Download as a PDF

CLIN. CHEM. 31/1, 5-13 (1985)
Uremic Toxins, and Their Effect on IntermediaryMetabolism
Michael R. Wills
In the late stages of chronic renal damage the functional
mass of the kidney is reduced and there is progression to.
renal insufficiency, usually called uremia, in which all aspects
of renal function are affected. The complexity of the biochemical aspects of the syndrome of uremia is a manifestation of
the wide variety and nature of the individual disorders that
contribute to the pathogenesis of the final clinical syndrome.
One major feature is the retention of metabolic end products
and their effects, as toxins, on intermediary metabolism. The
retained end products, working singly or in combination,
probably affect metabolic pathways by some modification of
enzymic reactions. They act at the cell membrane level.
Although “middle molecules” have been incriminated as
uremic toxins, recent attention has also focused on trace
elements-especially
aluminum, which has been implicated
in the pathogenesis of two major disorders, osteomalacic
dialysis osteodystrophy and dialysis encephalopathy.
AddItIonal
aluminum
alopathy
mones
Keyphrases:
dialysis
trace
renal
function
patients
elements
chronic renal failure
“middle
osteodystrophy
.
kidney
uremia
molecules”
.
disease
encephhor-
lipoproteins
Besides being the dominant organs in the homeostatic
control of the constitution of the internal environment, the
kidneys are also major endocrine organs, secreting various
hormones that act on the respective target tissues. The
kidneys also indirectly influence other endocrine systems,
by affecting the clearance and degradation of other hormones. In reviewing the biochemical aspects of chronic renal
failure-with
specific reference to toxins-one must have a
clear concept of the role that the kidneys play as homeostatic organs. However, the complexity of the syndrome of
uremia is due to the role played by the kidneys, not only as
homeostatic but also as endocrine organs.
As homeostatic organs the kidneys are responsible for the
volume and the ionic and molecular
composition
of the
internal environment.
The concept of an internal fluid
environment
in which the cells of the body live and function
was introduced by Claude Bernard in 1878(1). The vital role
of the kidneys in maintaining
the constitution of that
environment
was subsequently recognized by Peters in 1935
(2), who said: “the kidneys appear to serve as the ultimate
guardians of the constitution of the internal environment.”
Excretion of metabolic end products, a major homeostatic
function of the kidneys, involves both glomerular ultrafiltration and renal tubular secretion. During the development
Departments of Pathology and Internal Medicine, University of
Virginia, Charlottesville, VA 22908.
Received August 8, 1984; accepted October 1, 1984.
of the nephron unit the tubular secretory systems evolved to
excrete, into an aqueous environment, waste products of
high relative molecular mass that could not escape from the
body by the process of simple diffusion (3). Maintaining
optimal conditions in the fluid environment of the cells is
vitally important; the failure of the kidneys to perform this
task is manifested in the clinical syndrome called uremia.
The term “uremia” was introduced in 1840 by Piorry and
l’H#{233}ritier(4). In their treatise on alterations in the blood
they proposed use of the general suffix “-emia” to denote the
blood compartment and qualifying it with a specific prefix to
denote the presence of an abnormality in that compartment.
Thus in their terminology “uremia” literally means “urine
in the blood,” which reflects their view that the toxic
manifestations
of renal failure were a form of poisoning of
the blood, a consequence of reabsorption of urine. Today,
uremia is used clinically to describe a complex syndrome
that has many interrelated features.
In the specific context of toxins, the word “uremia” is used
to describe the state associated with the retention of nitrogenous metabolic end products and is characterized by an
increased concentration
of urea in the blood. Despite a
considerable amount of time and effort devoted to a search
for the uremic toxin, no one individual compound has, so far,
been incriminated. It may well be that no one uremic toxin
will ever be identified as the toxin. The clinical syndrome of
uremia should be recognized as a composite problem, involving all of the body’s systems and reflecting biochemical
alterations
in all aspects of the constitution
of the internal
environment.
In this view the alterations that produce
uremia would reflect not only accumulation of metabolic
end products but also associated changes in water, electrolyte, and acid-base homeostasis; disturbances in endocrine
and nutritional
status; and associated abnormalities
in
metabolism of fat, carbohydrate, and protein.
High concentrations
in serum of the end products of
protein catabolism are generally considered to be a major
feature of the uremic state; some of the intermediate breakdown products are also believed to accumulate and play a
role in the development of toxicity. Organic
substances
known or reported to accumulate in uremic blood include:
urea, creatinine, guanidines and related compounds, uric
acid, creatine, certain amino acids, polypeptides, polyamines, cyanate, indican (indoles), hippuric acid, phenols
and conjugates of phenol, phenolic and indolic acids and
their conjugates, organic acids of the tricarboxylic acid cycle,
aliphatic amines, guanidine bases, pseudouridine, acetoin
and 2,3-butylene glycol, j3-hydroxybutyrate, glucuronic acid,
carnitine, myoinositol, “middle molecules,” sulfates, and
phosphates (5). Given the improvement in clinical symptoms in uremic patients on institution of adequate therapy
with dialysis, and the associated decrease in the concentraCLINICAL CHEMISTRY, Vol. 31, No. 1, 1985
5
tions of these retained organic metabolic end products, there
is no doubt that the retained metabolites do play an important role in the pathogenesis of the clinical syndrome of
uremia. The retained organic metabolic products probably
play a major role as toxins in the pathogenesis of uremia,
either working singly or in combination.
In this review I will deal with those compounds that have
been of major interest in the past as toxins or are currently
of interest.
Urea
Among the specific compounds incriminated and investigated as uremic toxins, urea has received considerable
attention. The increase in the urea concentration in blood is
perhaps the most striking abnormality of the body fluids in
renal failure, although not the most important functionally.
Urea is formed only in the liver and may be regarded as
the end product of protein catabolism, whether the protein
originates from the diet or the tissues. Patients with acute
renal failure show a relatively good correlation between the
severity of the illness and the concentration of blood urea
nitrogen. In chronic renal failure, however, the concentration of creatinine in serum seems to be a better index of the
severity of the degree of failure, particularly
when the
patient is on a low protein diet.
The role of urea in the pathogenesis of the clinical
syndrome of uremia has been controversial. In 1827 an early
clinical chemist, Dr. John Bostock, who did the chemical
analyses for Dr. Richard Bright, reported that in patients
with chronic renal failure the serum “was found to consist in
part of an animal matter possessing peculiar properties
which seemed to approach those of urea” (6). Although
Bright recognized the markedly increased concentrations of
urea in patients with chronic renal failure, it is interesting
that he considered that it “may be but in part a cause of
general derangement
of the system” (7). As has been subsequently demonstrated, the administration
of urea to normal
subjects in amounts sufficient to raise their blood concentrations to values similar to those in patients with chronic
renal failure is associated only with thirst and polyuria, and
none of the other clinical manifestations
of uremia. In
patients with chronic renal failure, therefore, most of the
available evidence supports the proposal that an increased
urea concentration in blood does not itself have a major toxic
effect. In support of this proposal, Merrill et al. (8) hemodialyzed chronic uremic patients against solutions with high
urea concentrations and noted an excellent clinical response
even though the blood urea concentration was unaltered.
At one time, a high urea concentration in the blood was
implicated in the pathogenesis of the “dialysis disequilibrium syndrome.” This syndrome is characterized by the
development of headache, confusion, muscle twitching, progressing occasionally to convulsions, coma, and (rarely)
death, either during or towards the end of an otherwise
biochemically successful dialysis. In the mechanism proposed by Kennedy et al. (9), an osmotic gradient was
created, during dialysis, between the brain tissues, the
cerebrospinal fluid, and extracellular fluid compartments,
such that water would shift into the first of these; this event
would be associated with an increase in intra-cranial pressure, which would account for the features of the syndrome.
