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Protein Electrophoresis in Clinical Diagnosis
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Protein Electrophoresis in
Clinical Diagnosis
David F Keren
Hodder Arnold
A MEMBER OF THE HODDER HEADLINE GROUP
First published in Great Britain in 2003 by
Hodder Arnold, a member of the Hodder Headline Group,
338 Euston Road, London NW1 3BH
http://www.hoddereducation.com
Whilst the advice and information in this book are believed to be true and
accurate at the date of going to press, neither the author[s] nor the publisher
can accept any legal responsibility or liability for any errors or omissions
that may be made. In particular (but without limiting the generality of the
preceding disclaimer) every effort has been made to check drug dosages;
however it is still possible that errors have been missed. Furthermore,
dosage schedules are constantly being revised and new side-effects
recognized. For these reasons the reader is strongly urged to consult the
drug companies’ printed instructions before administering any of the drugs
recommended in this book.
2 3 4 5 6 7 8 9 10
What do you think about this book? Or any other Hodder Arnold title?
Please send your comments to www.hoddereducation.com
To my wonderful family
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Contents
Preface ix
Acknowledgements xi
1 Protein structure and electrophoresis 1
2 Techniques for protein electrophoresis 15
3 Immunofixation, immunosubtraction, and immunoselection techniques 33
4 Proteins identified by serum protein electrophoresis 63
5 Approach to pattern interpretation in serum 109
6 Conditions associated with monoclonal gammopathies 145
7 Examination of urine for proteinuria 217
8 Approach to pattern interpretation in cerebrospinal fluid 259
9 Laboratory strategies for diagnosing monoclonal gammopathies 285
10 Case studies for interpretation 304
Index 395
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Preface
This text presents the use of protein electrophoresis resulted in better detection of bisalbuminemia by
of serum, urine, and cerebrospinal fluid in clinical laboratories using capillary zone electrophoresis.
diagnosis. It is a revision of two previous books on That technique also has enhanced our ability to
this subject with several substantive and many detect genetic variants such as α1-antitrypsin
trivial changes. The title has been changed from inhibitor deficiencies (PiZZ and PiSZ) as well as
High-Resolution Electrophoresis and Immuno- benign variants.
fixation: Techniques and Interpretations to the In addition to technical advances in the general
present one in recognition of the fact that the infor- detection and quantification of specific proteins, the
mation in this book may be useful to individuals techniques available to identify the monoclonal
who interpret a wide variety of electrophoretic gels proteins have also changed significantly. Immuno-
(high-resolution or not). subtraction using capillary zone electrophoresis was
There have been several significant changes in the not even mentioned in the second edition. Now,
field since the second edition of High-Resolution immunosubtraction by automated technology is
Electrophoresis and Immunofixation: Techniques used in many laboratories that employ capillary
and Interpretation was published in 1994 by zone electrophoresis. It is highly efficient, but suf-
Butterworth-Heinemann. That text was largely fers from a lack of sensitivity and flexibility when
written in 1993, making it 10 years old at the time compared with immunofixation. This is discussed
of publication of this book. At the time the second in the present text. For laboratories using some of
edition was written there was no high-resolution the newer semi-automated gel-based techniques,
technique available that provided automated or semi-automated immunofixation is now available.
semi-automated methods for laboratories with a Beyond the instruments, there are new reagents
large clinical volume of testing. There are now available such as the ‘Penta’ (pentavalent) reagent
several products presented, with examples. Some of that one company supplies as a potential screen for
them are gel-based, others use capillary zone monoclonal proteins. Immunoselection was also
electrophoresis. Some automated gel-based systems just touched on in the second edition. Now that g
have achieved an excellent degree of resolution that heavy chain disease is more readily recognized, a
allows efficient performance of high-quality tech- broader discussion of immunoselection is provided.
niques by even moderately sized institutions. At the Advances have also been made in reagents avail-
time of the second edition, capillary zone electro- able for measurement of monoclonal free light
phoresis itself was quite new with no methods avail- chains by nephelometry in both serum and urine.
able that had been approved for use in the clinical Recent publications indicate that measurement of
laboratory by the Food and Drug Administration. I free light chains in serum may be useful not only to
discussed it only briefly. Now two such instruments follow patients with known monoclonal free light
are already in place in many laboratories and there chains (Bence Jones proteins), but also to assist in
have been several publications about the advan- the difficult diagnoses of amyloid (AL) and even
tages, artifacts and limitations of these techniques. non-secretory myeloma. This is discussed in
Improved resolution especially of protein bands has Chapters 6 and 7.
