SYMPOSIUM ON AZOTEMIA
ROBERT P. M A C F A T E ,
EICHELBERGER,
PH.D.,
CLARENCE COHN, M.D., LILLIAN
JOHN A. D. COOPER, M.D.
P H . D . , AND
University of Illinois College of Medicine and Board of Health, City of Chicago; Medical
Research Institute, Michael Reese Hospital- Department of Surgery, University of Chicago; and Department of Biochemistry, Northwestern University Medical School, Chicago,
Illinois
CONTENTS
Page
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
Introduction
511
Nitrogen metabolism. Nonprotein constituents
512
Physiologic and pathologic factors affecting over-all nitrogen metabolism
514
Protein metabolism leading to urea formation
516
Metabolism leading to formation of creatine and creatinine
519
Metabolism leading to formation of uric acid
522
Discussion of methods for estimation of nonprotein nitrogen in blood and u r i n e . . . 525
Discussion of methods for estimation of urea in blood and urine
530
Discussion of methods for estimation of creatine and creatinine in blood and
urine
533
X . Discussion of methods for estimation of uric acid in blood and urine
530
X I . Sources of error in clinical chemical determinations and suggestions for increasing the accuracy of these methods
53!)
X I I . Appendix. Suggested methodology. Bibliography
541, 565
I.
INTRODUCTION
The results of the Pennsylvania survey16 and other investigations,180 conducted
to check the accuracy of the more common chemical measurements in clinical
laboratories, clearly revealed a need for reviewing the methodology employed.
The importance of using reliable, trustworthy methods in the clinical laboratory
cannot be overemphasized. Of equal import is the understanding of, and conforming to, good laboratory practice in the matter of measurements and procedures in general. Finally, a knowledge of the common sources of error and the
methods of proving accuracy and reliability is essential.
These considerations led to the presentation of the Symposium on Blood
Glucose.180 In addition to the methodology for blood glucose, the collection and
preservation of blood, the pipetting of blood, protein precipitation and the principles of colorimetry were included. These items will not be repeated in this
symposium.
Received for publication July 31, 1953.
Presented at a joint meeting of the College of American Pathologists and the American Society of Clinical Pathology, in Chicago, October 16, 1951.
D r . MacFate is Chief, Division of Laboratories, Board of Health of the City of
Chicago, and Assistant Professor of Pathology, University of Illinois College of Medicine; Dr. Colin is Director of the Department of Biochemistry, Michael Reese H o s p i t a l ;
D r . ICichelberger is Associate Professor of Biochemistry, Division of Orthopedics, D e p a r t m e n t of Surgery, University of Chicago; and Dr. Cooper is Associate Professor of
Biochemistry, Northwestern University Medical School.
511
'
512
MACFATE ET
VOL. 24
AL.
The truth of the concept that end-products must accumulate in the blood
when the kidneys fail to perform their function in the formation of urine was
established as early as 1823, when Prevost and Dumas156 working with dogs,
demonstrated a gradual increase of the urea content of the blood following extirpation of the kidney. Previous to this, in 1773, Rouelle184 had isolated urea
from the urine. In 1799, Fourcroy 95 ' 96 recognized the importance of the excretion of urea by the kidney, although he based his statement on the abundance
of the urea rather than an actual knowledge of its functional importance. Bright,44
in 1830, first described the disease named in his honor. A little more than a year
later, Christison62 reported the finding of increased urea concentrations in the
blood of patients with this disease. In 1833, Wilson195 ascribed the symptoms
of uremia to the retention of urea in the blood. Since that time, the compound
urea has received much attention in studies of kidney function and kidney disease.
A detailed history is beyond the scope of the present work. In the late 1800's,
the concept was developed that the kidney acts as a delicate chemical laboratory
in addition to its function as a filtering apparatus. However, as a diagnostically
and prognostically valuable phenomenon, only the disturbance in nitrogen elimination proved fruitful. It was natural to study nitrogen retention in the same
medium in which cast's and albumin were studied. Little progress was made until
methods for the analysis of blood and urine were perfected.
In 1883, Kjeldahl123 introduced his method for the determination of nitrogen
in biologic materials. This replaced the older Knop-Hufner hypobromite method
for urea, and did much to advance the study of nitrogen retention. However,
as A'on Noorden192 pointed out in 1892, the tedious affair of a classic metabolic
experiment, covering 8 to 14 days, excluded completely a more extensive application of this method of study.
Another advance was made by Strauss179 in 1902, when he determined the
„ nonprotein nitrogen of the blood serum after first removing the proteins by boiling with acetic acid. His concept of the nonprotein nitrogen was that of "retained
nitrogen" and he proved the increase of the retained nitrogen in different stages
and in different groups of nephritis. Again this method of study was not extensively used, mainly for 2 reasons: (1) the method was too laborious; and (2)
there was no satisfactory classification of the different types of nephritis.
Finally, the methodology was improved by Folin and Denis,82 culminating
in the work of Folin and Wu92 in 1919, when they detailed their system of blood
analysis. Since then, prominent among the tools for studying heart, vascular
and kidney diseases have been the determinations of the concentration in the
blood and the excretion rate of urea, uric acid, creatine and creatinine. A discussion of each of these follows, that is not meant to be exhaustive, but will
serve to review the outstanding features.
'
I I . N I T R O G E N METABOLISM. N O N P R O T E I N
CONSTITUENTS
The nonprotein nitrogenous constituents of the body fluids to be considered
are urea, uric acid, creatine and creatinine. The body fluids to be considered are
MAY 1954
AZOTEMIA
513
blood and urine. Since the concentrations of these nitrogenous constituents in
the blood depend on the function of the kidney, an accurate knowledge of what
the kidney does in the normal organism and the range of variability of its
work must be understood. Reduced to simple terms, the kidney normally eliminates excess water, salts and nitrogenous end-products. It is, indeed, in the
elimination of nitrogenous waste products that the kidney plays its main role,
since this function cannot be replaced by the activities of any other organ.
The capacity of the kidney to perform its various excretory functions varies
considerably for the different constituents involved. Thus, water is ordinarily
excreted in volumes of 1 to 3 liters per 24 hours. Similarly, the normal kidney
under average conditions eliminates 20 to 30 grams of urea per day with a maximum concentration of approximately 4 per cent. However, an extra 50 to 100
grams of urea can be excreted by the normal kidney, if required.
It has been established, by Richards and his associates,161 that the function
of the glomeruli of the kidneys is a simple ultrafiltration of the blood plasma,
and the fate of this filtrate is determined by the activity of the tubular epithelium.
The tubules reabsorb some substances completely, leave others strictly alone,
and compromise in various ways with the remainder of the constituents of the
filtrate. The true end-products, such as urea, creatinine and uric acid, which
are of no direct value to the body, are not all treated exactly alike. This variation may be attributed to either an unequal reabsorption of these substances
by the tubules or to the selective secretion of the product by the tubules.
Urea is formed in the liver from a metabolic cycle involving ornithine, citrulline and arginine, as will be detailed later. It is known from experimentation
that, on an average protein diet, 85 per cent of the total nitrogen excreted by
the kidneys is urea nitrogen, 4.5 per cent is creatinine nitrogen and approximately 2 per cent is uric acid nitrogen. Since urea is the main end-product of
nitrogenous metabolism and its excretion is one of the kidney's chief functions,
it seems logical that the kidney's ability to remove urea from the blood should constitute an index of renal function. This correlation of the urinary output of
urea, blood urea level and the urine volume, called the "blood urea clearance,"
was worked out quantitatively by Moller, Mcintosh and Van Slyke138 and others.
It constitutes one of the most reliable renal function tests available at this time.
Creatine found in the skeletal muscle in the body is almost wholly in combination with phosphoric acid. This phosphoric acid is easily split off when the compound is acidified, and possibly the same reaction takes place with muscular
activity. An understanding of the mechanism of formation of creatine and creatinine in the body under normal conditions of metabolism is still in the controversial
stage. We do have experimental evidence that these substances arise from arginine, glycine and methionine. 24,28,41
Tissue creatine is constantly undergoing dehydration to creatinine, which is
eliminated from the body as a useless metabolite. Tierney and Peters183 showed
that the presence or absence of creatinuria is governed by the serum creatine
concentration. Hoberman, Sims and Peters111 have stated that the nonenzymatic
dephosphorylation of creatine phosphate to creatinine takes place at a rate to
514
MACFATE ET
AL.
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account for the amount of creatinine formed and excreted per day. Albanese
and Wangerin 3 demonstrated that creatinine excretion is relatively constant
under tremendous variations in protein intake.
Uric acid is formed from the purines of food and from the purines of the body
by the breakdown of nuclear material and nucleotides. Some of this formed uric
acid is retained in the blood and the balance is eliminated in the urine as the
monourate salt. The late work of Benedict, Forsham and Stetten,18 following
the giving of isotopic uric acid labeled with N15, showed that some uric acid undergoes catabolic breakdown in man. Excretion is increased by muscular exercise
and by foods rich in purines.
With the use of more and more amino acids and other substances labeled with
N16, we will have more direct answers to all of the questions involving nitrogen
metabolism in the body. With new tools and new methods being developed,
more reliable results can be expected in the future.
I I I . PHYSIOLOGIC AND PATHOLOGIC FACTORS A F F E C T I N G
NITROGEN
OVER-ALL
METABOLISM
The protein nitrogen of the body exceeds by far the other nitrogen-containing
compounds, and the over-all metabolism of nitrogen is essentially synonymous
with protein metabolism. The nonprotein nitrogen metabolites, with which the
present symposium is largely concerned, represent for the most part end-products
of protein metabolism. Their concentrations depend upon their rate of formation
during protein catabolism and their rate of excretion, chiefly by the kidneys.
Factors that influence either of these processes will affect their level in the blood
and their concentration in the urine.
The greater portion of the body protein is located within the cells where it
functions in the structural and catalytic machinery of the living tissue. Protein
is no longer believed to be the inert structural element that was popularized by
Folin74 in his concept of endogenous and exogenous protein metabolism. Folin
regarded endogenous protein as largely metabolically inert in the normal individual, with its anabolism and catabolism limited to that which occurred during
"wear and tear" of the cells. In the modern view, which was introduced by Borsook and Keighley,43 the breakdown and resynthesis of body protein is thought
to occur continuously and at a relatively rapid rate. There is no relationship
between these processes and "wear and tear." The relative rates of the anabolic
and catabolic reactions are determined by a dynamic equilibrium between the
tissue and plasma proteins, the tissue amino acids and the plasma amino acids.
The tissue amino acids, in addition, are in equilibrium with the "metabolic
pool" where the intermediary metabolisms of amino acids, carbohydrates and
fat are joined through common metabolites in their breakdown schemes. This
concept is shown in Figure 1. All the reactions are reversible except those in the
final common pathway of oxidation to carbon dioxide and water. There is continual breakdown of the tissue and plasma proteins with the formation of tissue
amino acids and the simultaneous resynthesis of these proteins from the same
amino acid pool. The amino acids in the proteins of one tissue are being exchanged
MAY 1954
5.15
AZOTEMIA
at all times with those of other tissues through the plasma amino acids, with
which the tissue amino acids are in equilibrium. The amino nitrogen of one amino
acid may be used to form another amino acid in the metabolic pool, and thus
even the nitrogen of the amino acids is constantly being interchanged.
When the plasma amino acid concentration rises, as after ingestion of protein,
there is a tendency for the equations shown in Figure 1 to shift to the left. However, the body has little capacity for storage of nitrogen except during periods
of tissue growth, as in pregnancy or childhood, or during periods of protein replacement, as in convalescence following a wasting disease or after protein
stai'vation. As a result, there is an increase in deamination of the tissue amino
acids in the metabolic pool, with the formation of intermediates that may be
converted into fat or carbohydrate for storage in this form, or oxidized, supplying energy to the cell. The nitrogen that is split off during these processes may
be converted into urea, creatine or uric acid.
TISSUE PROTEIN
u
PLASMA PPOTFIN —»- TISSUE AMINO ACIDS ^
PLASMA AMINO ACIDS
Jf
/METABOLIC
FAT
=^=i= f
TOL
\
j
^CARBOHYDRATE
FINAL
COMMON
PATHWAY
COi+HtO* ENERGY
FIG. 1. The dynamic concept of nitrogen metabolism
From the above discussion, it can be inferred for normal adults that the nitrogen excretion in the urine should parallel the protein intake. This is in agreement
with experimental observations.74 In starvation or inadequate caloric intake, the
tissue proteins are called upon to supply energy after the stores of fat and carbohydrate have been exhausted. Nitrogen excretion continues in the urine even
though it exceeds in amount the intake in the diet.17
The growth hormone of the anterior pituitary129 and androgens121 have been
found to promote nitrogen storage and the synthesis of tissue protein. Insulin 55
decreases the rate of gluconeogenesis from protein in the depancreatized animal
and in patients with a deficiency of the endogenous secretion of the pancreas, as
in diabetes mellitus. It appears to have little effect on the normal individual.
Thyroxin may increase the rate of utilization of amino acids for energy production and may increase nitrogen excretion in the urine, especially if the heightened
caloric demands are not met with sufficient fat and carbohydrate in the diet.38
Some of the corticosteroids of the adrenal cortex augment nitrogen excretion in
both fasting and fed animals.132 Long, Katzin and Fry132 have interpreted this
516
MACFATE ET
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action as indicating that these hormones promote the conversion of protein
into carbohydrate. The increase in nitrogen excretion that accompanies hemorrhage, trauma, surgical shock, the febrile states of most acute infections, coronary occlusion and other conditions of "stress," has been explained by some on
the basis of an increase in the rate of secretion of the corticosteriods by the adrenal
cortex under the influence of a heightened adrenocorticotrophic hormone formation by the anterior pituitary in response to this "stress."
Because of the key position that the liver occupies in protein metabolism and
its singular ability to form urea, disturbances in hepatic function may affect
nitrogen metabolism and the concentrations of some of the nonprotein nitrogenous constituents in the body. The capacity of the liver to form urea is great, and
it is usually only in extensive damage, such as that seen in acute yellow atrophy,
that there is a significant decrease in urea concentration in the blood. As the blood
urea concentration decreases, the blood amino acid concentration increases in
this condition.36
Although the kidneys do not play a major role in protein metabolism, alterations in the rate of excretion of nitrogenous substances in the urine will affect
the concentration of nonprotein nitrogen in the blood. In the normal person, the
rate of nitrogen excretion is related, within certain limits, to the rate of urinaiy
flow.136 Diuresis will tend to increase the rate of excretion and decrease the concentration of urea in the blood. Conversely, with water deprivation, there is a
decrease in urinary flow with an increase in blood urea. If water deprivation
becomes extreme and is associated with an electrolyte imbalance, there will be
an increase in the rate of protein catabolism and an increased rate of urea formation, which will enhance the azotemia.
The ability of the kidney to excrete nitrogenous compounds is also affected
by pathologic conditions in which glomerular filtration is diminished. The nitrogen retention associated with acute and chronic glomerulonephritis is well
recognized.
I V . P R O T E I N METABOLISM L E A D I N G TO U R E A
FORMATION
Since urea, creatinine and uric acid are end-products of metabolism in man,
i.e., are formed in the body but are not utilized for anabolic or energy yielding
purposes, it is not quite correct to speak of the metabolism of these substances.
For this reason, the discussions that follow can only consider the metabolic
reactions responsible for the formation of these substances and their modes of
excretion. Although urea, creatinine and uric acid have different metabolic
pathways that lead to their formation, all these substances have in common the
kidney as the principal organ for excretion.
Urea, OC(NH2)2, is a small compound in which the nitrogen content constitutes almost 50 per cent of the total molecular weight. Hence, it is well suited
for being the chief end-product of protein metabolism as regards economy of
excretion of nitrogen. It is very soluble in water, freely diffusible across cell and
capillary membranes, and is believed to be equally distributed in the body water
of man. Pharmacologically, urea serves as a mild diuretic.
MAY 1954
AZOTEMIA
517
Since urea is the main end-product of protein catabolism, the formation of
urea is concerned with over-all body protein metabolism. This includes absorption, transportation, storage and excretion of amino acids and/or protein. Proteins in the food are enzymatically degraded in the gastrointestinal tract to
polypeptides and amino acids, in which form they are absorbed and transported.
An alteration in pancreatic or bowel function, with enzyme deficiencies or mucosal changes that impair absorption, can therefore limit the amount of protein
absorbed and made available for storage in tissue cells or for urea formation.
Plasma amino acids are in equilibrium with cellular amino acids and/or amino
acids in proteins in that the two are constantly interchanging. Thyroid hormone
in excess disturbs the equilibrium by accelerating cellular breakdown, hence,
makes available increased amounts of amino acids from which urea is formed.