Since then, the dialysis disequilibrium
syndrome has been
found to relate to changes in sodium concentration
during
dialysis (10) and can be prevented by the use of a high
sodium concentration in the dialyzing fluid (11).
Although urea may not itself be a major toxin in patients
with chronic renal failure, it is one of the retained metabolites known to act as enzyme inhibitors (see below).
6 CLINICAL CHEMISTRY, Vol. 31, No. 1, 1985
Creatinine
An increase in creatimne concentration in the plasma is
another diagnostic feature of chronic renal failure; and it is
roughly correlated with the degree of failure. A formula has
been proposed that allows for age and weight and predicts
the endogenous creatinine clearance ratio from the serum
creatimne concentration (12). Like urea, creatinine may
not, per so, play a role as a uremic toxin. Its retention is of
importance in the genesis of other toxic metabolites, a
consequence of an alteration in its normal metabolic pathway. Creatinine metabolism and excretion is altered in
patients with chronic renal failure. In normal individuals,
the amount of creatimne excreted is chiefly influenced by
lean body mass and diet. Patients with chronic renal failure
excrete less creatiine
in their urine than would normally
be expected. The serum creatiine
concentration increases
correspondingly in chronic renal failure, although an increasing proportion reportedly is cleared by an extrarenal
mechanism, and this accounts for the reduction in urine
excretion (13). The proposed extrarenal clearance mechanism involves two potential pathways: recycling of creatinine to creatine, irreversible degradation to products other
than creatine, or both. Any alteration in creatiine metabolism during chronic renal failure, with an extrarenal clearance route, is of particular interest in that it represents an
adaptative change in a metabolic pathway in response to the
long-term decrease in functional renal mass.
Uric and Oxalic Acids
Urate retention is one of the recognized biochemical
features of chronic renal failure. The uric acid in serum
increases in uremia, but correlates poorly with the increment in creatinine concentration (14). As overall renal
function deteriorates, the excretion and clearance of uric
acid by the functional renal remnant markedly increases
(14). An increase in the tubular secretion of uric acid and
incomplete reabsorption of the ifitered fraction-which
is
normally almost completely reabsorbed-would
account for
the lack of correlation with the increase in creatimne
concentration. Again: the changes in the tubular secretion
and reabsorption of uric acid represent adaptations to the
decrease in functional renal mass. These adaptative changes
in the remaining nephrons of the chronically diseased
kidney with respect to uric acid transport may be prompted
by a uricosuric factor in uremic serum (15, 16).
In addition to an adaptative change in the renal tubular
handling of uric acid there is also evidence that uremic
patients develop an alternative route for either the metabolism or the clearance of uric acid (17). The alternative route
involves the extrarenal elimination by the process of uricolysis (18), which takes place entirely in the intestinal tract, is
catalyzed by bacterial enzymes, and appears to become
increasingly important as the plasma uric acid concentration increases. Here again, this induction of alternative
routes for the handling of uric acid, either by variations in
renal tubular handling or the process of uricolysis, is of
interest because of what it tells us of biochemical adaptations in response to decreased functional renal mass.
Retention of oxalic acid is a recognized feature in patients
with chronic renal failure (19), and it reportedly is associated with the deposition of oxalate crystals in the myocardium
and renal tissues (20-22). Crystal deposition in these tissues
could be of importance in the genesis of some of the clinical
features of the syndrome of uremia. It is also of interest in
the overall pathogenesis of the clinical syndrome of uremia
that oxalic acid is among the retained metabolites that are
known
to act as enzyme inhibitors-in
this particular
effects on glucose uptake and utilization
(33).
Guanidinoacetic acid is a precursor of creatinine,
and its
concentration increases during chronic renal failure as a
direct consequence of the decreased clearance that can be
effected by whatever functional renal tissue is left. An
increase in guanidinoacetic acid potentially
leads to the
transfer of the amidine group of arginine to aspartate
instead of glycine, with the formation of guanidinosuccinic
acid and ornithine.
The urinary excretion of guanidinosuccinic acid increases
in chronic renal failure (34), and the increase has been
attributed to the development of an alternative pathway for
the detoxication
of ammonia and urea synthesis, which
involves guanidinosuccinic
acid. The mechanism for this
enhancing
instance, lactate dehydrogenase (23).
Myoinositol
Myoinositol has a specific effect on nerve conduction.
Some patients with chronic renal failure have a marked
increase in serum myoinositol concentration and also fail to
clear it at the normal rate after an oral load. Changes in
myoinositol concentration and clearance rate could result
from an impairment of the renal catabolism (24). Myoinositol is a precursor and constituent of a class of phospholipids,
the phosphoinositides, whose metabolism has been linked
with the functional activity of nerve. Rats with hypermyoinositolemia from oral loading reportedly have a significant
decrease in the velocity of sciatic motor-nerve conduction
(24). On the basis of these findings it was suggested that the
abnormally high myoinositol concentrations found in uremics may be a factor in the development and progression of
a polyneuropathy. Most of the evidence, however, would
now support the view that, although hypermyoinositolemia
may depress nerve conduction velocity (25) and its concentrations in serum are markedly increased in patients with
chronic renal failure, there is no indication that myoinositol
is the neurotoxin of uremia (26). This view was based on the
fact that there was no correlation between nerve-conduction
velocities and either serum or cerebrospinal fluid myoinositel concentrations: there was also no correlation between
electroencephalographic
changes and myoinositol concentrations in either of these two compartments (26).
Guanidines
and Related Compounds
Guanidines and related compounds have long been implicated as uremic toxins. Experimental animals, chronically
intoxicated with methylguanidine, reportedly lose weight at
a rate that suggests that it exerts a catabolic action (27). In
these animals the late stages of intoxication
with methylguanidine were associated with disturbances in the gastrointestinal tract, the cardiovascular system, the lungs, and
the central and peripheral nervous systems. The potential
importance of methylguanidine
as a toxin in uremia is that
it is a constituent of creatine (methylguamdoacetic acid) and
its derivative, creatinine.
Although the concentrations of guanidine, methylguanidine, and 1,1-dimethylguanidine
in serum may not be
markedly increased in uremic patients, the urinary excretion of methylguamdine
is significantly increased (28, 29).
There is also evidence that the rate at which methylguanidine is produced metabolically is increased in chronic renal
failure and that this is associated with increased concentrations in the tissues (30). Thus it was proposed that methylguanidine retention plays a role in the pathogenesis of the
uremic syndrome (31). If methylguanidine
is a uremic toxin,
it presumably exerts its toxic actions in association with a
preferential
distribution
in the intracellular
compartment.
Although some experimental studies suggest that methylguanidine is a uremic toxin, other evidence supports the
proposal that its toxic role remains to be defined. This
proposal is based on observations that high concentrations
of methylguanidine
caused no significant inhibition of oxygen uptake in vitro in tissue-respiration studies of slices of
rat cerebral cortex, kidney, and liver (32). The concentrations of methylguanidine
used in those studies were up to
four times those in serum of patients with chronic renal
failure. In studies of glucose uptake and utilization
by
preparations of rat diaphragm, in vitro methylguanidine
had a small enhancing effect (33) rather than a toxic
inhibitory action. Guamdine and guanidinoacetic
acid also
have been studied individually
and shown to have small
alternative
pathway
would
involve
repression
of normal
enzyme activity and either the activation of a dormant
enzyme or the appearance of a new enzyme. The alternative
metabolic pathway for urea synthesis in chronic renal
failure (35) is consistent with an increase in methylguanidine concentration. In subsequent studies Stein et al. (36)
reported increased concentrations of guanidinosuccinic acid
in serum and cerebrospinal fluid and confirmed its high
urinary excretion in patients with chronic renal failure.