x Preface
In the second edition, high-resolution agarose gels from our clinicians. Our current sign-outs are pre-
were preferred for detecting oligoclonal bands in sented in tabular form and readers are encouraged
the cerebrospinal fluid from patients whose differ- to use them if they wish.
ential diagnosis included multiple sclerosis. Now, A couple of concepts were removed from this
the recommendation is the use of isoelectric focus- edition. The technique of two-dimensional electro-
ing or an immunofixation technique to identify the phoresis, while useful in research has not caught on
bands as immunoglobulins. One company cur- in the clinical laboratory and is not discussed in
rently offers a semi-automated immunofixation this volume. Further, the indirect immunofluores-
method for detecting oligoclonal bands (O-bands) cence techniques reviewed in the previous texts has
in cerebrospinal fluid on unconcentrated fluid. been replaced by immunohistochemical and flow
There have also been improvements in the detec- cytometry studies.
tion of leakage of cerebrospinal fluid into nasal and Finally, in this text I recommend the use of the
aural fluids; these are discussed in Chapter 8. term monoclonal free light chain (MFLC) to
Beyond technical issues, there have been improve- replace the term Bence Jones proteins. It is both a
ments in our knowledge of proper utilization and practical and a technical improvement. Some indi-
interpretation of protein electrophoresis when viduals confuse the presence of an intact
searching for the presence of a monoclonal immunoglobulin monoclonal gammopathy in the
gammopathy. In 1998, the College of American urine with a Bence Jones protein. It is not. Bence
Pathologists Conference XXXII convened a panel Jones protein is a monoclonal free light chain and
of experts to provide recommendations for the has much greater significance to the patient’s diag-
clinical and laboratory evaluation of patients sus- nosis and prognosis than an intact immunoglobulin
pected of having a monoclonal gammopathy. monoclonal gammopathy in the urine. The term
These guidelines were published in 1999 and MFLC says exactly what we found (i.e. a mono-
provide an important framework that emphasizes clonal free light chain). So it is technically correct
the partnership of clinicians with laboratory and removes any possible ambiguity. This has the
workers. advantage that individuals will no longer use a
Urine evaluation has improved with the newer hyphen that Dr Henry Bence Jones never used on
technologies and has been complicated by some any of the papers or books that he wrote.
current procedures. For example, individuals I hope you enjoy this book, or at least find it
receiving a pancreas transplant may have the useful. Your comments and questions help to
exocrine pancreas drainage empty into the urinary provide a focus for making this text more relevant
bladder. As discussed in Chapter 7, this results in g- to the use of electrophoresis in clinical diagnosis.
migrating bands that may be mistaken for Please feel free to contact me via e-mail:
monoclonal gammopathies. They are exocrine [email protected].
pancreas secretions.
The method I suggested in the first edition and David F. Keren, MD
expanded in the second for tailored reporting with Ann Arbor, Michigan, USA
detailed sign-outs has been improved by feedback January 12, 2003
Acknowledgements
I wish to thank my family and especially my wife Jeffrey S. Warren provided helpful suggestions in
for their great patience with me as I worked reviewing selected portions of this book. Their
through this task. input kept many outrageous statements out of the
I am deeply grateful to the many people who final copy, and corrected several errors of omis-
helped in the development of the materials for this sion.
and previous editions of this book. They allowed Special thanks are due to Ron Gulbranson and
me to use their clinical material or illustrations to Debbie Hedstrom who prepared many of the elec-
give the reader a first-hand view of the potential trophoretic gels, electropherograms, and special
uses and limitations of electrophoretic techniques studies that are in the present book. They have had
in the clinical laboratory. Figures, Tables or spe- endless patience with me while I cluttered their
cific cases were kindly provided by Dr R. S. work area with capillary zone electropherograms
Abraham, Dr Francesco Aguzzi, Dr Arranz-Peña, and gels containing interesting cases.
Dr Gary Assarian, Cynthia R. Blessum, Dr Xavier Finally, I also thank the many individuals who
Bossuyt, Dr Arthur Bradwell, Dr. Stephen O. have taken the time to write, telephone or speak to
Brennan, Chrissie Dyson, Dr Beverly Handy, me personally about some aspect of my work.