Excess adrenocortical steroids and lack of insulin have a similar effect. Growth
hormone and testosterone, on the other hand, appear to shift the equilibrium in
the reverse direction by promoting nitrogen deposition in cells.
Aside from hormonal effects, other factors such as caloric and protein intake
normally have little influence on the storage of nitrogen in cells. However, with
a deficient protein or caloric intake, cellular protein will be lost because the equilibrium for the normal interchange of cellular and plasma amino acids will be
disturbed and cellular amino acids will be used as a source of energy. In contrast,
it is impossible to build up increased stores of body protein by an excess of protein in the diet. The excess of the absorbed amino acids will be deaminated and
the nitrogen excreted as urea. Only under special conditions, such as in the repletion of protein in a protein-depleted individual, can protein intake influence
protein deposition. It should be emphasized that it is the state of constant interchange of plasma for cellular amino acids that makes the nitrogen of both the
ingested and tissue-cell amino acids the immediate precursors of urea. For more
detailed discussion of the metabolism of protein and its degradation to urea,
refer to Peters and Van Slyke,148 Everett 68 and Schoenheimer.168
The liver is the sole site of urea formation. Prior to the demonstration that it
is the only organ that contains all enzymes necessary for the formation of urea,
Bollman, Mann and Magath 35 showed, in liverless nephrectomized dogs, that
there was no increase in blood urea concentrations. If other organs beside the
liver could synthesize urea, an increase in the blood urea concentrations would
have occurred. Krebs and Henseleit126 subsequently demonstrated that urea
was enzymatically formed in the liver from a metabolic cycle involving ornithine,
citrulline and arginine (Fig. 2). Recent work 125,160 concerned with this aspect
of metabolism has confirmed the original observation and in addition has revealed the sources of nitrogen and energy responsible for the reactions. The energy necessary for this cycle is derived from adenosine triphosphate (ATP).
Aspartic acid, formed as a result of transamination reactions between amino
acids and oxalacetic acid, appears to be the immediate source of nitrogen. There
is also evidence for some connection between this cycle and the tricarboxylic
acid cycle (the reactions responsible for the conversion of fat, carbohydrate and
protein to CO2 and H2O).
518
MACFATE BT
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Urea is excreted from the body mainly by the kidney. It is thought to be cleared
at the glomerulus at the nitration rate, but partly to diffuse back into the blood
during the tubular reabsorption of water.174 The rate of back diffusion is a function of the urinary flow. Therefore, the urea clearance does not measure any
discrete renal function, since the clearance must be something less than the
filtration rate. Increased serum levels of urea will be found in advanced bilateral
renal disease, regardless of its etiology, and in lower urinary tract obstruction.
In addition, extrarenal factors, such as shock, dehydration or sodium chloride
deficiencies that influence the glomerular filtration rate, likewise are associated
with diminished urea excretion.
It is the custom at present to estimate renal function on the basis of a single
determination of serum urea. In most cases there is no objection to such a procedure, since with bilateral renal disease and lower genitourinary tract obstruction with urinary retention, elevated levels of serum urea are encountered. Renal
function, however, is only one factor involved in the regulation of serum urea
levels. The concentration of urea in serum is the resultant of protein catabolism
versus anabolism as influenced by the caloric and protein intake, endocrine acTricarboxylic acid cycle
-^-
\
Aspartate
(amination)
Citrulline (+ATP)
Intermediate
NH3
lamination)
Arginine +
Carbamyl
glutamate
Urea
(+ATP) Ornithine
FIG. 2. Simplified scheme of mechanism of formation of urea
malate
MAY 1954
519
AZOTEMIA
tivity and body injury, in the presence of normal or abnormal renal function.
Addis and his associates1 demonstrated the direct effect of protein intake on
blood urea levels. In both normal persons and in those with renal insufficiency,
it is possible to obtain a 400 per cent variation in serum urea levels due to differences in protein intake, renal function being constant100 (Fig. 3). This finding
emphasizes the necessity for some knowledge of the food habits of ambulatory
individuals and of the nitrogen intake of parenterally fed patients when evaluation of serum urea levels is attempted. I t might be pointed out, by contrast,
that serum creatinine levels vary at a minimum with the protein intake.12
Blood Urea
mg./100ml.
Renal Insufficient
250
llO
ISO
20.
SO
35
US
230
175
I.
75
10
Protein Intake
Gm./day
Urea Clearance
ml./min.
1 0 0 **1 10
1
sols
FIG. 3. The influence of dietary protein
intake on blood urea concentrations. The1
data for the normals arc taken from Addis.
The data for the person with renal insufficiency are taken from Goldring and
Chasis'.100
V.
METABOLISM
LEADING TO FORMATION OP CREATINE AND
CREATININE
Since 1886, when creatine was first isolated by Chevreul, the origin and function of this substance has been the subject of continual investigation. Creatine
is a methylated guanidoacetic acid, and creatinine is its internal anhydride
(Fig. 4). The first important advances in the chemistry of these 2 related substances were made by Folin from 1905 to 19.1.5.74'84 The Folin methods for their
quantitative estimation in fluids and tissues led to the accumulation of a vast
amount of information. In 1927, Fiske and Subbarow,69 and also the Eggletons,66
showed that a creatine-phosphoric acid occurs in muscle tissue of mammals,
which breaks down on contraction of muscle and is resynthesized on expansion.
It was not until the studies of du Vigneaud and his co-workers63, 64 were published that the formation of creatine and creatinine could be visualized. In 1941,
520
MACFATE ET
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du Arigneaud and his associates65 presented a new concept: that transmethylation
is a general metabolic process. Using methionine containing the isotopic methyl
group, they gave experimental evidence to show that methionine serves as a
source of methyl groups for creatine synthesis. Furthermore, the methyl group
of the creatine, isolated from the tissues of the animals receiving the methionine
containing the isotopic methyl group, agreed closely in isotopic content with
that of creatinine in the urine.
Du Vigneaud, and his co-workers,66 showed that the pathway of a given methyl
group was more direct from methionine to creatine than from other compounds
structurally related to methionine. The methyl donating ability of a group of
compounds, using the growth of the rat as the criterion, showed that a high
degree of specificity exists. In experiments consisting of feeding choline and betaine containing heavy carbon, the presence of the isotopic methyl group was
'NH2
C=NH
rN.CH,
/NH
C = NH
^)N.CH3
CH 2
CH 2
COOH
Creatine
CO
Creatinine
FIG. 4. Structural formulas of creatine and
creatinine
found in the creatine of the tissues of the rats and also in the creatinine of the
urines, showing that choline and betaine can eventually find their way to creatine.29
The scheme for the biologic synthesis of creatine may be summarized as follows: The amidine group of arginine is transferred from arginine to glycine to
form guanidoacetic acid (Fig. 5). The first part of this reaction was shown chemically by Bergmann and Zervas.24 Bloch and Schoenheimer28 used the nitrogen
isotope to prove the last part. Borsook and Dubnoff41 performed the tissue-slice
technic. Methionine then donates a methyl group to the guanidoacetic acid to
form creatine.
Du Vigneaud and his associates 6266 have stated that the methylation of guanidoacetic acid is nonreversible. The methyl group of creatine is stable and cannot
furnish methyl groups to the body. Furthermore, Hoberman110 in 1947 found
that guanidoacetic acid, the physiologic precursor of creatine, is a regular constituent of blood serum. These studies ended the long search for the precursors
of creatine.
The methyl radical, attached to S or N in certain compounds, enters into
intermediary metabolism as a unit, being transferred from methionine without
MAY 1 9 5 4
521
AZOTEMIA
any exchange of hydrogen atoms. This is a late finding of Keller, Rachele and
du Vigneaud.120 These transmethylation studies were first carried out on the
rat, but similar metabolic routes are followed in the human. Simmonds and du
Vigneaud'71 found that by using labeled methyl groups of methionine in the diet
of man, the isotopic methyl group made its appearance in the creatinine of the
urine.
As recently as 1949, du Vigneaud and his associates,134 observed in the intact
rat, that from the isotopic methyl group administered in methionine, one quarter
of the tagged element appeared in the expired CO2 and one half was excreted in
the urine and feces. They concluded, therefore, that transmethylation to form
creatine was a more active process than the incorporation of methyl groups in
total body protein.
The scheme of the metabolic interrelationships of methionine, choline and
creatine as presented by du Vigneaud62 is shown in Figure 6. The methyl group
of methionine can be released for the methylation of guanidoacetic acid to creatine and, as a result, homocysteine is formed. The homocysteine can then accept
a methyl group from choline yielding methionine, which in turn can give up its
methyl group. Thus, the methyl groups of choline are available for creatine
synthesis. The irreversibility of the methylation of guanidoacetic acid is represented.
The formation of creatine and creatinine in the intact body is evidently the
work of enzymatic processes and therefore must take place in the tissues. Many
in vitro studies have been conducted, and the possible sites of creatine formation
have been evaluated. In general, creatine formation occurs in the skeletal muscles, liver and kidneys. Baker and Miller9 showed that creatine formation occurred in all tissues. The formation of guanidoacetic acid in man has been shown
HH2
I
/*H2 I
\ N - O H y COOH
LfFJSj
NH
I
CH,
• *
CH 2
„'
GUANIDO ACETIC ACID
""^
*
1
•
H-CH„COOH'
Methylation
„/
H-CMH-,Z
I
COOH
HHg
CcNH
N-CH„COOH
/
CH3
ARQININE
GLYCINE
CREATINE
F I G . 5. Biologic synthesis of creatine
Z
522
MACFATE JST
VOL. 2 4
AL.
by Borsook and his associates42 to be slow, and thus the rate of formation of
creatine in the body must also be slow. Bodansky33 has given us evidence that
the conversion of the guanidoacetic acid to creatine occurs in the liver.
Ever since the discovery of creatine and creatinine, many observers have
thought that creatinine is formed in the body by the dehydration of creatine.
Experimental evidence has been given pro and con. Recently Bloch, Schoenheimer and Rittenberg 30 fed creatine containing N16 to rats; creatinine containing
N 16 was isolated from the urine. This is direct proof that creatine is changed
into creatinine. If creatinine containing isotopic nitrogen is fed to rats, a large
percentage of the creatinine containing N 16 is excreted in the urine and the
creatine of the muscles does not contain the isotope. In other words, it seems
that the biologic transformation of creatine into creatinine is irreversible.
On the whole, much progress has been made during the past 10 years in solving at least partially the problems of creatine and creatinine metabolism. The
use of isotopic nitrogen and carbon has been the main key to the successes attained thus far, but the fact must not be overlooked that these conclusions were
reached under conditions of metabolism that were not exactly normal.
V I . METABOLISM L E A D I N G TO FORMATION O F URIC ACID
Uric acid represents the principal end-product of purine metabolism in man.
The purine precursors are present in all living tissue as components of the nucleic
acids, which in combination with basic proteins form the nucleoproteins found
in the nucleus and cytoplasm of all cells. Two purines, adenine and guanine
J*
C=NH
\
HH-CHgCOOH
CHj-NjCHg-CHg-OH
CH.
NHj,
Choline
Guanido-acetic acid
^NCH,C=0
SH
[ay
I
CH,
fz<r
HCNHg
c=o
s
I
_ cm
^
CH.
I '
HCNHg
CH.
Creatine
t«y
I
s
OH
0=0
V
0H
Hoaocyrtaino
t
Jtothionina
KN=C
/
" \
\
C=0
/
H ——
CH2
/
•
«
%
F I G . 6. Scheme of metabolic interrelationships of methionine, choline
and creatine
MAY 1954
AZOTEMIA
523
occur in the nucleic acids as phosphorylated glycosides. The sugar is a pentose,
either ribose or desoxyribose. The phosphorylated glycoside is termed a nucleotide. The glycoside is called a nucleoside (Fig. 7). There are enzymes present
in tissues that are capable of degrading the nucleic acids to nucleotides, nucleosides or the component purines, pyrimidines and pentose.
The purines present in the body arise in part from the diet. Brown and his
co-workers49 have demonstrated by the use of isotopically labeled compounds
that, following administration of adenine, its purine skeleton is incorporated
into both adenine and guanine in the tissue nucleic acids. Guanine, on the other
hand, was not incorporated into nucleic acids after administration.
PROTEIN
NUCLEIC
ACID
NUCLEOTIDES
H 3 P0 4
NUCLEOSIDES
PURINES
PYRIMIDINES
PENTOSE
F I G . 7. Stepwise hydrolysis of nuclcoprotcin
The body is not dependent upon an exogenous supply of purines. Early balance
studies indicated that purine synthesis occurred in the body,162 and recent investigations employing isotopically labeled compounds have established that
synthesis does occur and have permitted the precursors to be identified. It has
been found that 1 of the nitrogen atoms and 2 of the carbon atoms in the purine
skeleton are contributed by the amino acid glycine. Formate contributes 2 carbon atoms and the remaining 1 arises from bicarbonate. The source of the
other 3 nitrogen atoms is the metabolic pool153, 176 (Fig. 8).
There are enzymes present in the body that are capable of converting the
purines or their nucleosides into uric acid. This process involves deamination
and oxidation through the intermediates hypoxanthine and xanthine,118 as shown
in Figure 9. The distribution of the enzymes involved is erratic and varies from
species to species. The locus of uric acid formation in man is not known. The
human being is said to possess no adenase, and the conversion of adenine to
hypoxanthine is thought to occur by deamination of adenine while it is still
combined as a nucleoside. Uricase, an enzyme involved in the oxidation of uric
524
MACFATE ET
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acid to allantoin, is absent from human tissue.118 There is no clear-cut evidence
that uric acid is further metabolized in man."
Uric acid is found in low concentration in all of the body fluids, apparently
in a freely diffusible state. In the blood, the concentration in the cells is approximately half that in the plasma.199 However, the results are variable and depend
upon the analytic method used. Uric acid is also found in the cerebrospinal fluid,140
sweat,166 feces194 and body tissues.81
By the use of isotopically labeled uric acid, the size of the uric acid pool in
the normal adult has been estimated to vary from 900 to 1400 mg.99 From one
half to more than three fourths of the pool was found to be turned over each
day.
Uric acid is excreted almost entirely by the kidneys. The concentration in
the intestinal secretions is low. The current concept of renal mechanisms for
urate excretion assumes that there is complete filtration by the glomerulus and
subsequent reabsorption of all but 5 to 10 per cent of the filtered load by the
tubules. There is no evidence for tubular excretion of urates in man.174 The maximal tubular reabsorption rate (Tm) appears to be about 15 mg. per minute per
1.73 square meters of body surface.25 The renal mechanisms for urate excretion
do not appear to differ essentially from normal in the gouty individual.181 The
Tm is not saturated in either normal or gouty subjects and the appearance of
urates in the urine cannot be ascribed directly to the saturation of the Tm.
The urates do not differ in this respect from other substances such as ascorbic
acid,97 amino acids162 and phosphate,167 where excretion also occurs at levels
well below those required to saturate the Tm for these substances.
The uricosuric agents apparently increase urate excretion by the influence
that they exert in the tubular reabsorption mechanism for urate.104
Reports on the influence of purine intake on uric acid excretion are inconsistent.
Most investigators have found a direct relationship. Denis67 determined in the
N
CARBON.
DIOXIDE
I
GLYCINE
METABOLIC
POOL
FORMATE
FORMATE
w
METABOLIC
POOL
FIG. 8. Origin of the purine skeleton
MAY 1954
525
AZOTEMIA
normal that the concentration in the blood is not related to the amount of purines in the diet. In gouty patients, there may be an increase in the blood concentration with an increased intake. An increase in the protein and carbohydrate
content of the diet increases the excretion rate of uric acid.2, "• 158 Fats, starvation, exercise and acidosis tend to decrease the rate of excretion. 2,130 Salicylates 68, l06 cincophen and neocincophen,172 and aminophylline141 increase uric
acid excretion. Adrenocorticotrophin and the adrenocortical steroids have been
found, also, to increase uric acid excretion.177
In gout, there is a deposition of urates in cartilages and tendons around joints
and in tophi. Most patients with gout have hyperuricemia. The size of the uric
acid pool in gout has been found to be 4 to 15 times that in the normal person.18
The excretion of uric acid in gout appears to be normal except in patients with
renal damage, a common complication of the disease.
ADENINE
GUANINE
l
I
j
GUANASE
ADENASE
^
XANTHINE
HYPOXANTHINE
*-
XANTHINE
QX,0ASE
XANTHINE
OXIDASE
>
URIC ACID
FIG. 9. Origin of uric acid from purine precursors
In nephritis, the retention of uric acid is extremely variable. High values of
blood uric acid have been found in eclampsia, leukemia, polycythemia, pneumonia, cardiac decompensation, pernicious anemia in remission, severe diabetes,
chronic eczema and methyl alcohol poisoning.