These observations have been confirmed by other workers,
who reported that not only does guanidinosuccimc
acid
accumulate in chronic renal failure, there was also evidence
of increased production in these patients as compared with
normal individuals (37). However, the role of guanidinosucciic acid as a toxin in uremia remains to be clarified;
animal experiments gave (38) no evidence of guanidiniosuccinic acid being a uremic toxin.
Dimethylamine
In any review of the metabolites retained in the serum of
patients with chronic renal failure it should be recognized
that not all these compounds are derived either from a
uremia-induced derangement of the usual metabolic pathway or from endogenous tissue sources. An example is
diethylamine,
which increases in serum in uremia. Its
concentration in duodenal contents is also increased as
compared either with normal healthy subjects or with other
types of patients (39). This increase is attributable to the
uremic state per so, a consequence of uremia-induced
alterations in the bacterial flora of the gastrointestinal
tract. In
uremic patients, part of the choline is transformed by
bacteria in the gut to trimethylamine,
which is reabsorbed
and then either oxidized by trimethylanune
dehydrogenase
(EC 1.5.99.7) or demethylated to dimethylamine
(DMA) in
the liver (40). Dimethylamine
enters the circulation and is
excreted in bile and urine. The increased concentrations of
dimethylamine
and trimethylamine
in the breath of uremics are correlated with their classic “fishy odor” (41).
Possibly, alterations in the gastrointestinal flora as a direct
consequence of the uremic state are of importance in the
formation of various other metabolites, which then are
absorbed into the extracellular fluid compartment, resulting
in an increased circulating concentration of them, with
potential toxic and nutritional sequelae.
Parathyrin
(Parathyroid
Hormone) as a Uremic Toxin
Normally functioning kidneys play a major role in the
enzymic degradation and clearance of hormones from the
circulation, and because of this the kidneys may be regarded
as indirect endocrine organs. Hormone status is disturbed in
patients with chronic renal failure, and the associated
consequences may be regarded as features of the clinical
syndrome of uremia. An established biochemical feature of
uremia is a markedly increased concentration of carboxyCLINICAL CHEMISTRY, Vol. 31, No. 1, 1985 7
terminal
immunoreactive
parathyroid
hormone (i-PTH),
which not only reflects a disturbance in the rate of secretion
of parathyrin but also a decrease, or even a failure, in the
renal excretion of the peptide fractions resulting from
enzylnic degradation. This accumulation of i-PTH degradation products, together with the known actions of the
hormone, has led to the proposal that it may be a major
uremic toxin.
In 1977, Massry (42) proposed that many of the manifestations of the uremic state could be accounted for by an
excess of circulating parathyrin and that this polypeptide
could be an important uremic toxin. The manifestations of
uremia that were attributed to parathyrin included disorders of the central nervous system, soft-tissue calcification,
soft-tissue necrosis, bone disease, pruritis, hyperlipidemia,
anemia, and sexual dysfunction (43). The major potential
role of parathyrin as a uremic toxin is as a neurotoxin, and a
considerable amount of evidence supports this view. Avram
et al. (44) reported a significantly decreased motor-nerve
conduction velocity in uremic patients with above-normal
i-P’FH concentrations in their serum as compared with agematched uremic patients with normal or only slightly
above-normal i-PTH. There were no significant differences,
in these two groups of patients, between the mean values for
calcium and creatinine in serum. In subsequent studies the
same workers added further support for the role of parathyrin as a neurotoxin with their observations that, after
parathyroidectomy,
motor nerve conduction velocity increased in a group of uremic patients on maintenance
hemodialysis
(45).
In experimental animals, an excess of parathyrin reportedly increases peripheral nerve calcium with a simultaneous decrease in motor-nerve conduction velocity (46), and
these changes were reversed when the administration
of
parathyrin was stopped. Marked abnormalities in electroencephalographic patterns have been found in patients with
acute renal failure who have concurrent increases in i-PTH;
it was proposed that the abnormalities might be ascribed to
a direct effect of parathyrin
on the brain, causing an
increase in calcium content (47). The electroencephalographic abnormalities show a direct relationship with the
i-PTH values (48). In these studies, however, the significant correlation was with the values for the amino-terminal
i-PTH fragment, and not with the carboxy-terminal
fragment, in serum. Moreover, after six months of treatment
with 1,25-dihydroxycholecalciferol,
there was a marked
decline in the concentrations of the amino-terminal
i-PTH
fragment in serum, which was associated with a significant
change in the electroencephalogram toward normal in some
of the patients. Thus the currently available evidence would
support the concept that parathyrin or its immunoreactive
degradation products, or both, act as a neurotoxin in uremia.
An anemia of the normochromic normocytic type is almost always a feature of chronic renal failure. The kidneys
play a major role in erythropoiesis and in the incorporation
of iron into erythrocytes, by their production of erythropoietin. Nevertheless, the anemia of chronic renal failure has a
multifactorial
etiology; it is not simply the end result of a
decrease in the renal production of erythropoietin.
The
pathogenesis of the anemia of chronic renal failure involves
multiple factors, parathyrin among them. It is, however,
important to recognize that among these the uremic state
per se is of major importance; uremic serum has been
recently reported to be toxic and inhibitory to erythropoiesis
(52). The suppressive activity against erythroid colony
growth in uremic serum appeared to be contained in compounds with molecular masses ranging from 47000 to
>150 000 Da. The importance of these observations is that
8
CLINICAL CHEMISTRY, Vol. 31, No. 1, 1985
inhibitors in this molecular-mass range would not be removed by conventional dialysis techniques and would not be
classified as “middle molecules” (see below). An anemia of
the normochromic normocytic type is a recognized clinical
feature of patients with primary hyperparathyroidism.
Although the anemia of chronic renal failure is of a different
type, secondary hyperparathyroidism,
with an excess of
i-PTH, has been implicated as a factor in the pathogenesis of
the anemia found in these patients (42). In uremic patients,
subtotal parathyroidectomy,
with a resulting decrease in
serum i-PTH, is followed by significantly improved hemstological indices(49-Si). There is no evidence that an excess
of parathyrin has a direct toxic effect on erythropoiesis.
Rather, the improvement in the anemia after parathyroidectomy in uremic patientsseems to be related to a decrease
in the amount of fibrous tissue in their bone marrow-an
increase in which is known to be induced by an excess of
parathyrin.
Middle Molecules
Most investigationsinto the pathophysiology of uremia
have been directed towards the search for and isolation of
eithera singletoxicsubstance or a group of substances,the
retention of which would account for all of the clinical
features of the syndrome. The most recent development in
this search has been termed the “middle molecule” hypothesis, a concept that has developed as the direct consequence of
the failureto incriminate such small molecules as urea and
creatinine.