Margaret A. Jenkins, Carl R. Jolliff, Dr Jerry They have framed many questions about concepts
Katzmann, Dr Robert H. Kelly, Dr Joseph M. or omissions from previous work that I hope to
Lombardo, Dr A. C. Parekh, Dr Jeffrey Pearson, clarify in this text.
Dr Arthur J. Sloman, Dr Lu Song, Dr Tsieh Sun, Dr
E. J. Thompson, Dr Adrian O. Vladutiu and David F. Keren, MD
Dorothy Wilkins. Ann Arbor, Michigan, USA
Drs John M. Averyt, John L. Carey, III, and January 12, 2003
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1
Protein structure and
electrophoresis
The term ‘electrophoresis’ refers to the migration These are the D- and L-forms (Fig. 1.2). In proteins,
of charged particles in an electrical field. The the L-form is almost always present.1
success of electrophoresis in separating serum,
L-Serine D-Serine
urine and cerebrospinal fluid proteins into clini-
COOH COOH
cally useful fractions results from the heterogeneity | |
of the charges of these molecules. It is useful, there- H2N ⎯ C ⎯ H H⎯ C ⎯ NH2
| |
fore, to review briefly the structural features that CH2 CH2
result in the observed migration. | |
H2 COH H2 COH
not migrate in an electrical field. Of course, the most hydrophobic amino acids exist as zwitterions
charge on each amino acid depends on both the R (Fig. 1.3). However, when large numbers of
group and the pH of the solution in which it is hydrophobic amino acids occur in other molecules,
dissolved. such as occasional products of malignant plasma
By altering the pH of an aqueous solution, the cell clones, they may exhibit properties that cause
charge on an amino acid can be changed. In acidic solubility problems. Hydrophobic amino acid
solution, the amino acid accepts a proton on its content is not the only factor that relates to solu-
carboxylate group, resulting in a net positive bility problems. For example, the complex cold-
charge on the molecule (Fig. 1.3). The positively precipitating property of cryoglobulins (see
charged cation thus formed will migrate toward Chapter 6) is not thought to be related to an excess
the negative pole (cathode) in an electrical field. of hydrophobic amino acids.2
Conversely, in basic solution the ammonium group
gives up a proton, leaving the amino acid with a Aliphatic Amino Acids
net negative charge (Fig. 1.3). This negatively
Glycine Alanine Valine Leucine
charged anion migrates toward the positive pole | | | |
(anode) in an electrical field. The pH of the solu- H CH3 CH CH2
|
tion and the nature of the R group (Fig. 1.4) have H3C CH3 CH
an important effect on the migration of individual
H3C CH3
amino acids. For example, both aspartic acid and
glutamic acid at pH 7.0 or greater are always non- Isoleucine Serine Threonine
protonated (negatively charged) and consequently | | |
H⎯C⎯CH3 CH2 H⎯ C ⎯ OH
are referred to as aspartate and glutamate. At | | |
neutral pH, arginine, histidine and lysine are all CH2 H2COH CH3
|
protonated (positively charged). These differences CH3
in charge are important in determining the migra- (a)
tion of proteins during electrophoresis. Further, in
Aromatic Amino Acids
genetic variants such as a1-antitrypsin deficiency,
the substitution of a charged amino acid for a Phenylalanine Tyrosine Tryptophan
neutral amino acid, or the converse, will alter the CH2 CH2 CH2
migration of the variant form that allows us to
detect the abnormality. NH
The solubility of proteins also is related to their
amino acid composition. For example, tyrosine, OH
threonine and serine have hydroxyl moieties in
their R group, and readily form hydrogen bonds
with water. Similarly, asparagine and glutamine
Sulfur-Containing Amnio Acids
have amide R groups that are hydrophilic. In con-
trast, several amino acids are hydrophobic. Large Cysteine Methionine
(Æschines)
What is he else?
(Thyonichus)
A gentleman: a man
É
6. Lesquier, J. Les institutions militaires de l'Égypte sous les
Lagides (1911).
7. Kornemann, E. Ægypten und das (römische) Reich. In
Gercke and Norden's, Einleitung in die
AItertumswissenschaft (1912), pp. 272 ff.
8. Mitteis, L., und Wilcken, U. Grundzüge und
Chrestomathie der Papyruskunde (1912).
FOOTNOTES:
[77] Diodorus, xviii, 4.
[78] Omitting the shadowy Eupator, Philopator Neos, and
Alexander II.