VII. DISCUSSION OF METHODS FOR ESTIMATION OF NONPROTEIN NITROGEN IN
BLOOD AND URINE
An acceptable method for research and clinical laboratory estimation of any
constituent of the blood, urine or tissue, should have the following characteristics: It should be specific for the substance analyzed; be precise and capable of
duplication; be as simple in performance as possible; and should require minimum amounts of material so as to avoid the necessity for large amounts of blood,
since microtechnics facilitate laboratory diagnosis in children. The time requirements for performance must be at a minimum for routine purposes. In
the discussion of the methods that follow, where possible, these characteristics
of the various procedures will be considered.
The nonprotein nitrogen of the blood and urine should include all of the nitrogen-containing substances that are not proteins. However, by common practice,
the nonprotein nitrogen is defined as that group of nitrogenous compounds that
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are not precipitated by the usual protein-precipitating reagents. Present in the
blood are various nonprotein nitrogenous materials, such as certain of the lipids
that are partly precipitated with the coagulable proteins and partly escape precipitation, the extent of this occurrence depending upon the method used.
The nonprotein nitrogen of the serum or plasma is composed largely of urea,
ammonia, amino acids, uric acid, creatine and creatinine. That portion which
usually is not identified is called the "undetermined" nitrogen. These nonprotein
nitrogenous compounds, for the greater part, are products of protein metabolism.
Accordingly, the determination of their total concentration in the blood and
urine will provide knowledge concerning over-all protein metabolism. However,
a knowledge of the concentration of the individual constituents gives more
specific information, of far greater significance than the concentration of the
total nonprotein nitrogen. Where possible, the determination of the blood total
nonprotein nitrogen should be discarded and the determination of the more significant blood urea nitrogen substituted.
The determination of the total nonprotein nitrogen is reviewed for those who
find it impossible to discard this examination and for those few determinations
where these findings are absolutely essential. The nonprotein nitrogen of the
serum or plasma consists largely of urea and other protein metabolites. On the
other hand, the blood cells contain nonprotein nitrogenous constituents whose
complete identity is not known. Accordingly, the determination of the nonprotein nitrogenous constituents in the serum or plasma will have far greater significance, and will be more sensitive to metabolic changes, than will be the determination on whole blood.
The general procedure for determining the nonprotein nitrogen in blood and
urine is: (1) precipitation of the proteins, (2) conversion of nitrogen to ammonia
in the compounds remaining in solution and (3) determination of ammonia
nitrogen.
1. Precipitation of the Proteins
The first important published report on the separation of the proteins of blood
for the purpose of determining the nonprotein nitrogen was that of Ascoli8 in
1901, who acidified blood serum with acetic acid and then added sodium chloride.
Strauss179 in 1902 separated the proteins by the addition of acetic acid and heating the mixture. Von Jaksch191 in the same year precipitated the blood proteins
with phosphotungstic acid. In 1903, Umber184 used alcohol and phosphotungstic
acid. None of these procedures was entirely satisfactory.
In 1911, Obermayer and Popper144 again used acetic acid and heat. More
satisfactory in the same year was the work of Hohhveg,113 who used acetic acid,
monobasic potassium phosphate and sodium chloride. Foster94 in 1912, in his
work on uremic bloods, used alcohol, ammonium sulfate and heat. In the same
year, Folin and Denis82 published their first methods for total nonprotein nitrogen, urea and ammonia in blood. They used, as the precipitating agents, methyl
alcohol and an alcoholic solution of zinc chloride.
Greenwald101 •102 in 1915 introduced the use of trichloroacetic acid as the pro-
MAY 1954
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527
tein-precipitating reagent. He found that methyl alcohol precipitated some of
the amino acids. Further, methyl alcohol dissolved some of the nitrogenous
lipids that could hardly be considered as belonging to the same group of compounds as urea and the other protein metabolites. Trichloroacetic acid had none
of these disadvantages. In 191.6, Folin and Denis86 used phosphoric acid as the
precipitating agent.
Finally in 1919, Folin and Wu92 published their system of blood chemical
analysis that has served since that time as the basis for much of the advance in
blood chemistry. In this system, tungstic acid is used as the protein-precipitating
agent and is produced by the addition to the blood of 10 per cent sodium tungstate and % normal sulfuric acid. The blood filtrate thus obtained is almost
neutral in reactionand may be used for the determination of several constituents.
Tungstic acid does precipitate some of the nonprotein nitrogenous constituents
of blood, such as ergothioneine, although these materials are present in blood in
such small quantities that their loss is not of clinical significance.
At the present time, the most common protein precipitants are tungstic acid
and trichloroacetic acid. For a further discussion of protein precipitation, refer
to Sunderman and his associates.180
2. Conversion of Nitrogenous Compounds to Ammonia
The methods now in use for the determination of total nitrogen and nonprotein nitrogen in biologic materials are primarily modifications of the method
published by Kjeldahl123 in 1883. In this method, the material for analysis was
digested with hot sulfuric acid, the nitrogen being converted to ammonia, which
then united with the sulfuric acid to form ammonium sulfate. It was found,
however, that to ensure complete digestion, prolonged heating was required
and then, in some cases, not all of the nitrogen was completely converted. Improvements in the method have been mainly along the line of adding accelerators or oxidizing agents to ensure more complete and more rapid conversion
of the nitrogen to ammonia.
Gunning,103 Arnold and Wedemeyer 6 ' 7 and Folin and Denis82 used potassium
sulfate and copper sulfate, or pure copper filings, as accelerators. In many respects, this combination has not been improved upon since that time. However,
because silica dissolves from the digestion flask or tube, the method is generally
not suitable for direct nesslerization and colorimetric reading.
Mears and Hussey136 suggested the use of perchloric acid in addition to the
sulfuric acid. This method has the disadvantage that if not all of the perchloric
acid is consumed by the organic matter present, the excess perchloric acid will
destroy part of the ammonia formed by the digestion. Dupray 61 attempted to
rectify this by the use of phosphoric and perchloric acids, but without much
success.
Wong,197 following the suggestions of Huguet114 and Pittarelli,160 used potassium persulfate in his modification of the Arnold-Gunning digestion procedure.
He found that the addition of the salt near the end of the digestion was most
efficient, reducing the digestion time to about one third that of the original
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method. He further found that persulfate does not destroy ammonia as does
perchloric acid, it does not have the explosive tendency of hydrogen peroxide,
and it does not etch the digestion flask.
Van Slyke186 in his gasometric technic used phosphoric acid in place of copper
in the first stage of the digestion, and then used persulfate to complete the digestion. This procedure still removes much silica from the digestion tube and
the digest is not good for direct nesslerization. Koch and McMeekin124 introduced the use of 30 per cent hydrogen peroxide. It has the advantage of being
obtainable nitrogen-free, and thus not requiring a correction for the nitrogen
content of the reagent. Davenport 56 suggested the use of large volumes of 3 per
cent hydrogen peroxide. However, since most 3 per cent hydrogen peroxide
contains acetanilid, its use would require repeated blanks to correct for the acetanilid nitrogen. Other oxidizing agents, such as selenium dioxide, have been
suggested1 for the oxidation of both protein and nonprotein solutions.
For the routine determination of nonprotein nitrogen, the present method of
choice is to use 1:1 sulfuric acid and 30 per cent hydrogen peroxide. Complete
oxidation occurs with little dissolving of silica. The digest is suitable for any
method of determining the ammonia nitrogen content, and it is especially suitable for colorimetric procedures. This digestion, however, is the source of most
of the error in the method. It is absolutely essential that the oxidation be complete and that the excess hydrogen peroxide be destroyed. Directions must be
followed carefully.
3. Determination of Ammonia Nitrogen
The ammonia nitrogen present in the digest can be determined in 3 ways:
1. By distillation of the ammonia into a standard solution of acid, which is
then titrated to determine the excess acid and thus, by difference, the acid
required to neutralize the ammonia;
2. By use of the hypobromite reaction in which the ammonia nitrogen is
liberated as a gas, the volume being measured in a gasometric apparatus; and
3. By use of Nessler's reagent, a mercuric-potassium iodide complex, which
with ammonia and ammonium salts produces amber or orange-brown colored
compounds which can be measured colorimetrically or photometrically.
The exact procedure to be followed depends on the amount of nitrogen to be
determined. If more than 1 mg. of nitrogen is present in the sample, macromethods are best; if less than 1 mg. of nitrogen, use is made of micromethods.
Macromethods make use of the well-known Kjeldahl digestion flask. Micromethods generally use for digestion a test tube approximately 25 mm. in diameter and 200 mm. long. Distillation and titration is the most accurate method
for macrodeterminations. The error is usually within plus or minus 1 per cent.
However, distillation and titration may also be used for micromethods. Removal of the ammonia by aeration has been recommended,83 but with improvements in the apparatus, distillation has proved more rapid and more accurate.
Bock and Benedict32 have presented a suitable procedure for distillation directly
from the digestion flask. Pregl166 suggested the use of steam distillation. This
MAY 1954
AZOTEMIA
529
method appears to have no great advantage over direct distillation. The distillation in microprocedures is usually made into 0.01 or Jfo normal hydrochloric
acid. The reaction involved is:
NH4OH + HC1 -> NH4CI + H 2 0
Each milliliter of 0.01 N solution equals 0.14 mg. of nitrogen. Each milliliter of
3^o N solution equals 0.2 mg. of nitrogen.
The gasometric and colorimetric technics are used more often in micromethods
than is the method of distillation and titration.
Van Slyke's185 gasometric method is based on the following reaction:
2 NH, + 3 NaBrO -» 3 NaBr + 3 H 2 0 + N 2
The reaction is not complete, producing less nitrogen than the theoretic yield,
but under the controlled conditions of the test, and making an empiric correction, the error usually is within plus or minus 0.5 per cent. Many of the oxidizing agents and accelerators interfere with this method. Potassium persulfate
does not interfere, so the method of Wong197 is the choice for digestion in this
procedure.
The colorimetric method, making use of Nessler's reagent, is accompanied by
the greatest error; generally amounting to about 3 per cent. This is because of
errors of dilution, impurities in the reagents and errors inherent in the method
itself. Blank determinations must be made frequently on the reagents and corrections applied as necessary. The greatest potential error is based in the development of the color. Nessler's solution unites with ammonia and ammonium salts,
forming an amber or orange-brown-colored compound. An excessively large
amount of ammonia Avill cause this compound to precipitate. Under accurately
controlled conditions, comparison of the color with that produced by a known
concentration of ammonia (ammonium sulfate) will afford quantitative results
within about plus or minus 3 per cent.
Many modifications have been made in Nessler's reagent Avith regard to composition and compounding. The most satisfactory preparation is that devised
by Koch and McMeekin.124 Their modification produces a smaller number of
turbid solutions than previous preparations, and a precipitate of green mercurous
compounds never appears.
The technic of nesslerization is most important. Folin and Denis85 stated that
the Nessler's reagent should be added to the digestion mixture and the resulting
solution mixed quickly. Wong198 stated that the Nessler's reagent should be
allowed to fall into the solution in the digestion tube and then mixed. More
recent papers state only that the reagent is to be added rapidly with immediate
inversion. Hunter117 found that to obtain satisfactory results in the use of Nessler's
reagent, not only should the reagent be added rapidly, but it should be added
directly into the center of the solution and immediately mixed by inversion.
Errors as great as 20 per cent were obtained when the Nessler's reagent was
added slowly down the side of the digestion tube instead of rapidly and directly
into the center of the solution. In one of our laboratories it has been found that
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MACFATE ET
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placing the diluted digested mixture in a water bath at 25 C. before and after
nesslerization helps to stabilize the color produced.
Protective colloids have been suggested as stabilizers. Looney133 introduced
the use of gum ghatti. A more satisfactory method is that introduced by Gentzkow,98 who stabilized the color and the clarity of the nesslerized solution by the
addition of potassium persulfate and potassium gluconate. With this method,
Gentzkow found 490 to 510 m^ the most appropriate wavelength for spectrophotometric reading, rather than 415 m^i as suggested in the usual determination.
Early methods 82 ' 88 distilled off the ammonia from the digestion mixture and
then nesslerized the resulting distillate. Later investigators 32 ' 119 suggested direct
nesslerization. The Arnold-Gunning digestion mixture and its many modifications are usually not satisfactory for colorimetric work owing to the large amount
of silica dissolved. A method that does not require any digestion was introduced
by Rappaport and Eichhorn169 in 1947. Here, an alkaline hypobromite-boric
acid solution is added to the protein-free nitrate, and the excess bromine is
titrated iodimetrically.
For routine clinical use in determining the nonprotein nitrogen of blood or
serum, the Rappaport and Eichhorn159 method, that is, the iodimetric titration,
has much in its favor. However, the method of choice is precipitation of the proteins with tungstic acid, digestion with 1:1 sulfuric acid and 30 per cent hydrogen peroxide, followed by direct nesslerization under controlled conditions. The
resulting solution can be compared in a visual colorimeter with a suitable standard, or its absorptance (conversely, its transmittance) measured in a filter photometer or spectrophotometer.
The nonprotein nitrogen of the urine can be determined by the same technic
as blood or serum, with suitable changes in the amount of sample used.
V I I I . DISCUSSION O F METHODS F O R ESTIMATION O F U R E A I N BLOOD AND U R I N E
In most of the procedures for the determination of urea, advantage is taken
of an enzyme, urease, which converts urea to ammonia and carbon dioxide. The
reaction catalyzed by the enzyme is shown by the following equation:
OC(NH 2 ) 2 + 2 H 2 0 urease (NHOsCOa
>
After urease has acted on urea, it is possible to measure the ammonia or carbon
dioxide gasometrically, or measure the ammonia colorimetrically or titrimetrically.
1. Gasometric Technics
Gasometric methods are used mainly for research purposes at the present time.
The procedures are fairly rapid, accurate, and do not require standard solutions,
but are not suitable for routine purposes because they do not lend themselves
to large numbers of determinations. Two general types of gasometric methods
have been described.
a. Determination of ammonia or carbon dioxide formed after urease activity.147 • 186
MAY 1954
AZOTEMIA
531
Either the ammonia or carbon dioxide may be determined with equal facility
and accuracy and the test can be performed on either serum or urine. Incubation of the samples and urease is done in flasks prior to introduction into the gas
apparatus. The quantitations of the gases are best carried out in the Van Slyke
manometric gas apparatus.
b. Determination of nitrogen after treatment of sample with hypobromite.™1 The
reaction of urea and hypobromite is likewise carried out in a separate flask with
gas volumes being measured in the Van Slyke manometric gas apparatus. Hypobromite reacts with urea according to the following equation:
OC(NHs)a + 3 NaBrO + 2 NaOH -» Na»CO, + 3 NaBr + 3 H 2 0 + N 2
Hypobromite, however, is less specific for urea than urease, in that other nonprotein nitrogenous constituents of serum or urine may also react if the conditions are not optimal. Not only may urea values be high owing to nonspecificity
of the reaction, but results may also be low, since the method has the disadvantage of not recovering all the nitrogen from the urea.
2. Colorimetric Methods
Colorimetric methods for urea estimations are either direct or indirect in
nature.
The direct colorimetric methods are based on a reaction between urea and some
organic compounds to yield a colored product. The degree of color formed is
proportional to the amount of urea present. Three direct methods are in use at
the present time.
a. Reaction of urea with diacetyl monoximc or diacetyl.11-142, l46 With this procedure, an aliquot of a protein-free filtrate of serum is heated with diacetyl
monoxime in the presence of concentrated hydrochloric or sulfuric acid. The
yellow color formed by the interaction of urea and diacetyl monoxime is intensified by the addition of potassium persulfate. The reaction is fairly specific in
that other physiologic compounds, which react with diacetyl monoxime, tend
to give rise to a red rather than a yellow color. In addition, the interfering substances are not found in significant concentrations in serum or urine. The presence
of ammonia does not interfere in that it causes no additional color, and thus the
method can be applied directly to urine. It is important that the tubes be kept
out of direct sunlight before and after heating, as light causes rapid fading of the
color.
Natelson and his associates'42 improved the method by demonstrating that
potassium persulfate and the oxime were not necessary for the reaction, since the
same degree of yellow color developed if the test was performed by heating a
protein-free filtrate with an acid diacetyl solution. However, all the reactions
Avith diacetyl or its monoxime yield a color that follows Beer's law for only a
limited concentration of urea. With increasing concentrations, there is a gradual
deviation from Beer's law. In addition, the photolability of the compound is a
disadvantage. Still, the ease, simplicity and rapidity of performance of the procedures are in their favor.