The middle molecule hypothesis is that a group of unknown compounds with molecular masses in the range of
300 to 1500 Da are of importance as toxic metabolites in
uremia at relatively low concentrations (53, 54). The hypothesis
is based on (a) a comparison
of the clinical
results
obtained on using either hemodialysis or peritoneal dialysis
and, more specifically, (b) the relationship between the
number of hours of dialysis and the development of the
symptoms of neuropathy. The assumption underlying the
development of the middle molecule hypothesis is based on
the difference between the molecular permeability of the
cellulosemembranes used in hemodialysis equipment as
compared with that of the peritonealmembrane. The latter,
which isknown to leak protein, would presumably allow the
removal of retained toxic metabolites
with a higher molecularweight than those removed by dialysis across a cellulose
membrane. The removal of toxic compounds of higher
molecular mass would account for the better clinical results,
as assessed by the alleviation of the symptoms of uremia,
obtained with the peritoneal dialysis technique. A major
feature of the middle molecule hypothesis is the concept that
these unknown molecules exert their toxic effects at relatively low concentrations. Reportedly, middle molecules
only accumulate in quantities that are sufficient to be
measured in patients with advanced chronic renal failure,
as assessed by an endogenous creatinine clearance rate of
less than 11 mL/min (55).
Several recent studies have been aimed at identifring the
middle molecules. One procedure revealed up to 10 identifiable subpeaks in the middle-molecule range in uremic sera,
urine, and erythrocyte hemolysates (56). The technique
used was a modification of a two-stage chromatographic
procedure, involving use of a molecular sieve followed by
ion-exchange chromatography (57). Differences were seen in
the distribution of middle molecules among the three different materials studied (56). Although the concentrations of
the middle molecules were increased in the serum of patients with uremia, the values for erythrocytes of patients
with the syndrome were similar to those for normal subjects.
Thus Chapman et al. (56) concluded that “caution is necessary in interpreting results of middle molecule analyses.”
One biochemical aspect of their findings to which they drew
attention is that if any of the species they identified is
subsequently shown to be a uremic toxin, the differences in
their distribution among the compartments of the body are
of potential importance in the choice of an appropriate
mathematical model for optimizing dialysis therapy.
Compounds in the middle-molecule range are said to be in
higher concentrations in the serum of”sick” uremic patients
than in an asymptomatic group of uremics (58,59), although
there is a considerable overlap between the data on individual middle-molecule fractions for the two groups (60). These
authors could not correlate the accumulation of any specific
middle-molecule fraction with the occurrence of any specific
uremic symptom in the “sick” group of patients. In contrast,
using a two-stage chromatographic technique-gel
permeation followed by anion-exchange chromatography-one
group of workers (61) has correlated the amplitude of one
specific middle-molecule peak with the occurrence of uremic
neuropathy. After successful renal transplantation,
uremic
middle molecules rapidly disappear from serum, even more
rapidly than the creatiine
concentration declines (62).
Collectively
these observations
add support
to the concept
that compounds in the middle-molecule range may indeed
play a role as uremic toxins.
A major problem in the development and understanding
of the potential toxic nature of middle molecules has been
the failure of various attempts to define both their chemical
and biological nature. Menyh#{225}rt
and Gr#{243}f
(63) proposed that
a large group of unknown peptides with limited diffusibility
through hemodialysis membranes constituted most of the
uremic middle molecules. They investigated the molecular
composition
of uremic middle molecules in the 500-5000
range of relative molecular mass. Using cation-exchange
column chromatography, they separated three fractions of
serum containing these molecules. Subsequent one-dimensional paper chromatography
of these fractions
demonstrat-
ed that each of them could be further resolved into at least
seven ninhydrin-positive
subfractions. The qualitative amino acid composition of the subfractions differed from each
other and from those of known peptides with which they
were compared. Some of these peptides appeared to be
completely removed from uremic serum by hemodialysis;
others were only partly removed or were completely unaltered by this treatment.
The kidneys play a major role in the enzymatic degradation and clearance of hormones from the circulation. A
disturbance in the normal clearance patterns of hormones
potentially could play a role in the pathogenesis of the
uremic syndrome. According to this concept, retention of
either the hormones themselves or of their subsequently
renal-cleared peptide degradation products could be regarded as potential uremic toxins, the unknown peptides reported by Menyhart and Gr#{243}f
(63).
Although the clinical evidence I have mentioned supports
the middle-molecule
hypothesis,
in the pathogenesis
of the
clinical syndrome of uremia their precise role still remains
to be clarified. The middle-molecule hypothesis currently is
used to refer to serum solutes in the relative molecular mass
range of 500 to 2000 Da. In a recent study, uremic ultrafiltrates were fractionated by means of gel ifitration and then
further investigated by combined analytical techniques:
“high-performance”
liquid chromatography, liquid chromatography, gas chromatography,
mass spectrometry, and
isotachophoresis
(64). These studies led the authors to
propose that gel filtration was inappropriate as an analytical technique for determination
of middle molecules, be-
cause they found that ultrafiltrate
fractions in the middlemolecular-mass range also contain a considerable amount of
substances of low molecular mass, such as carbohydrates,
organic acids, amino acids, and ultraviolet-absorbing
solutes. The earlier work on middle molecules was largely
based on gel ifitration studies, so they recommended reconsideration of the importance of low-molecular-mass
compounds and their role as uremic toxins. Other workers (65)
have also concluded that “despite the extensive studies in
this field, results are still confusing concerning the role of
so-called ‘middle molecules’ in uremic toxicity.”
They (65)
drew specific attention to the fact that a major problem in
this field of investigation has been the differences between
the analytical techniques used by different groups of workers, which has made it impossible to inter-compare the
reported middle-molecule
fractions. They proposed that “it
appears necessary to develop a chromatographic reference
system which would be the basis for the evaluation
of the in
vitro and in vivo effects of the various molecules ranging in
the still poorly defined M.W. fraction” (65). It can only be
concluded
at this time
that,
until
the chemical
identity
of
the retained uremic middle molecules is known and their
biological toxicity has been demonstrated by experimental
in vivo and in vitro studies, their role as uremic toxins is
only hypothetical. Chemical identification and characterization of middle molecules will probably require mass spectrometry but this will have to await further
advances in this field (66).
Retained Uremic Metabolites as Enzyme
technological
Inhibitors
A potential role of the retained serum metabolites as
toxins in the pathogenesis of the uremic syndrome is as
enzyme inhibitors. Urea, in concentrations comparable to
those found in uremia, significantly
inhibits monoamine
oxidase (67). This enzyme has a major role in the destruction
of both serotonin and catecholamines and in the regulation
of amine metabolism in nervous tissue, and its inhibition by
urea could be of significance in the pathogenesis of certain
clinical features of the uremic syndrome. Urea also inhibits
oxygen uptake by brain slices (68), but only at very high
concentrations as compared with those in blood and after a
3-h lag period. Urea and creatinine reportedly inhibit glucose uptake and utilization in rat diaphragm in vitro; there
was enhancement of the inhibitory effect when these two
compounds were used together in the system (69). These
latter findings are consistent with the proposal that the
retained metabolites have a cumulative enyzme inhibitory
effect in vivo.
Phenolic acids affect cerebral metabolism, as measured by
the rate of respiration and anaerobic glycolysis of guinea pig
brain slices, and they also inhibit the activity of some
selected enzymes (70). The enzymes studied were the decarboxylases of 3,4-dihydroxyphenylalanine,
5-hydroxytryptophan, and glutamic acid; aspartate aminotransferase; 5’nucleotidase; amine oxidase; and lactate dehydrogenase.
Many aromatic acids, especially those with an unsaturated
side chain, depressed enzyme reaction rates. The concentrations of phenolic acids used by Hicks et al. (70) were higher
than those found in uremic serum, but they proposed that
the lower concentrations of phenolic acids in uremic serum
might possibly exert an effect by virtue of having been
present for a longer time than the relatively high concentrations used in their enzyme inhibition studies. Alternatively,
it could be that these compounds exert a cumulative inhibitory effect in vivo.
