David Kachelhofer
and Ms Linda Brent for excellent assistance in the
preparation of the manuscript.
References
1 Lublin FD, Reingold SC. (1996) De®ning the clinical
course of multiple sclerosis: Results of an international
survey. Neurology 46: 907 ± 911.
Urine MBPLM in MS
JN Whitaker
245
2 The IFNB Multiple Sclerosis Study Group. (1993)
Interferon beta-1b is effective in relapsing-remitting
multiple sclerosis. I. Clinical results of a multicenter,
randomized, double-blind, placebo-controlled trial. Neu-
rology 43: 655 ± 661.
3 Paty DW, Li DKB, UBC MS/MRI Study Group, IFNB
Multiple Sclerosis Study Group. (1993) Interferon beta-
1b is effective in relapsing-remitting multiple sclerosis.
II. MRI analysis results of a multicenter, randomized,
double-blind, placebo-controlled trial. Neurology 43:
662 ± 667.
4 Jacobs LD et al. (1996) Intramuscular interferon beta-1
alpha for disease progression in relapsing multiple
sclerosis. Ann Neurol 39: 285 ± 294.
5 Johnson KP et al. (1995) Copolymer 1 reduces relapse rate
and improves disability in relapsing-remitting multiple
sclerosis: Results of a phase III multicenter, double-blind,
placebo-controlled trial. Neurology 45: 1268 ± 1276.
6 Noseworthy JH, O'Brien PC, Mayo Clinic-Canadian
Cooperative MS Study Group. (1997) The Mayo Clinic-
Canadian Cooperative study of sulfasalazine (Salazopyr-
in EN) in active multiple sclerosis: preliminary report.
Neurology 48: A340.
7 Weiner HL et al. (1993) Double-blind pilot trial of oral
tolerization with myelin antigens in multiple sclerosis.
Science 259: 1321 ± 1324.
8 Andersen O et al. (1996) Linomide reduces the rate of
active lesions in relapsing-remitting multiple sclerosis.
Neurology 47: 895 ± 900.
9 Karussis DM et al. (1996) Treatment of secondary
progressive multiple sclerosis with the immunomodu-
lator linomide: A double-blind, placebo-controlled pilot
study with monthly magnetic resonance imaging evalua-
tion. Neurology 47: 341 ± 346.
10 Whitaker JN, McFarland HF, Rudge P, Reingold SC.
(1995) Outcomes assessment in multiple sclerosis clin-
ical trials: A critical analysis. Multiple Sclerosis 1: 37 ±
47.
11 Bedell BJ, Narayana PA, Wolinsky JS. (1997) A dual
approach for minimizing false lesion classi®cations on
magnetic resonance images. MRM 37: 94 ± 102.
12 Rudick R et al. (1997) Recommendations from the
National Multiple Sclerosis Society Clinical Outcomes
Assessment Task Force. Ann Neurol 42: 379 ± 382.
13 Whitaker JN, Snyder DS. (1982) Myelin components in
the cerebrospinal ¯uid in diseases affecting central
nervous system myelin. Clin Immunol Allergy 2: 469 ±
482.
14 Whitaker JN. (1998) Myelin basic protein in cerebrosp-
inal ¯uid and other body ¯uids. Mult Scl 4: 16 ± 21.
15 Cuzner ML, Norton WT. (1996) Biochemistry of demye-
lination. Brain Pathol 6: 231 ± 242.
16 Morell P, Quarles RH, Norton WT. (1989) Formation,
structure and biochemistry of myelin. In: Siegel G,
Agranoff B, Albers RW, Molinoff P, (eds). Basic
neurochemistry Raven Press: New York 109 ± 136.
17 Roach A et al. (1983) Characterization of cloned cDNA
representing rat myelin basic protein: absence of expres-
sion in brain in shiverer mutant mice. Cell 42: 799 ± 806.
18 Campagnoni AT. (1988) Molecular biology of myelin
proteins from the central nervous system. J Neurochem
51: 1 ± 14.
19 Kamholz J, deFerra F, Puckett C, Lazzarini RA. (1986)
Identi®cation of three forms of human myelin basic
protein by cDNA cloning. Proc Natl Acad Sci USA 83:
4962 ± 4966.
20 Moscarello MA. (1990) Myelin basic protein: A dynami-
cally changing structure. In: Hashim GA, Moscarello M,
(eds.) Dynamic Interactions of Myelin Proteins John
Wiley: New York 25 ± 48.
 Urine MBPLM in MS
JN Whitaker
246
21 Hashim GA, Eylar EH. (1969) The structure of the
terminal regions of the encephalitogenic basic protein
from bovine myelin. Arch Biochem Biophys 135: 324 ±
333.
22 Ramwani JJ, Epand RM, Moscarello MA. (1989) Second-
ary structure of charge isomers of myelin basic protein
before and after phosphorylation. Biochemistry 28:
6538 ± 6543.
23 Chou FC-H, Chou C-HJ, Shapira R, Kibler RF. (1976)
Basis of microheterogeneity of myelin basic protein. J
Biol Chem 251: 2671 ± 2679.
24 Deibler GE, Martenson RE. (1973) Chromatographic
fractionation of myelin basic protein. Partial character-
ization and methylarginine contents of multiple forms. J
Biol Chem 248: 2392 ± 2396.