532
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b. Reaction of urea with alpha-isonitrosopropiophenone.i Aliquots of the material to be analyzed are heated in the dark with alpha-isonitrosopropiophenone
in the presence of strong acid for 1 hour. A photolabile red compound is formed
with urea; the color is compared with standards in a visual colorimeter or read
in a photometer. It is claimed that the reagent is more specific for urea than is
diacetyl, in that fewer physiologic compounds react with the phenone. However, the photolability and time of heating, in addition to urine values that
are about 3 per cent too high, are disadvantages to be considered by those contemplating its use.
c. Reaction of urea with xanthydrol.^ • 67,128 Xanthydrol unites with urea to
form an insoluble compound (dixanthyl urea) that dissolves to give rise to a
yellow color when treated with 50 per cent sulfuric acid. The degree of color
formed from the interaction of the precipitated xanthydrol and sulfuric acid is
proportional to the amount of urea present in the aliquot analyzed. However,
6 to 24 hours are required for precipitation. The method appears to be simple
and accurate, and it has been used to measure as little as 2 micrograms of nitrogen.
The indirect colorimetric methods for estimating urea use Nessler's reagent to
determine the ammonia formed from the action of urease on urea. The use of
Nessler's reagent has a number of disadvantages, which include a tendency for
turbidity to develop and a small color differential for varying amounts of ammonia, in addition to variations in quality of color, between specimens and standards. However, the procedure is simple and convenient to carry out.
A number of variations for the indirect colorimetric method have been worked
out in an effort to overcome the disadvantages mentioned above. Three general
technics follow.
1. Deproteinization, urease action, distillation of the ammonia and nesslerization of the distillate (Folin and Svedberg91). This method is neither rapid nor
does it permit large numbers of determinations to be made. In addition, a loss
of about 10 per cent of the ammonia occurs during the distillation process. By
altering the conditions of distillation, Gentzkow claimed to have prevented this
loss.98
2. Deproteinization, urease action and direct nesslerization (Karr119). Gum
ghatti is used as a stabilizing and dispersing agent in an effort to prevent turbidity formation (Looney133). However, turbidity frequently develops in spite of
the presence of gum ghatti. Furthermore, the necessity of pouring the incubated
specimen into tubes to develop the color and, in turn, pouring of the colored solutions into colorimeter tubes, is time-consuming and inconvenient.
3. Diluted serum, urease action, deproteinization, and nesslerization in the
presence of persulfate and gluconate (Gentzkow98). Gentzkow improved nesslerization methods in 2 ways. He eliminated turbidity formation by the addition of
potassium persulfate and potassium gluconate. He showed that the optimum
wavelength for reading the color photometrically was 490 to 510 m/j. At the
present time, this method is the reaction of choice for estimating urea colorimetrically.
MAY 1954
AZOTEMIA
533
3. Titrimetric Methods
This type of method is dependent also on the conversion of urea to ammonium
carbonate by urease activity. The ammonia formed is liberated by addition of
alkali (potassium carbonate) and is trapped by absorption in standard acid or
boric acid. These in turn are titrated with standard alkali or acid, respectively.
The methods are simple in that no deproteinization of serum is necessary, and
specimens with high values of urea are easily determined (no repetitions are
necessary as with colorimetric methods, since there is an excess of alkali or boric
acid present).
a. Aeration method (Van Slyke and Cullen188,189). Serum is treated with urease
in the presence of buffer in a special test tube. After 15 minutes' incubation, potassium carbonate is added and the ammonia aerated into acid in another tube.
Originally, the ammonia was trapped in standard acid and this was back-titrated
with standard alkali. Thus, 2 standard solutions were needed. With the introduction of boric acid as the absorbing agent only 1 standard, the titrating acid,
is needed.108 Sobell and his associates175 have devised a micromethod which, however, has the disadvantages of requiring a cumbersome set-up and 45 minutes of
time to complete the aeration.
b. Microdiffusion method (Conway54). Serum in the outer chamber of a Conway unit is treated with urease and buffer. The mixture is then made alkaline
with potassium carbonate and the freed ammonia diffuses into the center well
where it is absorbed in boric acid solution. This is titrated dh-ectly with standard acid. The method is simple, convenient and very accurate. The microana'ytic aspects are a definite advantage. An ultramicro adaptation has been
described.122 These procedures avoid aeration difficulties. The one disadvantage
is the time required, 90 minutes, for diffusion to occur. However, the advantages
far outweigh the disadvantages.
4- Determination of Urea in Urine
The direct colorimetric methods can be applied directly to diluted urine, if
no protein is present. The specificity of the direct methods obviates any interference by ammonia or other substances.
The gasometric, colorimetric or titrimetric procedures that depend on conversion by urease of urea to ammonium carbonate, require the performance of
2 determinations if the preformed ammonia is not removed. Where it is not removed, both the ammonia present in urine as a result of renal tubular activity
and the ammonia resulting from urease activity, must be estimated. Thus,
total ammonia nitrogen (after urease activity) minus the "preformed" ammonia
nitrogen equals the urea ammonia nitrogen (urea nitrogen).
I X . DISCUSSION O F METHODS FOR ESTIMATION O F C R E A T I N E AND C R E A T I N I N E I N
BLOOD AND
URINE
The importance of creatine and creatinine biologically is unchallengeable, yet
the quantitative measurements of these substances in the fluids of the body have
534
MACFATE ET
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24
been unreliable, mainly for 2 reasons: the low concentrations in which these substances exist in the body, and the nonspecificity of the Jarfe" reaction. It therefore became increasingly evident that the chemical methods for the determination of these substances needed revision, with the result that some important
improvements have been made recently by workers in this field.
For a long time it was realized that the alkaline picrate method of Folin,75-77
published in 1914, which was the first accepted quantitative method for creatine
and creatinine, has the defect of being nonspecific. Hunter115 in 1928 listed some
40 substances, such as glucose, formaldehyde and creatinine-like nitrogenous
compounds that react with alkaline picrate. Even with this finding, the alkaline
picrate method with modifications is the one commonly accepted today as the
"standard" method for creatine and creatinine in body fluids. Furthermore, the
introduction of photoelectric colorimeters and spectrophotometers for measuring color intensities has vastly improved accuracy in these methods.
Arguments over the presence of chromogenic material in blood brought about
the studies of Miller and Dubos.137 With the aid of a specific bacterial enzyme,
they devised a method for creatine and creatinine determination which showed
that from 80 to 100 per cent of the chromogenic material of serum is true creatinine.
It must be realized that all the methods in the literature cannot be reviewed
in this brief space. The methods of today, of which a more detailed account is
given, were selected because in the analysis of the fluids of the body, the advantages of simplicity and accuracy must be given paramount consideration by
the clinical worker.
The first attempt to devise quantitative methods for creatinine was made by
Heintz109 in 1849. His method was based on the isolation of creatinine as creatinine zinc chloride. Modified methods based on this principle remained in use
for more than 40 years. It was not until 1904 that the colorimetric method of
Folin72 was introduced. His method soon displaced all others. It was based upon
the red color reaction of creatinine with alkaline sodium picrate, and was especially useful for the determination of large quantities of creatinine. Ten years
later, in 1914, Folin76 published a revised method for the determination of small
quantities of creatinine. At the same time, the first satisfactory quantitative
method for determining creatine appeared in Folin's researches. This consisted
in converting creatine, by appropriate treatment with acid, into creatinine and
then determining the total creatinine ("preformed" creatinine plus creatinine
from creatine) by colorimetric methods. The creatine in terms of creatinine
equivalent was thus determined by difference. This is the procedure still followed.
In 1911, Walpole193 determined creatine by the diacetyl reaction, which consisted of treating a slightly alkaline solution with a few drops of 1 per cent diacetyl solution. Creatine gives a pink color; creatinine does not react.
As far back as 1916, Hunter and Campbell116 found that the picric acid used
in the alkaline picrate methods for creatine and creatinine determinations must
be of the highest possible purity. Folin and Doisy87 showed that not infrequently
the chromogenic substances that were attributed to the body fluids were already
MAY 1954
AZOTEMIA
535
in the picric acid reagent. Therefore, now as then, the picric acid used in the
color reactions must be pure and the solutions used must be fresh and protected
from light, heat and dust. Benedict21 has developed a method for the purification of picric acid.
Most of the methods now used for the determination of creatine and creatinine
are modifications of the classical alkaline picrate method published in 1919 by
Folin and Wu.92 Borsook40 in 1935 presented a modification that consisted of
absorbing the creatinine on Lloyd's reagent and, while in this state, washing
with acid until free of impurities. The creatinine is then removed from Lloyd's
reagent by the same alkaline picrate in which the color is developed. Creatine
was also determined by this method, after autoclaving for 20 minutes at 30
pounds pressure and then estimating the total creatinine.
In 1950, Hare107 gave a further modification of the Borsook method. Her procedure is adapted to small quantities of material and is of especial value in studies on infants and small animals. Five-tenths milliliters of serum is used and,
after precipitation of the proteins with 20 per cent trichloroacetic acid, the filtrate is treated with saturated oxalic acid and Lloyd's reagent. Following centrifugation and decantation, sodium picrate is added to the mixture for color development. After centrifugation, the supernatant is poured into cuvets and
the density of color read in a spectrophotometer. The most likely source of error
in this technic is the protein that might be adsorbed. Care must be taken that
the serum or urine filtrates are completely protein-free.
In 1942, Peters146 published a modification of the alkaline picrate method.
His method is an adaptation of the Folin and Wu92 colorimetric technic to the
photoelectric colorimeter. By means of standard solutions of creatinine zinc
chloride, a standard curve can be made that remains constant for any given
colorimeter. The curve is rectilinear, within the limits of accuracy of the procedure, with concentrations of creatinine only up to 5 mg. per 100 ml. If the
reading of creatinine on the curve exceeds 5 mg. per 100 ml. in the analysis of
urine or plasma, either a smaller aliquot must be taken for analysis or the tungstic
acid filtrate must be diluted with water.
Only 2 important modifications of the original Folin-Wu method have been
introduced by Peters: the use of a more dilute picric acid solution, and the omission of hydrochloric acid for the dehydration of creatine. Saturated picric acid
was not used because the solubility of picric acid varies with temperature. Therefore, a picric acid solution containing 11.75 Gm. per liter was used.
Bonsnes and Taussky37 have modified the alkaline picrate method still further.
They found that the amount of colored creatinine compound formed in these reactions was independent of the concentrations of the picric acid and was greatest at a low concentration of alkali. Further, the color formed was found to be
directly proportional to the concentrations of the creatinine only when the concentrations were very low. As a result of these findings, this method uses 0.04 M
picric acid and 0.75 N sodium hydroxide to develop the Jaffe" reaction. Brod and
Si rota46 then modified the Folin-Wu precipitation of the proteins in this method.
Steinitz and Turkand,178 working in the field of creatinine metabolism, used
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MACFATE ET
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the method of Popper, Mandel and Mayer154 for plasma and urine creatinine.
Here, picric acid and immersion in boiling water are used to precipitate the proteins from serum or plasma. Sodium hydroxide is added to the filtrate to develop
the color.
Many workers, aware that the alkaline picrate methods for creatine and creatinine suffer from the defect of being nonspecific, have described other procedures.
Among these have been the specific enzymatic methods of Miller and Dubos,137
which have been of real help in instances of the presence of chromogenic substances. These authors demonstrated that 80 to 100 per cent of the chromogenic
material in serum and plasma was true creatinine, 100 per cent in urine, and 30
to 50 per cent in the red blood cells. From their findings, if the analysis of only
serums and urines is to be considered, the alkaline picrate methods are accurate.
Barrett 13 in 1936 described a qualitative reagent for creatinine based on the
use of Nessler's reagent. Barclay and Kenney10 in 1947 devised a quantitative
nephelometric method using the Barrett reagent. A protein-free filtrate of serum
or urine is treated with a Nessler solution containing an added amount of potassium iodide, adjusted especially for this method. A precipitate appears, which
remains dispersed for at least 30 minutes. The method is claimed to be specific
by the authors.
The identification of creatine and creatinine in biologic material in amounts
down to 1 microgram may be successfully carried out by the technics of partition
chromatography on paper, using the methods of Dent. 59, 60 The movements of
creatinine are specific enough to permit its recognition. These methods have not
been extended beyond the qualitative detection of these compounds. It is of interest that creatine can be detected in the presence of large amounts of creatinine. One hope for the future is that this qualitative method will be supplemented
by a quantitative one.
The detailed spectrophotometric adaptations for the determination of creatine
and creatinine in plasma and urine by Fister70 must be mentioned. Also, the
alkaline picrate adaptations based on the methods of Folin and Wu,92 Pitts, 151
Hoffman,112 Peters,146 and Phillips,149 and the alkaline 3,5, dinitrobenzoic acid
adaptations based on the methods of Bolliger,34 Benedict and Behre,22 Langley
and Evans,127 and Andes.4 It should be emphasized that if any of these methods
are to be used, the original publications should be thoroughly studied. The
method of choice for clinical use is the Peters146 modification of the Folin and
Wu92 method.
X. DISCUSSION OF METHODS FOR ESTIMATION OF URIC ACID IN BLOOD AND URINE
Probably the first method for the determination of uric acid in body fluids was
the famous string test of Garrod. In 1848, he demonstrated the presence of uric
acid in the blood of gouty patients by allowing blood containing a string to stand
for several days after acidification with acetic acid. Microscopically, visible
crystals of uric acid appeared on the thread in cases of hyperuricemia.
During the remainder of the nineteenth century, more suitable methods were
devised that depended upon the isolation of the uric acid as an insoluble salt
MAY 1954
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537
from body fluids and its subsequent quantitative determination. Direct weighing and titration with potassium permanganate or iodine were the methods most
widely used. These were all macromethods, which required relatively large quantities of uric acid and were successfully applied only in the analysis of urine.
In 1913, Folin and Macallum89 proposed a new method for the estimation of
uric acid in urine, which was followed shortly by adaptation of the method to
blood by Folin and Denis.83 This method, which forms the basis of most of the
modern ones, depends upon the blue color that is produced when uric acid and
phosphotungstic acid are mixed in an alkaline solution.
Following the publication of the original methods, Folin and numerous other
investigators modified the reagents and procedures in an attempt to correct several shortcomings. These included the lack of specificity of the color reagent, the
inability to recover quantitatively uric acid added to blood, urine or proteinfree filtrates of blood, and the instability, turbidity and deviation from the BeerLambert law of the color produced. The complete solution of these problems has
not yet been achieved, which is attested by the legion of methods that have been
introduced into the literature.
The chromogens, occurring in body fluids, that interfere with the determination, have been identified, at least in part, and include methyl-substituted uric
acids, which arise in the metabolism of xanthines, phenolic compounds, ascorbic
acid, glutathione, ergothioneine, cystine and glucose.27 Folin and Denis83 recognized that the reaction of their original color reagent with phenols was caused
in large part by molybdate that was present as a contaminant in the sodium
tungstate commercially available. Folin 78 ' 80 ' 90 subsequently published a number
of methods for purifying tungstate and was eventually successful in having the
chemical industry prepare molybdate-free tungstate suitable for use without
further purification.
Although the effect of phenols was largely eliminated, the other chromogens
continued to produce color. Changes in the composition of the color reagent have
made it somewhat more specific. These changes, which have consisted largely
in a reduction of the phosphoric acid-sodium tungstate ratio, were advocated by
Folin,80 Blauch and Koch,27 Brown48 and others. Benedict20 introduced the use of
arsenophosphotungstic acid, claiming greater specificity. Benedict's color reagent
and modifications of Folin's phosphotungstic acid reagent are both in current
use.
The early color reagents and procedures were so seriously influenced by chromogens that preliminary isolation of the uric acid was attempted by precipitation as the silver salt. Folin and Wu92 used silver lactate in acid solution for the
precipitation and acidified sodium chloride for redissolving the uric acid from
the precipitate. Benedict19 used an ammoniacal solution of silver lactate for the
precipitation and redissolved the uric acid in sodium cyanide. Newton143 removed the chromogens by precipitation with silver chloride in highly acidic
solution. The uric acid remained soluble.
In an attempt to devise a still more specific method, Blauch and Koch27 in
1939 introduced the use of uricase, an enzyme that specifically destroys uric
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MACFATE ET
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acid. In their method, the total color value of a tungstic acid filtrate of blood
was determined, using a phosphotungstic acid reagent. Another sample of filtrate was treated with uricase to destroy the uric acid and the residual color,
due to chromogens, was determined. The "true" uric ac'd content was obtained
by subtracting the chromogen value from the total color value. The original
uricase method has been modified by Bulger and Johns,51 Mull,139 Silverman and
Gubernick,170 Block and Geib,31 and Wolfson, Levine and Tinsley196 for determination of uric acid in whole blood or plasma. Shaffer169 and Buchanan, Block
and Christman 50 have used the enzymatic method in the determination of urinary uric acid.