Aromatic compounds retained in uremia may exert an
enzyme inhibitory action by a summation effect in vivo; both
aromatic and aliphatic amines can cause enzyme inhibition
CLINICAL CHEMISTRY, Vol.31,No. 1, 1985 9
However, compared with the phenolic acids, the
amines were less effective inhibitors of glutamic acid and
dihydroxyphenylalamne
decarboxylases. Amines cross the
blood-brain barrier more readily than do acids (72) and
therefore may be more effective in vivo than in vitro. The
increased concentrations of aromatic amines in serum in
uremia approximately correspond with the increase in blood
urea nitrogen concentrations.
Although the precise biochemical role and the potential
mechanisms of the retained metabolites as enzyme inhibitors in the etiology of the clinical syndrome of uremia
remain to be clarified, these compounds clearly do accumulate in the extracellular-fluid
compartment, and many of
them can inhibit enzymes. Nevertheless, collectively they
may exert their actions at a final common site. In one review
of uremia, a unifying hypothesis for the toxicity of uremia
was proposed, with derangements in membrane transport as
the final common pathway (5). This hypothesis was based on
a perspective of the wide range of known and potential toxic
metabolites retained in uremic serum. Skeletal resistance to
the action of exogenous parathyrin is a recognized feature of
the disturbance in calcium homeostasis in patients with
uremia. Wills and Jenkins (74), who studied parathyrininduced bone resorption by using an in vivo organ culture
system, reported that some known retained uremic metabolites, including phosphate, when tested individually, inhibited the calcium-mobilizing
action of the hormone on bone,
but the inhibitory effect of many of the individual metabolites was only significant at concentrations higher than
those found in the serum of uremic patients. Serum collected
from uremic patients prior to hemodialysis totally inhibited
the calcium-mobilizing
action of parathyrin; that collected
from the same patients just after dialysis was not inhibitory.
Thus they (74) proposed that if the uremic metabolites have
an inhibitory effect on the action of parathyrin, it must be a
cumulative phenomenon, which potentially could involve
either the blockage of membrane receptors to parathyrin or
hormone-induced effects on cell membrane transport.
(71).
Aluminum
The potential role of aluminum as a toxic agent in
patients with normal renal function is controversial. Still, it
has been established that in chronic renal failure there is an
increased total body burden of aluminum and that this is
associated with toxic sequelae involving bone, brain, and
erythropoietic tissues (75). Aluminum salts are extensively
used in the therapeutic management of the hyperphosphatemia that is a relatively constant feature of patients with
chronic renal failure. Aluminum
is absorbed from the
gastrointestinal tract. In normal subjects, after an oral load
its concentration in serum increases, followed by its increased excretion in the urine (76). Increased aluminum
concentrations in the serum of some patients with chronic
renal failure was first reported in 1970 (77). It is now
established that hyperaluminemia
may occur in patients
with end-stage chronic renal failure and is associated with
the accumulation of aluminum in various tissues. Hyperaluminemia and the associated toxic sequelae may occur in
patients on treatment with either hemodialysis or peritoneal dialysis and in some patients who have not been dialyzed
but are being treated with orally administered aluminum
salts (75, 78). The high values for aluminum in serum and
tissue result from the intestinal absorption of aluminum
salts taken by mouth and from passage of aluminum across
the dialysis membrane. The aluminum
content of the dialyzing fluid obviously
also depends on the Al content of the
water used as the solvent. Some domestic tap-water contains aluminum in high concentration, either naturally or
10 CLINICAL CHEMISTRY, Vol. 31,No. 1,1985
because aluminum has been added as a flocculant in the
purification process. Acid rain markedly increases the “natural” aluminum content of water.
Aluminum is now recognized as a major, if not the major,
toxic factor in the pathogenesis of a progressive fatal neurological syndrome in patients with chronic renal failure that
was first reported in 1972 (79). The syndrome was later
termed “dialysis encephalopathy” or “dialysis dementia,”
and these patients showed increased amounts of aluminum
in the brain, muscle, and bone tissue (80). Aluminum
potentially exerts its neurotoxic action by inhibiting dihydropteridine reductase (EC 1.6.99.7) (81). Such inhibition
would result in a decrease in the tetrahydrobiopterin,
tyrosine, and neurotransmitters
in brain. The neurotoxicity of
aluminum may also involve alterations in the major postsynaptic enzymes of cholinergic neurotransmission
(82).
Although the precise mechanism possibly remains to be
defined, much evidence indicates that aluminum is neurotoxic for patients whose functional renal mass is decreased.
The latter leads to a reduction in the normal renal clearance
of aluminum with a consequent increase in its concentration
in serum and body tissue.
Bone pain, as a consequence of metabolic bone disease, is
a common symptom in patients with chronic renal failure
who are receiving long-term treatment with intermittent
hemodialysis.
The progressive
metabolic
bone disease in
these patients is usually termed “dialysis osteodystrophy,” a
term that distinguishes it, and some aspects of its pathogenesis, from renal osteodystrophy in the undialyzed patient.
The osteomalacic component of dialysis osteodystrophy is a
major clinical problem because it is associated with a high
incidence of fractures. An increase in the Al content of bone
in some patients with end-stage chronic renal failure was
first reported in 1970 (83). Only years later was an association reported between the occurrence of dialysis encephalopathy and osteomalacia: exposure to high amounts of aluminum was a factor common to both of those complications in
uremic patients (84, 85). Clearly, aluminum plays a major
etiological toxic role in one particular type of osteomalacic
dialysis osteodystrophy (75), a type that usually occurs in
patients on dialysis treatment but may also occur in nondialyzed patients (86). The mechanism for the disordered
bone formation induced by an excess of aluminum remains
to be clarified. It may involve a disturbance either in the
formation of calcium apatite or in the bone-mineralization
process (75,87). There is also evidence from in vitro studies
that suggests that aluminum may affect the activities of the
bone enzymes, acid and alkaline phosphatase, and modiI
their response to parathyrin and 1,25-dihydroxycholecalciferol (88).
In patients on dialysis treatment who develop aluminum
toxicity the major source of the aluminum is the tap-water
used to prepare the dialysate. In addition there is also some
intestinal absorption from the aluminum-containing
phosphate-binding gels; in some patients this route appears to
have been dominant. The driving force for aluminum transfer during dialysis appears to be the effective concentration
gradient between the dialysate aluminum and the free
diffusible aluminum fraction in serum (89). Transfer of
aluminum from the dialyzing fluid across the dialyzing
membrane appears to occur even when concentrations of the
metal in the fluid are low (90, 91). The major portion, if not
all, of aluminum in blood is bound to serum proteins and an
as-yet-unidentified low-Mr species (91). The identification of
the latter may be of importance in understanding the
mechanisms of tissue accumulation and consequent toxicity.
Although aluminum has attracted much attention in
recent years in this regard, it is important to recognize that
other trace metals in the serum and total body may be
affected and may also play a role in the pathogenesis of some
of the clinical features of uremia. Uremic patients show an
increased nickel concentration in serum, but this does not
appear to be associated with a corresponding increase in
tissues, in contrast to the hyperaluminemia
of chronic renal
failure (92). As for other trace metals and their potential
effects in chronic renal failure, it is important to recognize
that deficiency states may also play a contributory role in
the pathogenesis of the clinical syndrome of uremia. Zinc
deficiency is a feature of uremia (93) and has been implicated as a major cause of the impairment in cellular immunity
which is seen in these patients (94). The rates of sodium and
potassium transport in vitro are normally influenced by the
extracellular concentration of zinc; the cellular membrane
transport of sodium reportedly is defective in leukocytes and
erythrocytes from uremic patients. A low serum zinc concentration, however, does not account for the defect in membrane sodium transport in uremia (95), nor is it associated
with an intracellular deficiency (95).