25 Deibler GE et al. (1975) The contribution of phosphor-
ylation and loss of COOH-terminal arginine to the
microheterogeneity of myelin basic protein. J Biol Chem
250: 7931 ± 7938.
26 Baldwin GS, Carnegie PM. (1971) Isolation and partial
characterization of methylated arginines from the en-
cephalitogenic basic protein of myelin. Biochem J 123:
69 ± 74.
27 Wood DD, Moscarello MA. (1989) The isolation, char-
acterization, and lipid-aggregating properties of a citrul-
line containing myelin basic protein. J Biol Chem 264:
5121 ± 5127.
28 Fannon AM, Moscarello MA. (1990) Myelin basic protein
is affected by reduced synthesis of myelin proteolipid
protein in the jimpy mouse. Biochem J 268: 105 ± 110.
29 Whitaker JN et al. (1992) An immunochemical compar-
ison of human myelin basic protein and its modi®ed,
citrullinated form, C8. J Neuroimmunol 36: 135 ± 146.
30 Martin R et al. (1991) T-cell recognition of a citrulline-
containing myelin basic protein. Neurology 41: 257 ±
258.
31 Whitaker JN, Chou CHJ, Chou FCH, Kibler RF. (1979)
Antigenic regions for the humoral response to myelin
basic protein. Mol Immunol 16: 495 ± 501.
32 Day ED, Potter NT. (1986) Monoclonal and polyclonal
antibodies to myelin basic protein determinants. J
Neuroimmunol 10: 289 ± 312.
33 Fallis RJ, Raine CS, McFarlin DE. (1989) Chronic
relapsing experimental allergic encephalomyelitis in
SJL mice following the adoptive transfer of an epitope-
speci®c T cell line. J Neuroimmunol 22: 93 ± 105.
34 Sakai K et al. (1988) Characterization of a major
encephalitogenic T cell epitope in SJL/J mice with
synthetic oligopeptides of myelin basic protein. J
Neuroimmunol 19: 21 ± 32.
35 Offner H et al. (1989) T cell determinants of myelin basic
protein include a unique encephalitogenic I-E-restricted
epitope for Lewis rats. J Exp Med 170: 355 ± 367.
36 Warren KG, Catz I, Steinman L. (1995) Fine speci®city of
the antibody response to myelin basic protein in the
central nervous system in multiple sclerosis: The
minimal B-cell epitope and a model of its features. Proc
Natl Acad Sci USA 92: 11061 ± 11065.
37 Ota K et al. (1990) T-cell recognition of an immunodo-
minant myelin basic protein epitope in multiple sclero-
sis. Nature 346: 183 ± 187.
38 Bashir RM, Whitaker JN. (1980) Molecular features of
immunoreactive myelin basic protein in cerebrospinal
¯uid of persons with multiple sclerosis. Ann Neurol 7:
50 ± 57.
39 Gibson BW, Gilliom RD, Whitaker JN, Biemann K. (1984)
Amino acid sequence of human myelin basic protein
peptide 45-89 as determined by mass spectrometry. J Biol
Chem 259: 5028 ± 5031.
40 Whitaker JN et al. (1995) Urinary myelin basic protein-
like material as a correlate of the progression of multiple
sclerosis. Ann Neurol 38: 625 ± 632.
41 Stoner GL. (1984) Predicted folding of b-structure in
myelin basic protein. J Neurochem 43: 433 ± 447.
42 Zhou SR, Whitaker JN. (1990) An idiotype shared by
monoclonal antibodies to different peptides of human
myelin basic protein. J Immunol 145: 2554 ± 2560.
43 Zhou S-R et al. (1998) A cross-reactive anti-myelin basic
protein idiotope in cerebrospinal ¯uid cells in multiple
sclerosis. Neurology 50: 411 ± 417.
44 Whitaker JN, Bashir RM, Chou C-H, Kibler RF. (1980)
Antigenic features of myelin basic protein-like material
in cerebrospinal ¯uid. J Immunol 124: 1148 ± 1153.
45 Percy AK, Goodwin J, Whitaker JN. (1996) Myelin basic
protein-like material in urine: a developmental pro®le.
Ann Neurol 40: 297.
46 Whitaker JN et al. (1994) Correlation of clinical features
and ®ndings on cranial magnetic resonance imaging with
urinary myelin basic protein-like material in patients
with multiple sclerosis. Ann Neurol 35: 577 ± 585.
47 Whitaker JN, Chou CHJ, Chou FCH, Kibler RF. (1977)
Molecular internalization of a region of myelin basic
protein. J Exp Med 146: 317 ± 331.
48 Whitaker JN. (1984) Indicators of disease activity in
multiple sclerosis: Studies of myelin basic protein-like
materials. Ann NY Acad Sci 436: 140 ± 150.
49 Kurtzke JF. (1983) Rating neurologic impairment in
multiple sclerosis: An expanded disability status scale
(EDSS). Neurology 33: 1444 ± 1452.
50 Giovannoni G, Green A, Keir G, Thompson EJ. (1996)
Urinary myelin basic protein-like material as a correlate
of the progression of multiple sclerosis - Letter to Editor.
Ann Neurol 40: 128 ± 129.
51 Whitaker JN. (1987) The presence of immunoreactive
myelin basic protein peptide in urine of persons with
multiple sclerosis. Ann Neurol 22: 648 ± 655.