[79] Bouché-Leclercq, Histoire des Lagides, ii, p. 278; Plut.,
Antony, 54; Dio Cassius, xlix, 41.
[80] Pensées, vi, 43 bis. Ed. Havet; cf. Bouché-Leclercq, Hist. des
Lagides, ii, p. 180, n. 1.
[81] Gesammelte Schriften, iv, p. 256.
[82] Idyll, xvii. (Translation of Calverley.)
[83] See Droysen, Geschichte des Hellenismus,2 iii, pp. 262 ff.
[84] Plut., Aratus, xv.
After this rapid survey of the political history of the Seleucids, I wish
to devote the remainder of this chapter to considering the domestic
policy of the dynasty. I shall point out that Seleucus and his
successors continued Alexander the Great's work of founding city-
states in Asia, and that, having to deal with priestly communities and
feudal lords as well as with the occupants of the widespread royal
domains, they refused to exempt from their direct control any lands
not placed under the jurisdiction of a city-state. I shall discuss briefly
the internal structure of the city-states and more at length their
relation in theory and fact to the monarch. This will finally bring up
for examination the policy of Antiochus IV, in whose reign the
internal as well as the external development of the Seleucid empire
culminated.
Seleucus had been advanced to high position in Alexander's service
after Alexander had disclosed his purpose of fusing the
Macedonians, Greeks, and Iranians into a new cosmopolitan race.
The promotion obtained by him during the course of the bitter
struggle which this policy occasioned suggests that he made
Alexander's point of view his own. This surmise as to the attitude of
Seleucus is confirmed by the fact that he took his place, after
Alexander's death, with those who upheld the cause of Alexander's
family, and left it for his Babylonian satrapy only when it became
clear that to remain meant to perish without accomplishing anything.
Seleucus was the only one of the great Macedonian captains who did
not take Alexander's death as a license to discard their Iranian
wives. The Bactrian maiden assigned to him at the great Susian
marriage—Apama, daughter of Spitamenes—became his queen and
the mother of his heir, Antiochus. The Seleucid dynasty was,
accordingly, half Iranian from the start; and for the policy which it
inherited from Antigonus and Alexander, and which it prosecuted
vigorously till the death of Antiochus IV in 164 b.c., namely, the
Hellenization of Asia, it had as warrant the practice of its idealized
founder.
Of the Greek cities which Seleucus planted in his realm, fifty-nine are
named by Appian.[108] They lay especially in Syria, in the district
between the Euphrates and the sea which he sought to make a
second Macedonia. His son Antiochus, to whom he gave the
administration of the eastern satrapies in 293 b.c., was particularly
active in developing cities on the Greek model in that region; and to
him and his son Antiochus Theos belongs the honor of establishing
the urban habits of Hellenic life in the interior of Asia Minor. In the
arm of their realm which reached through this peninsula to the rear
of the Greek strip on the Anatolian coast, new cities sprang up under
their auspices by the score. Nor did the movement come to an end
with the reign of the second Antiochus in 246 b.c., though it
probably weakened at that time. Over two generations later, in the
reign of Antiochus IV, it was again resumed actively and directed
especially into Palestine, which had been newly added to the realm.
The most striking feature in the internal policy of the Seleucids is the
attempted transfer into Asia of the urban form of life theretofore
characteristic of Hellas. Evidently, these monarchs believed with
Aristotle, Alexander, and, we may add, Polybius, and, indeed, all
Greeks, that men who did not live in cities were uncivilized men.
Evidently, too, they thought it wise and possible to civilize their
dominions.