Conclusion
The biochemical disturbances-and
in particular the role
as toxins-in
the pathogenesis of
the syndrome of uremia are complex. The complexity is also
manifested by the variety and nature of the individual
disorders that make their individual contributions to the
features seen in the final clinical syndrome. The complexity
is a reflection of the importance of the homeostatic
and
endocrine roles of the kidneys in maintaining the constitution of the internal environment. In any review of uremia it
is apparent that none of the body systems is spared. The
constitution of the internal environment is disturbed not
only by the retention of metabolic end products but also by
the effects of the retained metabolites
on intermediary
metabolism. In addition to the many metabolites that are
retained in the body fluids as a consequence of chronic renal
failure there are major disturbances in total body electrolyte
composition and the distribution of body water (96, 97) and
hormone-control mechanisms (98). Brennan et al. (96) reported that in patients with end-stage chronic renal failure
there were significant increases in total body sodium, chloride, water, and extracellular
fluid volume. These changes
in total body water and sodium are linked both with
alterations in their respective hormonal control mechanisms and with the clinical problem of hypertension in
uremia. The role of the changes in extracellular
fluid
volume and electrolytes in the causation of some of the other
biochemical and metabolic disturbances in the syndrome of
uremia is not clearly defined.
The complexity, interactions, and consequences of the
uremic state and the potential role of toxins are exemplified
by a consideration of the disorder in carbohydrate and
insulin metabolism in chronic renal failure. Patients with
chronic renal failure have a diminished ability to handle a
glucose load, the degree of glucose intolerance correlating
roughly with the severity of the uremia (3). The disturbance
in glucose metabolism is the end result of the summation of
several interrelated factors, including toxins. The dominant
feature in the disturbance would appear to be an impairment of the insulin-dependent transfer of glucose into extrahepatic tissues and its subsequent utilization, with a defect
in insulin synthesis or release (or both) playing a relatively
minor role. The primary defect in the sequence appears to be
an insensitivity of peripheral tissue to insulin, probably a
defect either in intracellular
metabolism or in the glucose
transport system (99). The importance of the disturbance in
carbohydrate and glucose metabolism is not in any way the
of the retained metabolites
direct result of variations in blood glucose concentrationhyperglycemia is rare-rather,
it is ascribable to the important etiological role it potentially plays in the disturbance in
lipoprotein metabolism in uremia. The precise cause of the
dyslipoproteinemia
of chronic renal failure is not clear,
although the disorder in carbohydrate metabolism is probably a major factor. The severity of the dyslipoproteinemia
has shown no consistent correlation with the nature of the
underlying renal disease, the degree or duration of renal
failure, or the diet. The patterns of the disturbance in
lipoprotein
metabolism
in patients with chronic renal failure take several forms: an increase in the concentration and
cholesterol content of very-low-density
lipoprotein; an increase in the size and triglyceride content of low-density
lipoprotein particles; a decreased concentration of cholesterol in high-density lipoprotein; and an increased prevalence
of an electrophoretic
subclass (late pre-/3-lipoprotein)
of
very-low-density lipoprotein (100, 101). The importance of
the dyslipoproteinemia of uremia is that it is related to the
accelerated atherogenesis and premature coronary artery
disease that is a clinical feature of patients with chronic
renal failure, particularly those being treated by long-term
intermittent
hemodialysis.
Uremia is a multi-system clinical and biochemical problem that still requires extensive
clarification. It would seem
unlikely, in the final analysis, if any one individual toxin
will ever be identified as “the” toxin; rather, the syndrome of
uremia probably will be attributed to a cumulative effect in
vivo, to which all of the retained metabolites contribute. In
the process of evolution the kidneys have developed, during
the past 600 million years, into complex organs, sharply in
contrast to the simple open-ended tubes that drained waste
products from the coelomic cavity of our early remote
ancestors (3). As complex, sophisticated homeostatic and
endocrine organs the kidneys play the dominant role in the
maintenance of the internal environment. The failure to
remove metabolic waste end products from the urine, their
consequent retention in the blood and tissue compartments,
and their potential cumulative action as toxins are major
features in the pathogenesis of the clinical syndrome of
uremia.
References
1. Bernard C. Lecons sur les Phenomnes
di Is Vie Commune aux
Animaux et aux Vegetaux, 1, Balli#{233}re,
Paris, France, 1878.
2. Peters JP. Body Water, Balli#{233}re,
Tindall and Cox, London, U.K.,
1935.
3. WiI1 MR. Metabolic Consequences of Chronic Renal Failure, HM
+ M Publishers, Ltd., Aylesbury, Buckinghamshire,
U.K., 1978.
4. Piorry PA, L’H#{233}ritier
D. Trait#{233}
des Alterations du Sang, Bailhere, Paris, France, 1840.
5. Schreiner GE, Winchester JF. Uremia-1978
perspective. Clin
Nephrol 11, 52-55 (1979).
6. Bostock J. Observations on the chemical properties of urine,
Quoted by R Bright in Reports of Medical Cases, Selected with a
View of Illustrating
the Symptoms
and Cure of Diseases by a
Reference to Morbid Anatomy,
1, Longman, Rees, Orme, Brown and
Green, London, U.K., 1827, pp 75-85.
7. Bright it Reports of Medical Cases, Selected with a View of
Illustrating the Symptoms and Care of Diseases by Reference to
Morbid Anatomy, 2, Longman, Rees, Orme, Brown and Green,
London,
U.K.,
1831.
JP, Legrain M, Hoigne R. Observations on the role of
urea in uremia. Am J Med 14, 519-520 (1953).
9. Kennedy AC, Linton AL, Eaton JC. Urea levels in cerebrospinal
fluid after hemodialysis. Lancet i, 410-411 (1962).
10. Wakim KG, Predominance of hyponatremia over hypo-osmolality in simulation of the dialysis disequilibrium
syndrome. Mayo
Clinic Proc 44, 433-460 (1969).
11. Port FK, Johnson WJ, Klass DW. Prevention of dialysis dis8. Merrill
CLINICAL CHEMISTRY, Vol. 31, No. 1, 1985
11
equilibrium syndrome by use of high sodium concentration in the
dialysate. Kidney mt 3, 327-333 (1973).
12. Cockcroft DW, Gault MR. Prediction of creatimne clearance
from serum creatinine. Nephron 16, 31-41 (1976).
13. Mitch WE, Collier VU, Walser M. Creatinine metabolism in
chronic renal failure. Clin Sci 58, 327-335 (1980).
14. Gresham GE, Keller MD. Hyperuricemia and chronic renal
disease. J Chronic Die 23, 755-762 (1971).
15. Danovitch GM, Weinberger J, Berlyne GM. Uric acid in
advanced renal failure. Clin Sci 43, 331-341 (1972).
16. Danovitch GM. Uric acid transport in renal failure. A review.
Nephron 9, 291-299 (1972).
17. Richet G. Someaspectsof uric acid metabolism in chronic renal
failure. In Nutrition in Renal Disease, GM Berlyne, Ed., Livingstone, Edinburgh, U.K., 1968, pp 133-139.