The inability to recover uric acid added to whole blood or plasma is due in
part to the adsorption of uric acid on the protein precipitate produced during
deproteinization. Because of the insolubility of zinc urate, the Somogyi precipitation with zinc hydroxide carries down all of the uric acid. In the use of the
original Folin-Wu tungstic acid precipitate, the acid should be added very slowly,
and the mixture allowed to stand 15 to 20 minutes before filtration. Haden's
modification,106 using N/12 sulfuric acid, recovers most of the added uric acid.
Heating of the mixture before filtration, advised by Pucher,167 was not found to
be effective in increasing the recovery of uric acid by Bulger and Johns. 51
Rogers163 and Christman and Ravwitch 53 have suggested that the failure to
recover completely uric acid added to protein-free filtrates, when the silver precipitation method is used, may be due to destruction of uric acid by light in the
silver precipitate. This effect may be minimized by working rapidly during the
isolation of the silver urate and avoiding strong light.
Brown48 found, when the quantity of cyanide in the color reaction was increased, that there was a better recovery of uric acid added to protein-free
filtrates. Block and Geib31 found in the direct method that glutathione added to
a pure solution of uric acid, ergothioneine, or uric acid plus ergothioneine, resulted
in a definite increase in color production. They also found that ergothioneine
added to uric acid caused a depression in the uric acid color. When all 3 substances were present, the color was not the summation of color due to the individual components, but rather the resultant of the depression caused by the
ergothioneine and elevation produced by glutathione. Impure uricase was thought
to be the cause of the failure to recover uric acid added to filtrates. Amino acids
have been shown to inhibit color production.
The instability of the final color and high blank has been found to be due to
an excessive concentration of cyanide and alkalinity of the solution. The turbidities that appeared in earlier methods were eliminated by using color reagents
without lithium salts and incorporation of urea in the solution.78
The lack of proportionality between color intensity and uric acid concentration was shown by Brown47 to be due in part to the practice of heating the solution during development of the color. Brown's observations were ignored for
many years, but in his last revision, Folin eliminated heating. Even in the absence of heating, the Beer-Lambert law is obeyed only with low concentrations
of uric acid.48
MAY 1954
539
AZOTEMIA
The only other method for uric acid in general use is based upon the observation of Schlieper in 1848 that uric acid reduced potassium ferricyanide. The reaction occurs rapidly and at low temperatures. Methods have been devised by
Flatow,71 Br0chner-Mortensen,45 and Bulger and Johns, 61 employing this principle. The latter investigators found that there were non-uric acid substances
present in protein-free filtrates of plasma that reduced ferricyanide at pH 11
and at low temperatures, and they used the uricase method to determine "true"
uric acid. Their values were higher than those of Blauch and Koch,27 which they
explain on the basis of substances present in tungstic acid filtrates that inhibit
color development in the latter method.
Because of the uncertainties that still exist in the estimation of uric acid, the
methods currently in use are legion. At least 18 different methods or modifications of methods have been introduced since Folin and Denis devised their procedure. Although the uricase methods probably give more accurate values for
uric acid in blood, plasma and urine, there is disagreement even between these
methods, probably largely because of the color reaction employed. The added
steps that this method involves must be weighed against its advantages for use
in clinical estimations. Many laboratories have eliminated primary isolation of
silver urate and employ direct methods with the thought that once normal
values are established for a particular method, it will serve to demonstrate abnormally high values. The difficulties encountered in the methods used are considerably lessened if estimations are made on serum or plasma instead of whole
blood. Most of the chromogens that interfere with the color reactions are found
in the red cell.
The methods for whole blood or plasma, in common use in laboratories, include those of Folin,80 Benedict,20 Blauch and Koch,27 Block and Geib,31 and
Brown.48 For urine, the methods used are those of Benedict and Franke, 23
Christman and Ravwitch, 53 Folin and Wu,93 and Buchanan, Block and Christman,60 with the precautions in the latter method advised by Bien and Troll.26
A simple direct method for uric acid in plasma or serum such as
that of Brown48 is an intelligent compromise for clinical work. The estimation
of urinary uric acid is rarely required of routine clinical laboratories, but an
adaptation of Brown's method is suitable.
X I . S O U R C E S O F E R R O R I N CLINICAL CHEMICAL
DETERMINATIONS
AND S U G G E S T I O N S FOR I N C R E A S I N G T H E ACCURACY
THESE
OF
METHODS
For convenience in discussion, it is possible to classify the common causes
of error in clinical chemical laboratories into 6 main categories. This plan has
been utilized in the following analysis.
Chemical laboratory errors may result from:
1. The improper collection ami preservation of blood samples. Unless the proper
anticoagulant is selected and correct amounts used, analyses will be in error.
For example, by using ammonium oxalate as an anticoagulant, abnormally
high nonprotein nitrogen and urea (where ammonia formation after urease
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24
action is measured) values are obtained. Anticoagulants such as oxalate or
citrate increase the osmotic pressure of plasma and cause a shift of water from
cells to plasma. The resulting plasma dilution may amount to as much as 15
per cent and thus lower the concentration of each plasma constituent by that
amount. Fluoride cannot be used as a preservative for glucose if the estimation
of urea by any of the methods employing urease is to be used; the fluoride ion
inhibits the activity of the enzyme and leads to low urea values. Furthermore,
if serum should remain on the clot for an unduly long period of time prior to
its removal, abnormal values can be expected for a number of serum constituents. Thus, serum glucose will decrease and serum potassium and phosphate
will increase unless serum is removed from the clot within an hour after the
blood is drawn. Hemolyzed serum yields incorrect elevated values for potassium and phosphate. If blood samples for pH are not drawn and stored under
oil, analytic results are falsely high.
2. The use of inaccurate, out-moded methods. Even with precise standards
and faultless technic, the use of nonspecific methods can result in values that
are grossly in error. Thus, the use of a glucose method that measures all reducing substances present in blood or serum may yield a value that masks the true
concentration of glucose. For example, the true serum glucose in a case of hypoglycemia may be 40 mg. per 100 ml. of serum, a significantly low value. If the
serum glucose had been estimated by the Folin-Wu procedure, which includes
nonglucose reducing substances, a result of 65 mg. per 100 ml. may be obtained.
Iii similar fashion, no one should depend on the factory calibration of photoelectric units. The use of such instruments makes one liable to errors that may
arise from improper calibrations, in addition to making one dependent on
methods that may be out-moded or nonspecific. Colorimeter makers are not
biochemists, capable of judging the merits and disadvantages of the various
available procedures.
3. The use of inaccurate standards. I t is obvious, that since all colorimetric
procedures are dependent on a "curve" made from a standard solution or by
direct comparison with a standard, the absolute value of any unknown will
vary directly with the true value of the standard solution used. When titrimetric technics are employed, the absolute values of the unknowns are dependent again on the reference solution used to standardize the titrating
solution. As a starting point for all quantitative work, accurate standard solutions are imperative and must be renewed before they deteriorate. Standard
solutions should be run daily with all methods. This procedure insures that the
colorimetric reactions are proceeding correctly and that titrimetric ones are
properly standardized. In addition, the running of unknowns in duplicate or
on successive days performs a similar function.
4- Technical errors. Improper filters in colorimeters, and errors in weighing,
diluting and pipetting lead to unreliable results in any analysis. Technical
errors occasionally occur in the best laboratories, since the human element
can never be eliminated from any test. Constant vigilance and reliable personnel will keep these at a minimum.
MAY 1954
AZOTEMIA
541
5. Use of dirty glassware. Dirty glassware causes unpredictable errors in the
estimation of the constituents of serum or blood. Thus, if unclean cuvets are
used for measuring light absorption in a photoelectric instrument, part of the
absorption may result from dirt on the cuvets.
6. Untrained personnel. At the present time, with increased use of microtechnics, the importance of well-trained personnel is becoming increasingly
necessary. In contrast to pathology per se, where the ultimate diagnosis is
made by a pathologist and technical help serves only to prepare specimens for
microscopic examination, the clinical chemistry technologist performs the
tests and is responsible for the results obtained. Given methods, the performance of the technologist determines both accuracy and errors.
To avoid the types of errors outlined above, it is essential that clinical chemistry laboratories be headed by persons whose primary training is in biochemistry. As a specialized branch of the clinical pathology laboratory, a qualified,
trained individual is necessary if improvement in accuracy and reliability is to
continue. It is becoming increasingly obvious that it is impossible for any one
individual to keep abreast of all the developments in pathology, histopathology, bacteriology, parasitology, immunology, hematology and clinical chemistry. A function of the biochemist must be a knowledge of all the problems of
chemistry. In this way, the possible sources of error in any given procedure
may be spotted with a minimum of effort. Even the obtaining of clean glassware has its problems.
The use of standard solutions prepared by various official and unofficial
groups is only one step in improving the general level of clinical chemistry
determinations. This procedure serves to check on the ability of a technologist
to prepare accurately and care properly for standard solutions and to check
on commercially prepared calibration curves where these are in use. The major
problems of the selection and use of accurate methods, of proper collection and
preservation of blood samples, of securing trained technologists and of obtaining supervisory personnel trained in biochemistry still remain.
A new development during the past few years deserves mention. Smaller
hospitals, realizing their limitations, are sending specimens to large hospitals
and teaching institutions for the more difficult chemical tests. In this way,
technologists in the smaller laboratories perform only a limited variety of determinations, relieving both technical and supervisory personnel of the responsibility for a number of infrequently run tests where errors could creep in.
The large institutions, with more supervisory personnel, are in a position to
perform tests frequently and are organized to cope with the tests not run by
the smaller institutions. In the long run, it is felt that this type of arrangement
is the one that will result in more accurate clinical chemistry. The small laboratories are expert in a few, the large laboratory in many technics.
X I I . A P P E N D I X . SUGGESTED
METHODOLOGY
In the selection of methods for the nonprotein nitrogenous constituents of
the blood and urine, consideration has been given to the differences in labora-
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MACFATE ET
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24
tory routine, the volume of work performed, and the training of the average
laboratory staff. The methods here suggested are the usual macrotechnics with
the simplest possible procedures. To eliminate added steps, all methods use
the standard tungstic acid filtrate.
Under certain conditions, methods other than those suggested here may be
more suitable to some particular situation. Micromethods may be used. Refer
to the discussion of various methods in the text above.
An attempt has been made to standardize reagents and equipment. The
Gentzkow 98 method of stabilizing Nessler's solution has been adapted to the
Koch-McMeekin124 modification of this reagent, so that the latter may serve
for the determination of both nonprotein nitrogen and urea. A digestion tube
graduated at 21 and 30 ml. is suggested131 to replace the tube graduated at 35
and 50 ml. A 40-per cent saving in reagents is accomplished in determining the
nonprotein nitrogen. These same tubes may be used for the determination of
urea nitrogen by the suggested method, since both standards and unknowns
can be diluted to 21 ml., just as well as to 20 or 25 ml. Every effort has been
made to simplify the determinations and to fit them into the routine clinical
laboratory. Full notes and suggestions have been added to assist the technologist. Methods are given, first, for the visual (subjective) photometer, and
second, for the objective photometer or the spectrophotometer.
METHOD 1. NONPROTEIN NITROGEN
Visual Photometer Procedure
Principle. The nitrogen content of a protein-free filtrate is determined by
the micro-Kjeldahl method, in a modified digestion tube, using sulfuric acid
and 30 per cent hydrogen peroxide for the digestion, as recommended by Koch
and McMeekin.124 The ammonia formed is determined by direct nesslerization
of the digestion mixture, using a water bath at 25 C. to stabilize the color
reaction.131 Color measurement is performed with light of wavelength 490 to
510 niju, to minimize interference from substances other than ammonia present
in the filtrate, as recommended by Gentzkow.98
Reagents. (Isolate all reagents from ammonia fumes.)
1. Sodium lungstate, 10 -per cent solution. Dissolve 50 Gm. of reagent sodium
tungstate in distilled water and dilute to 500 ml. The solution can be used
indefinitely.
2. Sulfuric acid, 2/3 N solution. Add 10 ml. of concentrated, reagent sulfuric
acid, nitrogen-free (sp. gr. 1.84), to approximately 500 ml. of distilled water.
Mix thoroughly. Titrate against a standard solution of alkali, or against weighed
quantities of anhydrous, reagent sodium carbonate. Calculate the further
dilution necessary to make a 2/3 N solution of acid. Dilute and again titrate.
Adjust if necessary. The solution can be used indefinitely.
3. Stdjuric acid, 30 per cent solution. Slowly add 150 ml. of reagent sulfuric
acid, nitrogen-free, to 350 ml. of distilled water in a 1-liter pyrex beaker, stirring
well. If the solution becomes too hot, cool before adding all of the acid. The
solution can be used indefinitely.
MAY 1954
AZOTEMIA
543
4. Nessler's reagent (Koch-McMeekin modification124)
a. Sodium hydroxide solution, concentrated, carbonate-free. In a 1000-ml.
beaker or wide-mouth flask of resistance glass, dissolve 300 Gm. of reagent
sodium hydroxide (pellets) in 275 ml. of distilled water. Stopper or use a tightfitting cover and allow to stand several days until the sodium carbonate settles
out, leaving a clear solution of sodium hydroxide, practically free from sodium
carbonate. Obtain the clear solution by decantation or by filtration through an
alundum or fritted silica filter, or by filtration through specially prepared filter
paper. (See Technical note 1.)
Technical note 1. Cut a, "hardened" filter paper to fit a Biichner funnel, 3 or 4 inches
in diameter. In a flat dish, treat the paper with a small amount of the sodium hydroxide
solution, warmed slightly. After a few minutes, pour off the sodium hydroxide and wash
the paper, first with absolute alcohol, then with dilute alcohol, and finally with large
quantities of distilled water. Place the paper on the Biichner funnel and apply gentle
suction- until the greater part of the water has evaporated, but do not dry so
that the paper curls. Now pour concentrated alkali upon the middle of the paper, spread
it with a glass rod, making sure that the paper, under gentle suction, adheres well to the
funnel. Draw the solution through the paper with suction.
b. Sodium hydroxide, 10 per cent solution. Carefully measure 5 ml. of the
concentrated carbonate-free sodium hydroxide solution and transfer to a 100-ml.
volumetric flask. Dilute to 100 ml. with distilled water. Titrate 20 ml. of this
solution against an acid solution of known strength. Calculate the volume of
concentrated sodium hydroxide solution necessary to make 2500 ml. of 10 per
cent solution. Dilute this amount of concentrated sodium hydroxide, cool and
again titrate to check the concentration.
c. Potassium mercuric iodide solution. Grind 75 Gm. of potassium iodide in
a large mortar. Add 62.5 Gm. of iodine and grind with the iodide. Add about
150 to 200 ml. of distilled water and grind until all the iodide and iodine are in
solution. Transfer the solution quantitatively to a 2-liter Erlenmeyer flask.
Add 75 Gm. of mercury and shake with a rotary motion. A one-hole stopper
will prevent splashing. If the flask becomes too hot, cool by immersing in water
from time to time, as required. Continue shaking until the supernatant liquid
has lost all of the dark brown color (due to iodine) and becomes a light greenish
yellow. Filter the solution through a double thickness of filter paper. The
solution is heavy and it is well to use a nickel or porcelain cone in the funnel
to prevent tearing of the paper. Wash the filter paper with distilled water,
collecting the washings and adding to the filtrate. Add distilled water until the
volume of the filtrate is about 450 ml. Mix the filtrate thoroughly and test by
adding 1 drop to a 1 per cent solution of soluble starch on a spot plate. Usually
no free iodine is present. To the filtrate add 1 drop of a solution of 2.5 Gm. of
iodine and 3 Gm. of potassium iodide in 10 ml. of water. Mix thoroughly and
again test. Continue the addition of the iodine solution until a very faint test
for free iodine is obtained on the spot plate. Dilute the filtrate to 500 ml. with
distilled water and mix thoroughly.
d. Working solution. To 2440 ml. of the 10 per cent sodium hydroxide solu-
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MACFATE ET
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VOL. 24
tion, add the 500 ml. of iodide solution and mix thoroughly. A slight cloudiness
may form, but this will settle on standing. Use the clear supernatant fluid
after 2 or 3 days. The solution can be used indefinitely.