It is not easy to form a definite impression of the political situation in
the Seleucid realm when the Macedonians first took possession, but
cities in the Greek sense seem to have been entirely absent. This
does not mean that towns were lacking altogether, since, even if we
disregard administrative centres like Babylon, Susa, Ecbatana,
Persepolis, we may use the term "town" properly of places like
Bambyce in Syria, fourteen miles west of the Euphrates. There, in a
fertile, well-watered valley, stood a famous temple of Atargatis, the
Syrian goddess. It was a wonderful establishment, as readers of
Lucian know, with its wide approaches, its obelisk-like phalli, its
sacred fish-pond, its shocking rites. At its head were emasculated
priests, who not only conducted the ceremonies and directed a far-
reaching proselytism, but also governed the community of temple
slaves (hieroduli) who tilled the land of the neighborhood for their
own support and that of the church. This clerical form of local
organization had been carefully fostered by the Persians. We all
know how in reorganizing Judæa they put the common people, who
were there simply to pay tithes, under the control of the high priest,
elders, and Levites, and made the so-called law of Moses the civil
law of the land. They proceeded in similar fashion throughout Asia
Minor. There Seleucus found scores of towns, big and little, to which
the description, given by Strabo[109] of the sacred city of Ma at
Comana in Cappadocia, is applicable: "In itself it is," he says, "a
notable city, but most of its inhabitants are god-possessed, or
temple slaves. They are all of Cataonian stock and are subject
generally to the authority of the king, but are under the immediate
control of the priest. He is lord at once of the temple and of the
temple slaves, of whom there were more than six thousand,
including men and women, at the time of my visit. Attached to the
temple is much land, of which the priest enjoys the revenues, and
there is no one in Cappadocia of higher dignity than he except the
king." Similar to this was the temple of Zeus at Venasa with its three
thousand temple slaves and its land which yielded to its priest an
annual income of twenty thousand dollars (fifteen talents); the
temple of Zeus Asbamæus near Tyana, of Apollo in Cataonia, Mater
Zizimene near Iconium, and Artemis Perasia in Castabala. Similar,
too, was the temple of Ma in Pontic Comana with its swarming mart,
its wide acres, and its six thousand temple slaves, of whom the
young women, here as elsewhere, were sacred prostitutes; the
temple of Anaitis in Zela, of Men in Cabira, of Selene in Iberia, of the
Great Mother at Pessinus, of Zeus at Olba, and of other gods in
other places scattered through Phrygia, Pisidia, and Lydia, as well as
Palestine, Syria, and Babylonia. The Seleucids must have found the
body of their empire thickly studded with religious communities,
each subject to its own code of divine law, each dominated by a
masterful and long-petted theocracy. Under this baleful rule the
whole country had settled gradually down during the Persian time in
progressive political and economic stagnation.
Apart from the mountains and the deserts, where tribal and nomadic
liberty reigned,—a constant menace to central government,—the
peoples of Asia lived in villages when the Macedonians came. Many
of these villages, villagers and all, were owned by princelings and
noblemen, who, if natives, had been undisturbed by the Persians, if
Iranians, had come into their possessions by reason of royal grants.
All through Syria and Asia Minor may be seen to-day the ruins of
"square towers" (tetrapyrgiæ) and manorial castles such as these
grandees built and fortified for defense against their neighbors and,
if need be, against the royal authority itself.[110] Those who built
them had apparently had little loyalty to the Persian king, but also
little inclination to obey his successor. They were, accordingly,
ejected right and left. Of the estates thus obtained, the Macedonian
kings could dispose at their pleasure. They formed again part of the
royal domain, which stretched in all directions at the edges of the
deserts and the mountains, among the temple lands, the feudal
fiefs, and administrative cities—a veritable archipelago of landed
property, tilled for the crown by myriads of royal serfs. Here was the
ὑλη the material, of which the Seleucids founded many of their city-
states.
The handle for reorganization which the priestly towns offered to the
new government was often the non-ecclesiastical part of the
population. That the temple was regularly the centre of local trade
and the scene of a recurrent bazaar tempted to its proximity money-
changers and the like. When the Greek immigration began this
element was naturally strengthened. It was, therefore, possible for
the Seleucids to give it an urban organization—a general assembly, a
council, and magistrates; and in this way to create a new city-state.
According to invariable Greek practice, however, such a city
controlled—with certain limitations—its own shrines. The great
temples of Apollo at Delphi and Delos, for example, were governed
by the citizens of those towns. Hence the natural policy for the
Seleucids, and the one which they in fact followed wherever
practicable, was to subordinate the high priests and clergy to the
adjacent urban authorities, thus solving the ecclesiastical question in
a way convenient for themselves and agreeable to European feeling.
Where this did not prove practicable, they often despoiled the
temple of its lands for the benefit of their followers. Thus the village
of Bætocæce was taken from the local temple of Zeus and given to a
certain Demetrius, and the sacred land of Zeus of Æzani was divided
into lots, which were assigned to cleruchs, subjected to a tax, and
attached to the financial jurisdiction of an adjoining city.[111] It is
simply another aspect of the same general policy when king after
king sought to lay "impious" hands upon the treasures stored up in
the temples of Bel, Anaitis, Atargatis, and Jehovah.