18. S#{248}rensen
LB. The elimination
of uric acid in man. Scand J Clin
Lab Invest 12, 1-2 14 (1960).
19. Zarembski PM, Hodgkinson A, Parsons FM. Elevation of the
concentration of plasma oxalic acid in renal failure. Nature (London) 212, 511-512 (1966).
20. Bennett B, Rosenblum C. Identification
of calcium oxalate
crystals in the myocardium in patients with uremia. Lab Invest 10,
947-955 (1961).
21. Bennington JL, Haber SL, Smith JV, et al. Crystals of calcium
oxalate in the human kidney. Am J Clin Pathol 41, 8-14 (1964).
22. Fanger H, Esparza A. Crystals of calcium oxalate in kidneys in
uremia. Am J Clin Pathol 41, 597-603 (1964).
23. Emerson PM, Wilkinson JH. Urea and oxalate inhibition of the
serum lactate dehydrogenase. J Clin Pathol 18, 803-807 (1965).
24. Clements Jr RS, DeJesusJr PV, Winegrad Al. Raised plasmamyoinositol levels in uraemia and experimental neuropathy. Lancet
i, 1137-1141 (1973).
25. Reznek RH, Salway JG, Thomas PK. Plasma-myoinositol
concentrations in uraemic neuropathy. Lancet 1, 675-676 (1977).
26. Blwnberg A, Esslen E, B#{252}rgi
W. Myoinositol-a
uremic neurotoxin? Nephron 21, 186-191 (1978).
27. Giovanetti S, Biagini M, Balestri PL, et al. Uraemia-like
syndrome in dogs chronically intoxicated with methylguanidine
and creatinine. Clin Sci 36, 445-452 (1969).
28. Stein IM, Perez G, Johnson R, Cummings NB. Serum levels
and urinary excretion of methylguanidine in chronic renal failure.
JLab Clin Med 77, 1020-1024 (1971).
29. Stein IM, Michlus MJ. Concentrations in serum and urinary
excretion of guanidine, 1-methylguanidine, and 1,1-dimethylguanidine in chronic renal failure. Clin Chem 19, 583-585 (1973).
30. Giovanetti 5, Barsotti G. Methylguanidine in uremia. Arch
Intern Med 131, 709-713 (1973).
31. Giovanetti 5, Barsotti G. Uremic intoxication. Nephron 14,
123-133 (1975).
32. Felgate RA, Taylor WH. The effects of methylguanidine on
tissue respiration in vitro. Clin Sci 41, 85-87 (1971).
33. Balestri PL, Rindi P, Biagini M, et al. Effects of uraemic serum,
urea, creatinine and methylguanidine on glucosemetabolism. Clin
Sci 42, 395-404 (1972).
34. Natelson 5, Stein I, BonasJE. Improvements in the method of
separation of guarndino organic acids by column chromatography.
Isolation and identification of guanidinosuccinic acid from human
urine. Microchem J 8, 371-382 (1964).
35. Cohen BD, Stein IM, Bonas JE. Guarudinosuccinic aciduria in
uremia. A possible alternative pathway for urea synthesis. Am J
Med 45, 63-68 (1968).
36. Stein IM, Cohen BD, Kornhauser RS. Guanidinosuccinic acid
in renal failure, experimental azotemia and inborn errors of the
urea cycle. N EngI J Med 280, 926-930 (1969).
37. Dobbelstein H, Edel HH, Schmidt M, et al. Guanidinbersteinsflure and Uramie. I. Mitteilung: Klimsche Untersuchungen. Klin
Wochenschr 49, 348-357 (1971).
38. Dobbelstein H, Grunst J, Schubert G, et al. Guanidinbersteins#{228}ure
and Ur#{228}mie.
II. Tierexperimentelle
Befunde. Kim Wo49, 1077-1083 (1971).
39. Simenhoff ML, Saukkonen JJ, Burke JF, et al. Amine metabolism and the small bowel in uremia. Lancet ii, 818-821 (1976).
40. Simenhoff ML, Saukkonen JJ, Burke JF, et al. Importance of
chenschr
12 CLINICAL CHEMISTRY, Vol.31, No. 1,1985
aliphatic amines in uremia. Kidney Int 13, S16-S19 (1978).
41. Simenhoff ML, Burke JF, Saukkonen JJ, et al. Biochemical
profile of uremic breath. N Engl J Med 297, 132-135 (1977).
42. Massry SO. Is parathyroid hormone a uremic toxin? Nephron
19, 125-130 (1977).
43. Massry SO, Goldstein DA. The search for uremic toxin(s) “X”“X” = PTH. ClinNephrol
11, 181-189 (1979).
44. Avram MM, Feinfeld DA, Huatuco AR. Search for the uremic
toxin. Decreased motor-nerve conduction velocity and elevated
parathyroid hormone in uremia. N EngI J Med 298, 1000-1003
(1978).
45. Avram MM, Morrow IP, Feinfeld D, et ah. Uremic syndrome in
man: New evidence for parathormone as a multi-system nuerotoxin. Clin Nephrol 11, 59-62 (1979).
46. Goldstein DA, Chui LA, Massry SG. Effect of parathyroid
hormone and uremia on peripheral nerve calcium and motor-nerve
conduction velocity. J Clin Invest 62, 88-93 (1978).
47. CooperJD, Lazarowitz VC, Arieff A!. Neurodiagnostic
abnormalities
in patients with acute renal failure. Evidence for neurotoxicity of parathyroid hormone. J Clin Invest 61, 1448-1455 (1978).
48. Goldstein DA, Feinstein El, Chui LA, et al. The relationship
between the abnormalities in electroencephalogram and blood
levels of parathyroid hormone in dialysis patients. J Clin Endocrinol Metab 51, 130-134 (1980).
49. Zingraff J, Drueke T, Marie P, et al. Anemia and secondary
hyperparathyroidism. Arch Intern Med 138, 1650-1652 (1978).
50. Barbour GL. Effect of parathyroidectomy on anemia in chronic
renal failure. Arch Intern Med 139, 889-891 (1979).
51. Podjarny E, Rathaus M, Kormts Z, et al. Is anemia of chronic
renal failure related to secondary hyperparathyroidism? Arch Intern Med 141, 453-455 (1981).
52. Freedman MH, Cattran DC, Saunders EF. Anemia of chronic
renal failure: Inhibition of erythropoiesis by uremic serum. Nephron 35, 15-19 (1983).
53. Babb AL, Popovich RP, Christopher TO, et al. The genesis of
the square metre-hour hypothesis. Trans Am Soc Artif Int Organs
17, 81-91 (1971).
54. Babb AL, Farrell PC, Uvelli DA, et al. Hemodialyzer evaluation by examination of solute molecular spectra. Trans Am Soc
Artiflnt Organs 18, 98-105 (1972).
55. Asaba H. Accumulation and excretion of middle molecules.
Clin Nephrol 19, 116-123 (1983).
56. Chapman GV, Ward RA, Farrell PC. Separation and quantifi-
of the “middle
molecules” in uremia. Kidney mt 17, 82-88
(1980).
57. FUrst P, Zimmerman
L, Bergstrom J. Determination of endogenous middle molecules in normal and uremic body fluids. Clin
Nephrol 5, 178-188 (1976).
58. Bergstrom J, Furst P, Zimmerman L. Uremic middle molecules
exist and are biologically active. Clin Nephrol 11, 229-238 (1979).
59. Asaba H, Alvestrand A, Bergstrom J, et al. Uremic middle
molecules in non-dialyzed azotemic patients: Relation to symptoms
and clinical biochemistries. Clin Nephrol 17,90-95 (1982).