5. Stock ammonium sulfate solution. Dissolve 4.7166 Gm. of anhydrous
reagent ammonium sulfate in N / 5 sulfuric acid and dilute to 500 ml. with the
same solution. (Prepare the N / 5 sulfuric acid by diluting 2.8 ml. of concentrated
reagent sulfuric acid, nitrogen-free, sp. gr. 1.84, to 500 ml. with distilled water.)
One milliliter of this solution contains 2 mg. of nitrogen. The solution can be
used indefinitely.
6. Standard nitrogen solution. Transfer 10 ml. of the stock ammonium sulfate
solution (reagent 5) to a 500-ml. volumetric flask. Add 90 ml. of N / 5 sulfuric
acid (see under reagent 5, above) and dilute to 500 ml. with distilled water. One
milliliter of this solution contains 0.04 mg. of nitrogen. The solution can be
used indefinitely.
7. Hydrogen peroxide, SO per cent solution. Merck's Reagent Superoxol is
suitable. (Buy the 3^-pound bottle and keep in the refrigerator. Transfer 1 or
2 ml. as needed to a smaller bottle for transporting between the refrigerator and
the digestion table. I t is convenient to use a 1-ounce bottle with a dropper wired
to the side. Return the unused portion to the refrigerator as soon as possible.
Keep in the small bottle. Do not return to the stock bottle.)
Special Apparatus
1. Digestion tubes. Pyrex No. 9820, 200 by 25 mm., graduated at 21 and 30
ml. (Laboratory supply houses, or the Corning Glass Works, Corning, New
York, will calibrate these tubes. They are also available from stock from the
Scientific Products Division, American Hospital Supply Corporation, Evanston,
Illinois.)
2. Paraffined corks. Obtain 2 or 3 dozen, highest quality X X X X corks, to fit
the tubes in item 1, above. Dip into hot melted paraffin until thoroughly
saturated. The outer coating of paraffin on the cork should not be too thick.
3. Glass beads. Solid, 2 to 3 mm. in diameter. One pound will last a long time.
Wash in acid, rinse well with distilled water, dry and keep in a clean bottle.
4- Wratten gelatin filter. No. 75. Cement a piece of this filter between circular
microscope coverglasses and use in the eyepiece of the visual photometer, or
cement between glass slides of suitable size and place over the light source in
the photometer. Remove any daylight-blue or other filters that may be otherwise used.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate. (See Technical note 2.)
Technical note 2. Note that a serum or plasma filtrate is made from 1 volume of serum
or plasma, 8 volumes of water, J.^ volume of 10 per cent sodium tungstate and }^ volume
of ji N sulfuric acid.131
Transfer 3 ml. of filtrate to a digestion tube. Add 1 ml. of the 30 per cent
solution of sulfuric acid and 2 glass beads. Attach the tube in a vertical position
MAY 1954
AZOTEMIA
545
to a clamp on an iron support, so that the bottom of the tube is about 4 or 5
inches from the table top. Heat carefully with a microburner until boiling begins,
keeping the flame in constant motion. Heat until dense white fumes fill the
tube and continue heating for at least 2 minutes until the dark solution begins
to lighten in color. This digestion is most important. Insufficient digestion is the
source of most of the error in the method. Heating is best done in a hood to remove
the fumes. If no hood is available, an inverted funnel or carbon filter may be
attached to a glass water-suction pump and suspended over the top of the
digestion tube. If a glass suction pump is not available, use one made of metal,
but lead the gas through a water bottle, first, to absorb the fumes. Rinse the
funnel after use.
Allow the digestion tube to cool for about one-half minute and add 2 drops
of 30 per cent hydrogen peroxide, dropping the peroxide directly into the digest.
Heat again until dense white fumes fill the tube, and continue heating for 1
minute. If the brown color of the solution fails to disappear, add more peroxide
and heat for a full minute. It is most important that the digestion be complete
and that the excess peroxide be decomposed. (A yellow precipitate, caused by
an excess of tungstic acid, may appear. It will dissolve on nesslerization.) Cool
the tube and dilute the contents to 21 ml. with distilled water. Place the tube
in a beaker of water or a water bath at approximately 25 C. (This temperature
is very important for the stabilization of the color reaction and the prevention
of cloudiness.)
Transfer 3 ml. of the standard nitrogen solution (reagent 6) to one digestion
tube and 5 ml. to another. Add 1 ml. of the 30 per cent solution of sulfuric
acid to each. Dilute the contents of each tube to 21 ml. with distilled water
and place in the water bath at 25 C.
After 10 to 15 minutes, when the mixtures in the tubes have stabilized at
approximately 25 C , add 9 ml. of Nessler's reagent to the first tube, allowing
the reagent to fall directly into the middle of the solution, mix at once by
inversion, using a paraffined cork, and return to the water bath. Add the Nessler's
reagent to each tube in turn, mix and return to the water bath. It is important
that the color be developed under uniform conditions.
Allow the stoppered tubes to stand in the water bath for 5 minutes and then
compare the unknowns in a visual photometer against the suitable standard
within the next 15 minutes, setting the standard at 20 mm. (See Technical
note 3.)
Technical note 3. T h e unknowns may be read from 12 to 30 mm. If the readings are lower or higher than this range, repeat the analysis, using an amount of filtrate
(or diluted urine) t h a t will bring the reading of the unknown as close as possible to 20
mm. Make proper adjustment in the calculations.
If the purity of the reagents is doubted, run a blank consisting of 3 ml. of
standard nitrogen solution and 1 ml. of digestion mixture. Heat as usual. Develop
the color and read against an unheated standard containing 3 ml. of standard
nitrogen solution and 1 ml. of digestion mixture. Use the regular calculations
for the 3-ml. standard. The nitrogen content should be 40 mg. per 100 ml.
546
MACFATE ET
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VOL.
24
Any increase in nitrogen content will be attributable to the reagents and must
be subtracted in all calculations on unknown solutions.
Calculation for blood or serum. If the 3-ml. standard was used and 3 ml. of
Folin-Wu filtrate was taken for analysis, the calculation is as follows:
Reading of standard
Reading of unknown
_ _ mg. of nonprotein nitrogen per 100 ml. of blood
or serum. (Calculate in whole numbers only.)
If the 5-ml. standard was used, substitute the factor 66.6 for the 40 in the
above equation.
Procedure for Urine
Collect a 24-hour specimen of urine and measure the volume. (See Technical
Note 4.)
Technical note 4- Occasionally there is some confusion with regard to the collecting
of urine over a stated period of time. At the beginning of the collecting period, have the
patient empty his bladder (catheterize if necessary) and discard this specimen. Collect
all urine passed during the collecting period, and at the end, have the patient again empty
his bladder (again catheterize if necessary) and add this specimen to those collected, if
any, during the period of time involved.
Place a measured quantity (25 or 50 ml. is convenient) in a beaker, acidify
with acetic acid (not too great an excess), and boil for a few minutes until the
protein has coagulated. Cool and return the urine quantitatively to the graduate
used in the original measurement. Rinse the beaker with a few drops of distilled
water, add to the urine in the cylinder and bring the volume of the urine back
to that originally measured by the addition of distilled water. Mix thoroughly
and filter. (Do not filter before making up to the original volume.)
For analysis, take a sample of protein-free urine which will contain about 0.1
mg. of nitrogen. The routine procedure is to make the analysis, as given below,
and repeat, if necessary, adjusting the amount of sample used.
Transfer 1 ml. of protein-free urine to a 100-ml. volumetric flask and dilute
to 100 ml. with distilled water. Mix thoroughly and transfer 1 ml. (2 ml. or
more if the urine is very dilute) to a digestion tube for analysis.
Add 1 ml. of the 30 per cent solution of sulfuric acid and 2 glass beads. Digest,
develop the color and compare in a visual photometer as given directly above
under Procedure for Blood or Serum.
Calcidation for urine. If the urine was diluted 1 to 100, as given above, and
the 3-ml. standard was used, the calculation is as follows:
Reading of standard
Reading of unknown
12
ml. of diluted urine used for analysis (usually 1 ml.)
_ mg. of nonprotein nitrogen per ml. of urine. (Calcidate to 1 decimal place
only)
If the 5-ml. standard was used, substitute the factor 20 for the 12 in the
above equation.
MAY 1 9 5 4
547
AZOTEMIA
-.*•
, . . „, ,
,
•
,
grams of nonprotein nitrogen exMg. per ml. X 24-hour volume in ml.
, ,
,_ , ,
, , • ,
——
= creted per day [Calculate to 1 decimal
J-UUU
7
7
\
place only.)
Normal findings. The normal concentration of nonprotein nitrogen, in a
person on an average diet and after a 12- to 14-hour fast, varies from 25 to 35
mg. per 100 ml. of whole blood and from 15 to 30 mg. per 100 ml. of serum.
The normal adult on an average diet excretes from 14 to 20 grams of nonprotein nitrogen per day. The actual amount is influenced by the diet and other
factors.
METHOD 2. NONPROTEIN NITROGEN
Objective Photometer and Spectrophotometer Procedure
Principle, reagents and special apparatus. Refer to Method 1, above.
Filter. Green.
Wavelength. 500 mji.
Calibration curve. Transfer the following amounts of standard nitrogen solution
to tubes graduated at 21 and 30 ml.: 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ml.
Add 1 ml. of the 30 per cent solution of sulfuric acid to each tube and dilute
each to 21 ml. with distilled water. Place in a beaker of water or a water bath
at 25 C. for 5 minutes. (The temperature is important for stabilization of
the color reaction and prevention of cloudiness.)
Add 9 ml. of Nessler's reagent to the first tube, allowing the reagent to fall
directly into the middle of the solution, mix at once by inversion, using a
paraffined cork, and return to the water bath. Add the Nessler's reagent to each
tube in turn, mix and return to the water bath. It is important that the color
be developed under uniform conditions.
Allow the stoppered tubes to stand in the water bath for 5 minutes and then
read in the photometer or spectrophotometer within the next 15 minutes, using
the blank or " 0 " tube as the reference liquid. (See Technical Note 5.)
Technical note 5. Distilled water may be used, also, as the reference liquid in those instruments where the blank or " 0 " tube will read no less than 90 per cent t r a n s m i t t a n c e .
However, be sure to use the same reference liquid for both standards and unknowns.
Prepare a calibration curve on coordinate paper, using the following figures
to correspond to the amounts of standard solution used above: 0, 10, 20, 40, 60,
80, 100 and 120 mg. of nonprotein nitrogen per 100 ml. of blood or serum, and
plotting against the corresponding readings in the photometer or spectrophotometer. (See Technical note 6.)
Technical note 6. Depending upon the instrument used, the type of filter, size of cuvet, etc., the last reading or two may not fit on the graph paper. In this case, draw the
curve as completely as possible and ignore those readings t h a t cannot be charted, i.e.,
readings below 10 on the instrument.
Prepare a new curve with each new lot of Nessler's reagent.
548
MACFATE ET AL.
VOL. 24
Procedure for Blood or Serum
Follow the procedure as given above in Method 1, Procedure for Blood or
Serum, except that 2 ml. of Folin-Wu filtrate (instead of the 3 ml. prescribed)
is taken for digestion, and do not prepare standard solutions. Develop the
color as detailed and allow the stoppered tubes to stand in the water bath at
approximately 25 C. for 5 minutes. (The temperature is important for stabilization of the color reaction and prevention of cloudiness.) Then read within the
next 15 minutes, in the photometer or spectrophotometer, using as the reference
liquid a blank containing 1 ml. of the 30 per cent solution of sulfuric acid, diluted
to 21 ml. with distilled water and 9 ml. of Nessler's reagent added. (See Technical
notes 5, 7 and 8.)
Technical note 7. If the photometer readings cannot be located on the calibration curve,
repeat the analysis, using such an amount of filtrate (or diluted urine) as will allow reading near the middle of the photometer scale. Make proper adjustment in the calculations.
Technical note 8. It is well each day to prepare a standard solution, as given under Calibration Curve, and check the photometer reading. If any significant difference appears,
make suitable corrections in the calculations.
Calculation for blood or serum. Read the value directly from the calibration
curve in terms of mg. of nonprotein nitrogen per 100 ml. of blood or serum.
Calculate in whole numbers only.
Procedure for Urine
Collect a 24-hour specimen of urine, (see Technical note 4), measure the
volume and deproteinize as given above in Method 1, Procedure for Urine.
Transfer 1 ml. of the protein-free urine to a 100-ml. volumetric flask and dilute
to 100 ml. with distilled water. Mix thoroughly and transfer 1 ml. (2 ml. or
more if the urine is very dilute) to a digestion tube for analysis.
Add 1 ml. of the 30 per cent solution of sulfuric acid and 2 glass beads, and
proceed as given above in Method 1 under Procedure for Blood or Serum.
Develop the color as detailed but do not prepare standard solutions. Allow the
stoppered tubes to stand in the water bath at approximately 25 C. for 5 minutes.
Then read, within the next 15 minutes, in the photometer or spectrophotometer,
using as the reference liquid a blank containing 1 ml. of the 30 per cent solution
of sulfuric acid, diluted to 21 ml. with distilled water and 9 ml. of Nessler's
reagent added. (See Technical notes 5, 7 and 8.)
Calculation for urine. Use the same curve as prepared for blood or serum. If
the urine was diluted 1 to 100, as given above, the calculation is as follows:
Value from the calibration curve in terms
mg of nonprotein nitrogen per ml.
of mg. of nonprotein nitrogen per 100 ml.
= of urine. (Calculate to 1 decimal •place
5 X ml. of diluted urine used for analysis
only)
(usually 1 ml.)
Gm. of nonprotein nitrogen exMg. per ml. X 24-hour volume in ml.
= creted per day (Calculated to 1 dec1000
imal place only)
Normal findings. Refer to Method 1, above
MAY 1954
AZOTEMIA
549
METHOD 3 . UREA
Visual Photometer Procedure
Principle. A protein-free filtrate is treated with urease to convert the urea to
ammonium carbonate. The ammonia is determined by direct nesslerization in
the presence of persulfate and gluconate as stabilizers, as recommended by
Gentzkow,98 but using the Koch-McMeekin124 Nessler's reagent. Color measurement is performed with light of wavelength 490 to 510 m,u, to minimize interference from substances other than ammonia present in the filtrate, as recommended by Gentzkow.98
Reagents. (Isolate all reagents from ammonia fumes.)
./. Sodium tungstate, 10 per cent solution. Refer to Method 1, Reagent 1.
2. Sulfuric acid, 2/3 N solution. Refer to Method 1, Reagent 2.
8. Urease solution. Shake 15 Gm. of permutit with 200 ml. of 2 per cent
acetic acid. Decant the supernatant fluid and discard. Wash the permutit 3
times with distilled water. To the damp permutit add 50 ml. of 0.001 N sulfuric
acid (0.03 ml. of concentrated reagent sulfuric acid, sp. gr. 1.84, diluted to 1
liter with distilled water) and 30 Gm. of jack bean meal. Shake gently for 30
minutes. Add 150 ml. of glycerol and mix thoroughly. Let stand overnight.
Filter through several thicknesses of gauze. Centrifugalize the filtrate until
essentially clear (an opalescent haziness remains). Store in the refrigerator.
The solution is active for at least 1 year.
4. Phosphate buffer solution. Dissolve 14 Gm. of reagent sodium pyrophosphate
and 2 ml. of 85 per cent reagent phosphoric acid in distilled water and dilute
to 250 ml. in a volumetric flask. The solution can be used indefinitely.
5. Stock ammonium sulfate solution. Refer to Method 1, Reagent 5.
6. Standard nitrogen solution. Refer to Method 1, Reagent 6.
7. Nessler's reagent (Koch-McMeekin modification). Refer to Method 1,
Reagent 4.
8. Potassium gluconate, 1 per cent solution. Dissolve 1 Gm. of reagent potassium
gluconate in distilled water and dilute to 100 ml. Store in the refrigerator. The
solution may be used for 1 week, only. Label the bottle with the date of preparation.
9. Potassium persulfate, 2.5 per cent solution. Dissolve 2.5 Gm. of reagent,
nitrogen-free potassium persulfate in distilled water and dilute to 100 ml. Keep
in the refrigerator at all times, removing only long enough to measure the
amount desired. The solution may be used for 1 week only. Label the bottle
with the date of preparation.
Special Apparatus
1. Test lubes. Graduated at 20, 21 or 25 ml. The digestion tubes graduated at
21 and 30 ml. (see Method 1, Special apparatus, item 1) are convenient to use.
Tubes graduated at 20 or 25 ml. may be used also, but be sure to use the same
tubes for all standard and unknown solutions.
2. Paraffined corks. Refer to Method 1, Special Apparatus, item 2.
3. Wratten gelatin filter, No. 75. Refer to Method 1, Special Apparatus, item 4.
550
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24
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate (see Technical note 2) and transfer 6 ml. to a test
tube or small flask. Add 0.4 ml. of phosphate buffer solution and 0.2 ml. of
urease solution. (See Technical note 9.)