The secularization of religious properties was a very difficult matter,
and it was not pushed at all times with equal vigor by the Seleucids.
When the monarchs were embarrassed by foreign or domestic
troubles, they had to conciliate the priests even to the point of
undoing what they had already done. How easily a reaction might
occur we can perceive from the case recorded in the splendid
inscription which Mr. Butler has recently found cut on the inner wall
of the great temple of Artemis at Sardis.[112] A certain
Mnesimachus, presumably a Macedonian officer or adventurer, had
got a huge fief from King Antigonus. It consisted of the village of
Tobalmura in the Sardian plain and its appurtenances, the villages of
Tandu and Combdilipia. On these he had to pay annual dues of £50
to the proper chiliarchy, or subdivision of the satrapy. Near by, in
Cinara was a kleros, or lot, on which he paid £3 yearly. The fief
consisted, in addition, of the village of Periasasostra, of which the
annual dues, payable to another chiliarchy, were £57. Close by, in
Nagrioa was again a kleros, on which he paid £3 7s. yearly. The fief
consisted, moreover, of the village of Ilu in the territory of the well-
known city of Attuda, of which the dues, paid annually to the city of
course, were £3 5s. The manor (aule) of the fief was in the village of
Tobalmura, and together with certain lodges held by bailiffs and
certain gardens and parks tilled by manorial serfs in Tobalmura and
Periasasostra, it had once been assigned to Pytheus and Adrastus,
likewise Macedonians; but later on it had seemingly come into the
possession of Mnesimachus. The happy holder of this great estate
enjoyed, doubtless, the total net yield of the manor and the lots,
and, in addition, as the possessor of the manor and lots, rights to
exact services in money, kind, and labor from the villeins of the
villages. Had he been so minded he might have settled down in
Lydia and become a baron like the Iranian nobles whom the
Macedonians dispossessed; and, doubtless, many Macedonians and
Greeks established themselves in Asia in this fashion. Mnesimachus,
however, wanted money rather than "rights"; and, turning to the
temple of Artemis in Sardis, he secured a loan of £1325 from the
temple treasurers. The inscription cut on the temple wall records the
sale to Artemis, with right to repurchase, of Mnesimachus's interest
in the fief. His inability to repay his loan resulted in the
aggrandizement of the temple.
Incidents of this sort were, doubtless, of frequent occurrence and
show what the Seleucids had to guard against. Their land policy
seems to have been prudent and far-sighted. They were bound to
deal cautiously with their gigantic domain, since from it came the
most valuable and stable portion of their revenues. On the other
hand, they found a limit set to the quantity which they could
profitably retain by the fact that, unlike the Ptolemies, they inherited
from the Persians a public service adequate, not to administer, but
simply to supervise administration. They did indeed increase the
number of their satrapies, without, perhaps, diminishing the number
of chiliarchies or hyparchies into which each satrapy was divided,
and they seem to have paralleled the general service by a distinct
fiscal service and by a distinct priestly service; but, none the less,
they had to leave the details of fiscal, judicial, and religious
administration to the villages. Though we are singularly ill-informed
as to how they organized the villages, it is conceivable that they
picked out certain persons, like the elders in the Egyptian villages,
and held them responsible for the taxes of the villagers and for their
general good behavior. These men would be rather hostages than
officials, and would be nominees of the central government rather
than of the villages. But we really know nothing of the facts, except
that the villagers had some means by which they paid their taxes
collectively.