60. Asaba H, Alvestrand A, Furst P, et al. Clinical implications of
uremic middle molecules in regular hemodialysis patients. Clin
Nephroi 19, 179-187 (1983).
61. Faguer P, Man N-K, Cueille G, et al. Improved separation and
quantification of the “middle molecule” b4-2 in uremia. Clin Chem
29, 703-707 (1983).
cation
62. Asaba H, Bergstrom J, Ftirst P, et al. The effect of renal
transplantation on middle molecules in plasma and urine. Clin
Nephrol 8, 329-334 (1977).
63. Menyhart J, Grof J. Many hitherto unknown peptides are
principal constituents of “uremic” middle molecules. Clin Chem 27,
1712-1716
(1981).
64. Schoots AC, Mikkers FEP, ClaessensHA, et al. Characterization of uremic “middle molecular” fractions by gas chromatography,
mass spectrometry,
isotachophoresis, and liquid chromatography.
Clin Chem 28, 45-49 (1982).
65. Navarro J, Contreras P, Touraine JL, et al. Are ‘middle
molecules’ responsible for toxic phenomena in chronic renal failure?
Nephron 32, 301-307 (1982).
66. Fenselau C. Mass spectrometry of middle molecules. Anal
Chem 54, 105A-116A (1982).
67. Giordano C, Bloom J, Merrill JP. Effect of urea on physiologic
systems. I. Studies on monoamine oxidase activity. JLab Clin Med
59, 396-400 (1962).
68. Lacefles PT, Taylor WH. The effect upon tissue respiration in
vitro of metabolites which accumulate in uremic coma. Clin Sci 31,
403-413 (1966).
69. Balestri PL, Rindi P, Biagini M, et al. Effects of uremic serum,
urea, creatiine and methylguanidine on glucose metabolism. Clin
Sci 42, 395-404 (1972).
70. Hicks JM, Young DS, Wootton IDP. The effect of uremic blood
constituents on certain cerebral enzymes. Clin Chim Acta 9, 228235 (1964).
71. Young DS, Wootton IDP. The retention of ammes as a factor in
uremic toxaemia. Clin Chim Acta 9, 503-505 (1964).
72. Schwerin P, Bessman SP, Waelsch H. The uptake of glutamic
acid and glutamine by brain and other tissues of the rat and mouse.
JBiol Chem 184, 37-44 (1950).
73. Morgan RE, Morgan JM. Plasma levels of aromatic amines in
renal failure. Metabolism 15, 479-481 (1966).
74. Wills MR, Jenkins MV. The effect of uraemic metabolites on
parathyroid extract-induced bone resorption in vitro. Clin Chim
Acta 73, 121-125 (1976).
75. Wills MB, Savory J. Aluminum poisoning Dialysis encephalopathy, osteomalacia and anaemia. Lancet ii, 29-34 (1983).
76. Mauras Y, Allain P, Riberi P. Etude de l’absorption digestive de
l’hydrocarbonate d’aluminum chez l’individu sain. Therapie 37,
593-605
(1982).
77. Berlyne GM, Ben-An J, Pest D, et al. Hyperaluminaemia from
aluminum resins in chronic renal failure. Lancet ii, 494-496(1970).
78. Alfrey AC. Aluminum intoxication. N Engi J Med 310, 1113-
1115 (1984).
79. Alfrey AC, Mishell JM, Burks J, et al. Syndrome of dyspraxia
and multifocal seizures associated with chronic hemodialysis. Trans
Am Soc Artif Intern Org 18, 257-261 (1972).
80 Alfrey AC, L.eGendre OR, Kaehny WD. The dialysis encephalopathy syndrome. Possible aluminum intoxication. N Engl J Med
294, 184-188, (1976).
81. Leeming RJ, Blair JA. Dialysis dementia, aluminium and
tetrahydrobiopterin
metabolism.
Lancet i, 556 (1979).
82. Marquis J. Aluminum neurotoxicity: An experimental perspective. Bull Environ Contam Toxicol 29, 43-49 (1982).
83. Parsons V, Davies C, Goode C, et al. Aluminium inbone from
patients with renal failure. Br Med J iv, 273-275 (1971).
84. Ward MK, Feest TO, Ellis HA, et al. Osteomalacic dialysis
osteodystrophy:Evidence for a water-borne aetiological agent probably aluminium. Lancet i, 841-845 (1978).
85. Parkinson IS, Ward MK, Feest TO, et al. Fracturing dialysis
osteodystrophy and dialysis encephalopathy. An epidemiological
survey. Lancet i, 406-409 (1979).
86. Kaye M. Oral aluminum toxicity in a non-dialyzed patient with
renal failure. Clin Nephroi 20, 208-211 (1983).
87. Thomas WC, Meyer JL. Aluminum-induced
osteomalacia: An
explanation. Am J Nephrol 4, 201-203 (1984).
88. Lieberherr M, Grosse B, Cournot-Witmer
G, et al. In vitro
effects of aluminum on bone phosphatases: A possible interaction
with bPTH and vitamin D metabolites. Calcif Tissue mt 34,280284 (1982).
89. Graf H, Stummvoll HK, Meisinger V. Dialysate aluminium
concentration and aluminium transfer during haemodialysis. Lan.
oct i, 46-47 (1982).
90. Kaehny WD, Alfrey AC, Holman RE, et al. Aluminum
transfer
during hemodialysis.
Kidney mt 12, 361-365 (1977).
91. Savory J, Wills MR. Dialysis fluids as a source of aluminum
accumulation. In Trace Elements in Rena! Insufficiency, GM Gerlyne, S Giovanetti, Eds., Karger, Basle, Switzerland, 1984, pp 1223.
92. Savory J, Brown S, Bertholf R, et al. Serum and lymphocyte,
nickel and aluminum concentrations in patients with extracorporeal hemodialysis. Ann Clin Lab Sci 14, 413-414 (1984).
93. Lindemann RB, Baxter DJ, Yunice An, et al. Serum concentration and urinary excretions of zinc in cirrhosis, nephrotic syndrome
and renal insufficiency. Am J Med Sci 275, 17-31 (1978).
94. Antomou LD, Shaloub RJ, Schechter GP. The effect of zinc on
cellular immunity in chronic uremia. Am J Clin Nutr 34, 19121917 (1981).
95. Michael J, Hilton PJ, Jones NF. Zinc and the sodium pump in
uremia. Am J Ciin Nutr 31, 1945-1947 (1978).
96. Brennan BL, Yasumura 5, Letteri JM, et al. Total body
electrolyte composition and distribution of body water in uremia.
Kidney mt 17, 364-371 (1980).
97. Mitch WE, Wilcox CS. Disorders of body fluids, sodium and
potassium in chronic renal failure. Am J Med 72, 536-550 (1982).
98. Bonomini V, 0mm 0, Stefoni S, et al. Hormonal changes in
uremia. Clin Nephrol 11, 275-280 (1979).
99. De Fronzo RA. Pathogenesis of glucose intolerance in uremia.
Metabolism 27, 1866-1880 (1978).
100. Norbeck HE. Serum lipoproteins in chronic renal failure. Acta
Med Scand 1, Suppl 649, 1-49 (1981).
101. Chan MK, Varghese Z, Moorhead JF. Lipid abnormalities in
uremia, dialysis and transplantation.
Kidney mt 19, 625-637
(1981).
CLINICAL CHEMISTRY, Vol. 31, No. 1, 1985
13