Technical note 9. The filtrate must not be too acid (not below about pn 6) or the urease
will not work. This is unusual, but should be remembered. A drop or two of N/10 sodium
hydroxide will usually correct the condition, or the filtrate may be neutralized, testing
with litmus or a similar indicator.
Mix well and let stand at room temperature (not below 20 C.) for 20 minutes.
Filter through a good grade of qualitative filter paper. ("Acid-washed" filter
paper may contain an appreciable amount of ammonia.)
Transfer 5 ml. of this final filtrate to a test tube graduated at 21 ml. (Tubes
graduated at 20 or 25 ml. may be used.) Transfer 2 ml. of standard nitrogen
solution to a similar tube and 4 ml. to another. Dilute the contents of all tubes
to 21 ml. with distilled water. (If tubes graduated at 20 or 25 ml. are used,
dilute to this volume.)
At this point, and not before, prepare the gluconate-persulfate-Nessler
solution. Mix together 1 part of the gluconate solution and 1 part of the persulfate
solution. Pour this mixture into 2 parts of the clear Nessler's reagent and mix
well. (The volume to be prepared depends upon the number of tests to be
made, each test and each standard requiring 4 ml. of the final mixture.) Use
this mixture within 15 minutes and discard the excess.
Add 4 ml. of the freshly prepared Nessler's mixture to the first tube, allowing
the reagent to fall directly into the middle of the solution. Immediately stopper
with a paraffined cork and mix by vigorous shaking. Add the Nessler's mixture
to each tube in turn and mix.
Allow the stoppered tubes to stand for 15 minutes and compare the unknowns
in a visual photometer against the suitable standard within the next 45 minutes,
setting the standard at 20 mm. (See Technical note 3.)
Certain preparations of glycerol urease may develop an appreciable amount
of ammonia. To determine this, add 2.64 ml. of standard nitrogen solution to
3.36 ml. of distilled water. Treat with buffer and urease exactly as though this
were 6 ml. of blood filtrate. Prepare the 2-ml. standard. Develop the color as
detailed above and allow the stoppered tubes to stand for 15 minutes. Compare
in a visual photometer within the next 45 minutes, setting the standard at 20
mm. Use the regular calculations for the 2-ml. standard. The urea nitrogen
content should be 17.6 mg. per 100 ml. If the urease-treated solution shows any
appreciable increase in urea nitrogen, subtract this value in all calculations on
unknown solutions.
Calculation for blood or serum. If the 2-ml. standard was used and 5 ml. of
the final filtrate was taken for analysis, the calculation is as follows:
Reading of standard
Reading of unknown
17
'
.. _ mg. of urea nitrogen per 100 ml. of blood or
serum. (Calculate in whole numbers only)
MAY 1954
AZOTEMIA
551
If the 4-ml. standard was used, substitute the factor 35.2 for the 17.6 in the
above equation.
Mg. of urea nitrogen X 2.14 = mg. of urea. (Results are usually reported in
terms of urea nitrogen.)
Procedure for Urine
Collect a 24-hour specimen of urine, or in a urea clearance test, collect urine
for the stated period of the test. (See Technical note 4.) Measure the volume.
Transfer 5 ml. of the urine to a 100-ml. volumetric flask and dilute to 100
ml. with distilled water. Mix thoroughly. Transfer about 1 Gm. of permutit to
a 100-ml. Erlenmeyer flask. Add about 10 to 20 ml. of the diluted urine. Shake
for 5 minutes. Allow the permutit to settle and filter the supernatant fluid or
separate by centrifugation.
Transfer 2 ml. of this preparation to a test tube or small flask. Add 15.4 ml.
of distilled water, 0.4 ml. of phosphate buffer solution and 0.2 ml. of urease
solution. (See Technical note 9.) Mix well and let stand at room temperature
(not below 20 C.) for 20 minutes. Then add 1 ml. of 10 per cent sodium tungstate
and 1 ml. of 2/3 N sulfuric acid. Mix thoroughly, let stand a few minutes and
filter through a good grade of qualitative filter paper.
Transfer 5 ml. of this final filtrate to a test tube graduated at 21 ml. (Tubes
graduated at 20 or 25 ml. may be used.) Transfer 2 ml. of standard nitrogen
solution to a similar tube and 4 ml. to another. Dilute the contents of all tubes
to 21 ml. with distilled water. (If tubes graduated at 20 or 25 ml. were used,
dilute to this volume.)
Prepare the gluconate-persulfate-Nessler solution, develop the color and
compare in a visual photometer, as given directly above under Procedure for
Blood or Serum.
Calculation for urine. If the urine was diluted as given above, and the 2-ml.
standard was used, the calculation is as follows:
Reading of standard
Reading of unknown
16
ml. of final filtrate used for analysis (usually 5 ml.)
= mg. of urea nitrogen per ml. of urine. (Calculate to 1 decimal place only.)
If the 4-ml. standard was used, substitute the factor 32 for the 16 in the
above equation.
Mg. per ml. X 24-hour volume in ml. _ Gm. of urea nitrogen excreted per day.
1000
(Calculate to 1 decimal place only)
Gm. of urea nitrogen X 2.14 = Gm. of urea
Normal findings. The normal concentration of urea nitrogen, in a person on
an average diet and after a 12- to 14-hour fast, varies from 9 to 15 mg. per 100
ml. of whole blood and from 8 to 18 mg. per 100 ml. of serum.
The normal adult on an average diet excretes from 12 to 16 Gm. of urea
nitrogen per day. The actual amount is influenced by the diet and other factors
552
MACFATE ET
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VOL. 24
METHOD 4 . UREA
Objective Photometer and Spectrophotometer Procedure
Principle, reagents and special apparatus. Refer to Method 3, above.
Filter. Green.
Wavelength. 500 m^t.
Calibration curve. Transfer 20 ml. of standard nitrogen solution to a small
flask and add 2 ml. of distilled water. Mix thoroughly and transfer the following
amounts of the solution to tubes graduated at 21 ml. (tubes graduated at 20
or 25 ml. may be used): 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 ml. Dilute the
contents of each tube to 21 ml. with distilled water. (If tubes graduated at 20
or 25 ml. are used, dilute to this volume.)
At this point, and not before, prepare the gluconate-persulfate-Nessler
solution as described above in Method 3, Procedure for Blood or Serum.
Add 4 ml. of the freshly prepared Nessler's mixture to the first tube, allowing
the reagent to fall directly into the middle of the solution. Immediately stopper
with a paraffined cork and mix by vigorous shaking. Add the Nessler's mixture
to each tube in turn and mix.
Allow the stoppered tubes to stand for 15 minutes and then read in the
photometer or spectrophotometer within the next 45 minutes, using the blank
or " 0 " tube as the reference liquid. (See Technical note 5.)
Prepare a calibration curve on coordinate paper, using the following figures
to correspond to the amounts of standard solution used above: 0, 5, 10, 20, 30,
40, 50 and 60 mg. of urea nitrogen per 100 ml. of blood or serum, and plotting
against the corresponding readings in the photometer or spectrophotometer.
(See Technical note 6.) Prepare a new curve with each new lot of stock Nessler's
reagent.
Procedure for Blood or Serum
Follow the procedure as given above in Method 3, Procedure for Blood or
Serum, except that 4 ml. of the final filtrate (instead of the 5 ml. prescribed) is
used for the development of the color. Use tubes graduated to the same volume
as used in the preparation of the calibration curve, and do not prepare standard
solutions. Develop the color as detailed and allow the stoppered tubes to stand
for 15 minutes. Then read within the next 45 minutes, in the photometer or
spectrophotometer, using as the reference liquid a blank containing 4 ml. of
the gluconate-persulfate-Nessler solution diluted with distilled water to the
same final volume as the unknowns. (See Technical notes 5, 7 and 8.)
Determine the urease blank by adding 3 ml. of standard nitrogen solution to
3 ml. of distilled water. Treat with buffer and urease exactly as though this
were 6 ml. of blood filtrate, as directed above in Method 3, Procedure for Blood
and Serum. Use 4 ml. of the final filtrate for the development of the color. Use
tubes graduated to the same volume as used in the preparation of the calibration curve. Develop the color and read in the photometer or spectrophotometer
as directed. Read the value directly from the calibration curve. The urea content
MAY 1954
AZOTEMIA
553
should be 20 mg. of urea nitrogen per 100 ml. If the urease-treated solution
shows any appreciable increase in urea nitrogen, subtract this value in all
calculations on unknown solutions.
Calculation for blood or serum. Read the value directly from the calibration
curve in terms of mg. of urea nitrogen per 100 ml. of blood or serum. (Calculate
in whole numbers only.)
Mg. of urea nitrogen X 2.14 = mg. of urea. (Results are usually reported in
terms of urea nitrogen.)
Procedure for Urine
Collect a 24-hour specimen of urine, or in a urea clearance test, collect urine
for the stated period of the test. (See Technical note 4.) Measure the volume.
Transfer 5 ml. of the urine to a 100-ml. volumetric flask and proceed as
given above in Method 3, Procedure for Urine, except that 4 ml. of the final
filtrate (instead of the 5 ml. prescribed) is taken for the development of the
color, use tubes graduated to the same volume as in the preparation of the
calibration curve and do not prepare standard solutions. Develop the color as
detailed and allow the stoppered tubes to stand for 15 minutes. Then read
within the next 45 minutes, in the photometer or spectrophotometer, using as
the reference liquid a blank containing 4 ml. of the gluconate-persulfate-Nessler
solution diluted with distilled water to the same final volume as the unknowns.
(See Technical notes 5, 7 and 8.)
Calculation for urine. Use the same curve as prepared for blood or serum. If
the urine was diluted as given above, the calculation is as follows:
8 X Value from the calibration curve
in terms of urea nitrogen per 100 ml. _ mg. of urea nitrogen per ml. of urine.
11 X ml. of final filtrate used for
(Calculate to 1 decimal place only.)
analysis (usually 4 ml.)
Mg. per ml. X 24-hour volume in ml. _ Gm. of urea nitrogen excreted per day.
1000
(Calculate to 1 decimal place only)
Gm. of urea nitrogen X 2.14 = Gm. of urea
Normal findings. Refer to Method 3 above.
METHOD 5. CREATININE
Visual Photometer Procedure
Principle. The determination is based upon the reaction between creatinine
and sodium picrate in alkaline solution to form a tautomer of creatinine picrate,
as detailed by Folin and Wu92 and Folin.72 A constant strength solution of
picric acid is used, as recommended by Peters.146 The color reaction is not
specific for creatinine, but the other chromogenic materials are low in serum
and plasma, and are absent from normal urine, thus making the method ideal
for these fluids.
554
MACFATE ET
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VOL. 24
Reagents
1. Sodium tungstate, 10 per cent solution. Refer to Method 1, Reagent 1.
2. Sulfuric acid, 2/3 N solution. Refer to Method 1, Reagent 2.
3. Picric acid solution. Dissolve 11.75 Gm. of reagent picric acid (for blood test)
in distilled water and dilute to 1 liter. The solution can be used indefinitely.
If reagent grade picric acid is not available, the commercial grade may be
purified21 as follows: Dry the commercial picric acid at a temperature of 80 to
90 C. Dissolve 100 Gm. of this dried material in 150 ml. of glacial acetic acid
with the aid of heat. Continue heating until the solution boils. Filter the hot
solution through a heated funnel and collect the filtrate in a dry beaker. Cover
the beaker with a watchglass and let stand overnight at room temperature. If
crystals fail to appear, seed with a small crystal of pure picric acid and let
stand for about 2 hours. Filter with suction on a Buchner funnel. Wash the
crystals with about 35 ml. of cold glacial acetic acid and apply suction until as
dry as possible. Heat at 80 to 90 C. until thoroughly dry and no odor of acetic
acid is present.
4- Sodium hydroxide, 10 per cent solution. Prepare 500 ml. of carbonate-free
sodium hydroxide solution as given in Method 1, Reagents 4(a) and 4(b). The
solution can be used indefinitely.
5. Stock creatinine solution. Dissolve 0.5 Gm. of reagent creatinine (or 0.805
Gm. of creatinine-zinc chloride) in 0.1 N hydrochloric acid and dilute to 500
ml. in a volumetric flask with the same solution. (Prepare the 0.1 N hydrochloric acid by diluting 4.2 ml. of concentrated, reagent hydrochloric acid,
specific gravity 1.19, to 500 ml. with distilled water.) Mix thoroughly. Add a
few drops of toluene as a preservative. One milliliter of this solution contains 1
mg. of creatinine. The solution may be used for 1 year if kept in the refrigerator.
Warm to room temperature before preparation of the dilute standards. Label
the bottle with the date of preparation.
6. Standard creatinine solution. To a 500-ml. volumetric flask, add 3 ml. of
stock creatinine solution, (Reagent 5), and 50 ml. of 0.1 N hydrochloric acid.
(See under Reagent 5, above.) Dilute to 500 ml. with distilled water and mix
thoroughly. Add a few drops of toluene as a preservative. Five milliliters of this
solution contains 0.03 mg. of creatinine. The solution may be used for 6 months.
Label the bottle with the date of preparation.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate (See Technical note 2) and transfer 10 ml. to a
plain tube or flask holding about 40 to 50 ml. To another tube or flask, add 5
ml. of standard creatinine solution and 15 ml. of distilled water, and to a third,
add 20 ml. of standard creatinine solution.
Transfer 25 ml. (or more as required) of the picric acid solution to a small
flask and add 5 ml. (or one-fifth volume) of 10 per cent sodium hydroxide. Mix
thoroughly. Use within 5 minutes. (See Technical note 10.)
Technical note 10. A flaky precipitate may form. Although this precipitate dissolves
readily when the picrate is added to the Folin-Wu filtrate, it makes difficult the accurate
measurement of the solution.
MAY 1954
AZOTEMIA
OOO
Add 5 ml. of the freshly prepared alkaline picrate solution to the blood filtrate
and 10 ml. to each of the creatinine standards. Mix and let stand for 20 minutes
in a beaker of water or water bath at 25 C. (To stabilize the color reaction.)
Compare the unknowns in a visual photometer against the suitable standard
within the next 2 hours, setting the standard at 20 mm. (See Technical notes 3,
11 and 12.)
Technical note 11. A better comparison of colors is obtained if cups with blue
glass bottoms arc employed, or if a blue or green glass filter is used for the light in the
colorimeter.
Technical note 12. If sufficient blood filtrate is not available to repeat the analysis when
the unknown solution is too concentrated in color to read against the strongest standard
(or in the objective photometer) add a measured quantity of a mixture of 2 volumes of
distilled water and 1 volume of alkaline picrate solution to a measured quantity of
the filtrate-picrate mixture as prepared above, continuing the addition until the unknown
solution approximates the color of the stronger standard solution, (or until it can be read
in the objective photometer). Note the volumes of the solutions mixed and again compare in the visual photometer (or read in the objective photometer). Make proper adjustment in the calculations.
Calculation for blood or serum. If the 5-ml. standard was used and 10 ml. of
Folin-Wu filtrate was taken for analysis, the calculation is as follows:
Reading of standard
Reading of unknown
'
_ mg. of creatinine per 100 ml. of blood or serum.
{Calculate to 1 decimal place only)
If the 20-ml. standard was used, substitute the factor 6 for the 1.5 in the
above equation.
Procedure for Urine
Collect a 24-hour specimen of urine (see Technical note 4) and measure the
volume. If any appreciable amount of protein is present, remove as directed in
Method 1, under Procedure for Urine. Transfer 1 ml. of the urine (2 ml. or
more if very dilute) to a 100-ml. volumetric flask. To a similar flask, transfer
1 ml. of stock creatinine solution, Reagent 5, above (not the "dilute" standard
creatinine solution).
To each flask add 1.5 ml. of 10 per cent sodium hydroxide and 20 ml. of
picric acid solution. Mix thoroughly and let stand for 20 minutes. Dilute to 100
ml. with distilled water and mix thoroughly. Compare in a visual photometer
within the next 2 hours, setting the standard at 20 mm. (See Technical notes
3 and 11.)
Calculation for Urine
Reading of standard
Reading of unknown
1
ml. of urine used for analysis (usually 1 ml.)
= mg. of creatinine per ml. of urine. (Calculate to 1 decimal place only)
Mg. per ml. X 24-hour volume in ml. _ Gm. of creatinine excreted per day.
1000
(Calcidate to 1 decimal place only.)
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Normal findings. The normal concentration of creatinine, in a person on an
average diet and after a 12- to 14-hour fast, varies from 1 to 2 mg. per 100
ml. of whole blood and from 0.9 to 1.7 mg. per 100 ml. of serum.