One way by which the Seleucids could relieve themselves of the
troubles of local administration and at the same time strengthen
their hold upon the country was to make grants of whole blocks or
complexes of their land, villages and villagers and all, to Macedonian
and Greek feudatories, like Mnesimachus, who of course became
responsible for the dues owing to the crown. But the evils of this
system had been discovered by the Persian kings when they found
their vassals more unruly than the villages. Hence the Seleucids
refused to give a clear title to those to whom they made grants of
portions of the royal domain, and in the case of Mnesimachus the
temple of Artemis, to which he sold his rights, had to secure itself in
the contract against the loss which it might sustain should the king
recall his grant. On the other hand, when the king sold outright parts
of the domain, as he frequently did, particularly in times of financial
distress, and the lands and villages sold became with their peasants
private property, he required that they should be added to the
territory of some city-state, it being a privilege highly cherished by
the purchasers that they should be given the liberty of deciding to
which city their property should belong. The Seleucid policy that land
should belong either to a city-state or to the crown was admirably
calculated to destroy priestly and feudal sovereignties, and it was
taken over by the Roman emperors, into whose patrimony in Asia
Minor passed many temple lands which had either escaped
secularization under their predecessors or had been regained by the
priests during the later, weaker days of the Seleucid dynasty.[113]
By a similar sale, or by a gift outright, the colonists who formed a
new city, or the old inhabitants of a village on being constituted
citizens of a free town, obtained full ownership of the lots of land
assigned to them. The citizens, in turn, had authority, subject of
course to the city's laws and the constitutions of the realm, over the
serfs when there happened to be any on their holdings. In this way
the king lost control of his peasants and his property; for the
foundation was not merely a new city, but at the same time a new
state. Its sovereign was not, or not simply, the king: it was the body
of its franchised inhabitants assembled in general assembly, and it
proceeded to manage its public affairs by means of discussion and
resolutions, by delegating functions to a council and magistrates,
and by determining its own domestic and foreign policies. The
language of public life was of course Greek. The code of public and
private law was, doubtless, drafted according to Hellenic models.
Gymnasia appeared and with them gymnastic and musical contests
—the most characteristic marks of Greek education. The deities they
honored were those whom they themselves chose: they chose native
gods and goddesses as well as Greek; above all they chose as their
chief city-god the living emperor.
The Seleucid empire was a state without a citizenship. If an Athenian
settled in it, he remained an Athenian, even if he became a satrap,
unless he were given citizen rights in some one of the free cities. In
other words, the empire had as many different citizenships as there
were different cities, and as many distinct states as there were
distinct citizenships.
Accordingly, each city could adopt whatever policy it pleased in the
matter of admitting foreigners, be they Greeks or Asiatics,
newcomers or natives, to its body politic. It might prohibit the
intermarriage of citizens with non-citizens altogether, or it might go
so far as to open its doors to bastards. It is, therefore, impossible for
us, without such sources of knowledge as the papyri afford for
Egypt, to speak in any general way of the extent and effects of racial
fusion in the Seleucid empire. Two things, however, seem clear: (1)
Intermarriage between citizens of different cities was of frequent
occurrence and, doubtless, of full legal propriety; (2) the great mass
of the agricultural population was not much affected racially by the
proximity of Greeks, Macedonians, Jews, or Iranians. The peasants
were practically serfs. Their social inferiority protected them against
assimilation by citizens. On the other hand, they were, doubtless,
much more deeply stirred by the European immigration than were
the fellahs in Egypt; for the influx into their land was much more
abundant and more spontaneous than was that into the valley of the
Nile. Here, too, Hellenism had much more effective agents for its
diffusion than it had there. For within the hundreds of city-states in
Asia we must presume that intermarriage was permitted among all
citizens, whether the elements which mingled with the Greeks were
Phrygians, Lydians, Syrians, Jews, Babylonians, or Iranians. We must
presume that at least the men knew in a fashion the Greek
language. They certainly tended to take Greek names, and in
documents of Delos dating from the second century b.c. we meet
with natives of Bambyce—now a polis and renamed Hierapolis, or
the Sacred City—who would be indistinguishable from native-born
Greeks were it not for the Semitic names of their wives. Indeed,
some of them may have been Greeks who had married Syrian
women. The various circles of Europeans in Syria, and, though to a
less degree, elsewhere in Asia, must have been surrounded at an
early date by a penumbra of half-breeds, by means of which the
sharp contrast of antagonistic civilizations was lessened. In these
circumstances Greek ideas and customs became a ferment which
stirred the peoples of Asia to the depths. The awakening of the
Nearer East was in progress in the third century b.c., and had the
Romans succumbed to Hannibal and the Greeks maintained their
prestige unimpaired for a century or two longer, the whole course of
history would have been changed.
The Greeks came to Asia "not to send peace but a sword." They
came to fill the continent up with cantankerous little republics where
formerly a dense multitude had lived in a state of political lethargy.
And curiously enough those who directed the dismemberment of
Asia into far more states than even mediæval Germany produced
were the rulers who had the responsibility for the government of the
whole region. How explain this anomaly?
The anomaly is more apparent than real. The ruler was not simply
the great landed proprietor of the cities' neighborhood; he was the
founder, or the descendant of the heroized founder, of most of them;
he was the "benefactor" or the "preserver" of them all. As such he
was deserving of their homage and entitled to their obedience. This
they could proffer in an unobjectionable manner once Alexander had
shown them the way. They had simply to make their proskynesis; to
elect him to membership in their circle of deities, furnish him with a
sacred precinct, temple, altar, image, procession, and contest, and
designate a priest to attend to the sacrifices and other matters
pertaining to his worship. This they did of their own volition during
the reign of the founder of the dynasty. Apotheosis without territorial
limitations Antiochus I demanded for Seleucus "after his departure
from the life among men," and the second Antiochus demanded it
for himself and his sister-wife during their lifetime as well as for their
"departed" father; so that just as in Egypt and for the same reasons
an imperial cult of the rulers dead and living was established
throughout not only the satrapies and hyparchies but also the cities
of the realm.[114]
The city had, accordingly, a dual character: it was at once both in
theory and fact a nation and a municipality. In the former capacity it
could grant or withhold allegiance to the king; in the latter it had
simply to obey. It had for example to pay tribute to him (phoros or
syntaxis), which might be viewed as a rent for the land assigned to
it, or as the price paid for military protection. It might have not
simply a priest of the king and a priestess of the queen, but also a
resident (epistates), who on occasion might also be phrurarch, or
commandant, of the royal garrison when it had one. The double
status of the city is further evidenced by the fact that its citizens
were subject not only to the laws which they themselves passed but
also to the mandates (prostagmata) which the king issued. In cases
of conflict there could be no doubt which was superior. The king was
in theory absolute as a god was absolute. He had, of course,
citizenship in no state, but was simply basileus, or king. This title,
attached to the name without an ethnicum, was the only one that
the early Seleucids used; but the later members of the dynasty,
beginning with Antiochus IV, added to it the title, such as Epiphanes,
or God Manifest, by which their peculiar office as gods was
indicated. Thereafter, on their coins and edicts the two titles
appeared, and the monarchs were thereby classified in the two
worlds to which they belonged—that of men and that of gods—as
completely as were citizens when to their names were added the
adjectival forms of their city's name. In either capacity they were
superior to the cities. On the other hand, the Seleucid god-kings had
to consider carefully the demands of their cities, since these, having
the means to organize resistance, could easily revolt. When they did
not get satisfaction they might choose some other god-king instead,
as the cities in Parthia and Bactria actually did; or they might secure
immunity from tribute, as did the cities in Asia Minor in the reign of
Antiochus I. Room was, accordingly, left for a large measure of
municipal liberty; and, in general, the activities of the citizens were
numerous and important. They had to attend to the maintenance of
order, the administration of justice, and the collection of taxes within
their several territories. Hence the cities gave a stimulus to political
interest and ambition such as Asia had never known before. They
occupied, in fact, a place in the Seleucid empire quite as important
as that of the municipalities in the early Roman empire, of which
they were, indeed, the prototypes.
The Roman empire, however, had not yet come into existence. It
was the Italian federation under Rome's leadership which defeated
Hannibal and won the battles of Thermopylæ and Magnesia. When
compared with this aggregate of incorporated and allied states, the
Seleucid empire demonstrated fatal weaknesses. Rome had,
perhaps, not many more citizens on her army list than there were
males of military age in the franchised population of the Seleucid
cities; and her public land, which, too, was her chief source of
revenue, was far inferior in extent and yield to the royal domain of
the Seleucids. Her advantages were twofold and their enumeration
will help us to understand the disabilities under which the Asiatic
monarchy labored. First, apart from the soldiers on Rome's army list
there were few males of military age in Italy; in other words, there
was no vast native population to hold in subjection. Second, Rome
could mobilize her forces much more easily, quickly, and completely
than could the Seleucids. The great distances, often of mountain and
desert, which separated the cities of Asia from one another; the
considerable trading, industrial, Asiatic, and otherwise unwarlike,
element in the free population of the Hellenic and Hellenized cities;
and the independence of the cities, particularly in the matter of
giving or refusing military aid to their suzerain, had no parallel in
Italy, where the territory was compact, the population mainly a
warlike peasantry, and the cities all bound to provide troops at the
call of Rome to the full extent of their power. Like the giant Antæus
in his trial of strength with Heracles, Rome with every fall renewed
her might from contact with her native soil. The Seleucids ruled over
a cosmopolitan, denationalized world. They had no native soil on
which to fall. It is of profound significance that there were and could
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