The normal adult on an average diet excretes from 1.4 to 1.8 Gm. of creatinine
per day. The excretion is constant for each person and is proportional to the
muscle mass. It is independent of ordinary variations in the diet.
METHOD 6 .
CREATININE
Objective Photometer and Spectrophotometer Procedure
Principle and reagents. Refer to Method 5, above.
Filter. Green.
Wavelength. 520 rmx.
Calibration curve. From the stock creatinine solution (Method 5, Reagent 5)
prepare 2 dilute standard solutions, as follows:
1. Standard creatinine solution, No. J. To a 200-ml. volumetric flask, add 1
ml. of stock creatinine solution and 20 ml. of 0.1 N hydrochloric acid. (See
Method 5, under Reagent 5.) Dilute to 200 ml. with distilled water and mix
well. Add a few drops of toluol as a preservative. One milliliter of this solution
contains 0.005 mg. of creatinine. The solution may be used for 6 months. Label
the bottle with the date of preparation.
2. Standard creatinine solution, No. 2. Prepare as in Standard No. 1, above,
but use 6 ml. of stock creatinine solution. One milliliter of this solution contains
0.030 mg. of creatinine.
To tubes or flasks that will hold about 20 ml., transfer the following amounts
of standard creatinine solution No. 1: 0, 1, 2, 4 and 8 ml. To similar tubes or
flasks, transfer the following amounts of standard creatinine solution No. 2:
2, 3 and 4 ml. Dilute each to 10 ml. with distilled water.
Transfer 50 ml. of the picric acid solution to a small flask and add 10 ml.
of 10 per cent sodium hydroxide. Mix thoroughly. Use within 5 minutes. (See
Technical note 10.)
Add 5 ml. of the freshly prepared alkaline picrate solution to each of the
standard solutions. Mix and let stand for 20 minutes in a beaker of water or
water bath at 25 C. (to stabilize the color reaction). Read in the photometer
or spectrophotometer within the next 2 hours, using the blank or " 0 " tube as
the reference liquid. (See Technical note 5.)
Prepare a calibration curve on coordinate paper, using the following figures to
correspond to the amounts of standard solutions used above: 0, 0.5, 1.0, 2.0,
4.0, 6.0, 9.0 and 12.0 mg. of creatinine per 100 ml. of blood or serum, and plotting
against the corresponding readings in the photometer or spectrophotometer.
(See Technical note 6.) Prepare a new curve with each new lot of picric acid
solution.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate (see Technical note 2) and transfer 10 ml. to a
plain tube or flask holding about 20 ml.
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Transfer 25 ml. (or more, as required) of the picric acid solution to a small
flask and add 5 ml. (or one-fifth volume) of 10 per cent sodium hydroxide. Mix
thoroughly. Use within 5 minutes. (See Technical note 10.)
Add 5.0 ml. of the freshly prepared alkaline picrate solution to the blood
filtrate and mix. Let stand for 20 minutes in a beaker of water or water bath at
25 C. (to stabilize the color reaction). Read in the photometer or spectrophotometer within the next 2 hours, using as the reference liquid a blank containing 5 ml. of distilled water and 2.5 ml. of freshly prepared alkaline picrate
solution. (See Technical notes 5, 7, 8 and 12.)
Special cuvets may be used and the volumes of filtrate and picrate may be
changed to suit the particular needs of the instrument, keeping the proportion
of 2 volumes of filtrate to 1 of picrate. For greatest accuracy, if the concentration
of creatinine is in excess of 5 mg. per 100 ml. of blood or serum, repeat the
analysis, using less nitrate.
Calculation for blood or serum. Read the value directly from the calibration
curve in terms of mg. of creatinine per 100 ml. of blood or serum. {Calculate to
1 decimal 'place only.)
Procedure for Urine
Collect a 24-hour sample of urine. (See Technical note 4.) Measure the
volume (V) and determine the specific gravity.
If the specific gravity is greater than 1.010, dilute the urine with distilled
water until the specific gravity is 1.010. Again measure the volume (V d ). Dilute
5 ml. of this diluted urine to 100 ml. with distilled water in a volumetric flask.
If the specific gravity is 1.010 or less, omit the first dilution (in which case V
and Vd are the same figures) and use 5 ml. or an aliquot appropriately greater
for dilution to 100 ml. with distilled water.
Use the diluted urine obtained by either of the above methods in the same
manner as serum for preparation of a Folin-Wu filtrate. (See Technical note 2.)
Analyze the filtrate for creatinine as given directly above under Procedure for
Blood or Serum.
Calculation for urine. Use the same calibration curve as prepared for blood or
serum.
Vd X value from the calibration curve
in terms of mg. of creatinine per 100 ml. _ mg. of creatinine per ml. of urine.
V X aliquot of urine diluted to 100 ml.
(Calculate to 1 decimal place only.)
(usually 5 ml.)
Mg. per ml. X 24-hour volume in ml. _ Gm. of creatinine excreted per day.
1000
(Calculate to 1 decimal place only.)
Normal findings. Refer to Method 5, above.
METHOD 7 . CREATINE AND CREATININE
Visual Photometer Procedure
Principle. The creatine is changed to creatinine by autoclaving the Folin-Wu
filtrate without the addition of acid, as recommended by Peters,146 or heating
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in a water bath with the addition of hydrochloric acid, as suggested by Pitts. 151
The preformed creatinine and the total creatinine are then determined as a
tautomer of creatinine picrate, as detailed by Folin and Wu92 and Folin.72
Creatine is determined by difference. A constant strength solution of picric
acid is used, as recommended by Peters.146 Other chromogenic substances may
be hydrolyzed, also, but these are at a minimum in serum and plasma.
Reagents. Refer to Method 5, above.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate. (See Technical notes 2 and 13.)
Technical note 13. Andes4 recommends the following procedure: Add the water to the
blood or serum, allow the mixture to stand 4 to 5 minutes, add the % N sulfuric acid and
finally the sodium tungstate. This filtrate may be autoclaved without discoloring.
Transfer 10 ml. of the filtrate to a test tube graduated at 10 ml. Cover the
mouth of the tube with tinfoil and heat in an autoclave at 115 to 120 C. for 20
minutes. (See Technical note 14.)
Technical note 14- If an autoclave is not available, prepare a 1 to 5 Folin-Wu filtrate
instead of the usual 1 to 10 dilution, reducing the amount of water used. To a test tube
add 5 ml. of filtrate and 1 ml. of 3 N hydrochloric acid. (25.2 ml. of concentrated, reagent
hydrochloric acid, specific gravity 1.19, diluted to 100 ml. with distilled water.) Cover
the tube with a loose-fitting glass bulb or piece of tinfoil. Place the tube in a boiling water
bath for 4 hours. Cool and add 1 ml. of 3 N sodium hydroxide. (Prepare carbonate-free
sodium hydroxide solution as given in Method 1, Reagent 4 (a), and dilute to the 3 N
solution.) Dilute the mixture to 10 ml. with distilled water and proceed as with the regular 1 to 10 nitrate which has been autoclaved.
Cool. If the volume of liquid is less than 10 ml., restore the volume to 10 ml.
with distilled water and mix.
Transfer 10 ml. of unheated Folin-Wu filtrate to another test tube. Proceed
with the determination of creatinine, on both the heated and unheated filtrates,
as given above in Method 5, Procedure for Blood or Serum, preparing the
standards and comparing in a visual photometer.
Calculation for blood or serum. If the 5-ml. standard was used and 10 ml. of
Folin-Wu filtrate was taken for analysis, the calculation is as follows:
Reading of standard
Reading of unknown
'
. _ mg. of creatinine per 100 ml. of blood or serum.
(Calculate to 1 decimal place only.)
If the 20-ml. standard was used, substitute the factor 6 for the 1.5 in the
above equation.
Total creatinine (in the heated filtrate) — preformed creatinine (in the
filtrate not heated) = creatinine obtained from creatine.
Mg. of creatinine X 1.16 = mg. of creatine. (Calculate to 1 decimal place
only.)
Procedure for Urine
Collect a 24-hour specimen of urine (see Technical note 4) and measure the
volume. If any appreciable amount of protein is present, remove as directed in
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Method 1, under Procedure for Urine. Transfer 1 ml. of urine (2 ml. or more if
very dilute) to a test tube and dilute to about 10 ml. with distilled water. Cover
the mouth of the tube with tinfoil and heat in an autoclave at 115 to 120 C. for
20 minutes. (See Technical note 15.)
Technical note 15. If an autoclave is not available, transfer 1 ml. of urine (2 ml. or more
if very dilute) to a test tube and dilute to about 5 ml. with distilled water. Add 2 ml. of
3 N hydrochloric acid. (See under Technical note 14.) Cover the tube with a loose-fitting
glass bulb or piece of tinfoil. Place the tube in a boiling water bath for 4 hours. Cool and
add 2 ml. of 3 N sodium hydroxide (see under Technical note 14).
Cool the heated urine and transfer, quantitatively, to a 100-ml. volumetric
flask, rinsing the tube with several portions of distilled water.
Transfer the same volume of unheated urine, as used above, to another 100-ml.
volumetric flask. Proceed with the determination of creatinine, on both the
heated and unheated urines, as given above in Method 5, Procedure for Urine,
preparing the standards and comparing in a visual photometer.
Calculation for urine.
Reading of standard
Reading of unknown
1
ml. of urine used for analysis (usually 1 ml.)
= mg. of creatinine per ml. of urine. {Calculate to 1 decimal place only)
Total creatinine (in the heated urine) — preformed creatinine (in the urine
not heated) = creatinine obtained from creatine. (See Technical note 16.)
Technical note 16. Should the total creatinine be less than the preformed creatinine,
report as negative for creatine. The loss will be due to the decomposition of non-creatinine chromogenic substances.
Mg. of creatinine X 1.16 = mg. of creatine. (Calculate to 1 decimal place only.)
Mg. per ml. X 24-hour volume in ml. _ Gm. of creatine excreted per day.
1000
(Calculate to 1 decimal place only)
Normal findings. The normal concentration of creatine, in a person on an
average diet and after a 12- to 14-hour fast, varies from 3 to 7 mg. per 100
ml. of whole blood. Much of this is not true creatine but other chromogenic
materials found in the blood cells. The normal concentration of creatine in the
serum varies from 0.17 to 0.70 mg. per 100 ml. in the male and 0.30 to 3.0 mg.
per 100 ml. in the female. This appears to be true creatine, for the most part.
(This variation between the findings on whole blood and serum again emphasizes
the importance of using serum in many of the common blood chemical determinations.)
The normal adult excretion of creatine is very low. Increased amounts, from
10 to 50 mg. per day, are found in children. Normal women during the puerperium
excrete small amounts.
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METHOD 8 . C R E A T I N E AND
CREATININE
Objective Photometer and Spectropholo?neter Procedure
Principle and reagents. Refer to Method 5, above.
Filter. Green.
Wavelength. 520 im*.
Calibration curve. Refer to Method 6, above.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate. (See Technical notes 2 and 13.) Transfer 10 ml.
of the filtrate to a test tube graduated at 10 ml. Cover the mouth of the tube
with tinfoil and heat in an autoclave at 115 to 120 C. for 20 minutes. (See
Technical note 14.) Cool. If the volume of liquid is less than 10 ml., restore the
volume to 10 ml. with distilled water and mix.
Transfer 10 ml. of unheated Folin-Wu filtrate to another test tube and
proceed with the determination of creatinine on both the heated and unheated
filtrates as given above in Method 6, Procedure for Blood or Serum, and read
in a photometer or spectrophotometer.
Calculation for blood or serum. Read the value directly from the calibration
curve in terms of mg. of creatinine per 100 ml. of blood or serum. {Calculate to
1 decimal place only.)
Total creatinine (in the heated filtrate) — preformed creatinine (in the
filtrate not heated) = creatinine obtained from creatine.
Mg. of creatinine X 1.16 = mg. of creatine. (Calculate to 1 decimal place
only.)
Procedure for Urine
Collect a 24-hour sample of urine. (See Technical note 4.) Measure the volume
(V) and determine the specific gravity. If the specific gravity is greater than
1.010, dilute the urine with distilled water until the specific gravity is 1.010.
Again measure the volume (V d ). If the specific gravity of the urine is 1.010 or
less, omit the above dilution (in which case V and Vd are the same figures).
Prepare a Folin-Wu filtrate, using 3 or more ml. of urine. (See Technical notes
2 and 13.) Transfer 5 ml. of the filtrate (or in the case of very dilute urine,
10 ml.) to a test tube. Cover the mouth of the tube with tinfoil and heat in an
autoclave at 115 to 120 C. for 20 minutes. (See Technical note 15, but use 5
ml. of the above filtrate.) Cool the heated nitrate and transfer, quantitatively,
to a 100-ml. volumetric flask, rinsing the tube with several portions of distilled
water. Dilute to 100 ml. with distilled water. Transfer the same volume (5 or
10 ml.) of unheated filtrate to another 100-ml. volumetric flask and dilute to
100 ml. with distilled water.
Proceed with the determination of creatinine on both the heated and unheated filtrates, as given above in Method 6, Procedure for Blood or Serum,
and read in a photometer or spectrophotometer.
MAY 1954
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Calculation for urine. Use the same calibration curve as prepared for blood or
serum.
Vd X value from the calibration curve
in terms of mg. of creatinine per 100 ml. _ mg. of creatinine per ml. of urine.
V X aliquot of filtrate diluted to 100 ml.
{Calculate to 1 decimal place only)
(usually 5 ml.)
Total creatinine (in the heated urine) — preformed creatinine (in the urine
not heated) = creatinine obtained from creatine. (See Technical note 16.)
Mg. of creatinine X 1.16 = mg. of creatine. (Calculate to 1 decimal place only.)
Mg. per ml. X 24-hour volume in ml. _ Gm. of creatine excreted per day.
1000
(Calculate to 1 decimal -place only)
Normal findings. Refer to Method 7, above.
METHOD 9. URIC ACID
Visual Photometer Procedure
Principle. The determination is based upon the reaction between uric acid
and a phosphotungstic acid reagent, without heating, in the presence of cyanide
and urea. Brown's48 uric acid reagent is used, since under the conditions specified,
it affords lower blanks, improves specificity and gives strict proportionality
between color intensity and uric acid concentration. Serum or plasma is necessary, since most of the chromogens that interfere with the color reaction are
found in the red cells.
Reagents.
1. Sodium tungstale, 10 per cent solution. Refer to Method 1, Reagent 1.
2. Sulfuric acid, 2/8 N solution. Refer to Method 1, Reagent 2.
3. Sodium cyanide, 12 per cent solution. Transfer 12 Gm. of reagent sodium
cyanide to a 250-ml. beaker and dissolve in 50 to 60 ml. of distilled water.
Transfer to a 100-ml. volumetric flask and dilute to 100 ml. with distilled water.
Handle this solution with extreme caution; it is very poisonous. Measure from a
buret, never from a pipet. Keep in the refrigerator. Bring the required amount of
solution to room temperature before use. Do not return unused amounts to the
stock bottle. The solution may be used for 2 weeks, only. Label the bottle with
the date of preparation.
4- Urea, 50 per cent solution. Dissolve 50 Gm. of reagent urea in distilled
water and dilute to 100 ml. with distilled water. The solution can be used
indefinitely.
5. Uric acid reagent (Brown48). Dissolve 100 Gm. of molybdate-free reagent
sodium tungstate and 20 Gm. of anhydrous reagent disodium hydrogen phosphate in about 150 ml. of distilled water in a 1-liter Erlenmeyer or Florence
flask with the aid of heat. (If any molybdate is present in the sodium tungstate,
even 0.001 per cent, the final reagent must be decolorized with bromine.) Mix
25 ml. of concentrated reagent sulfuric acid (sp. gr. 1.84) with about 75 ml. of
distilled water and pour the warm solution slowly and with continuous shaking
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into the solution in the flask. Boil gently for 1 hour, using as a condenser a
funnel holding a 200-ml. Florence flask partly filled with ice water. After boiling,
cool under running water, transfer to a 1-liter volumetric flask, dilute to 1 liter
with distilled water, and mix. The heated solution acquires a light greenishyellow tint which is considerably reduced on cooling and largely disappears as a
result of dilution. Decolorizing with bromine water is not necessary, except as
noted above. The solution can be used indefinitely.
6. Stock uric acid solution (Folin,19 modified). Transfer 0.6 Gm. of lithium
carbonate and approximately 150 ml. of distilled water to a 250-ml. Florence or
Erlenmeyer flask. Shake about 5 minutes until dissolved. Some insoluble material
may remain. Filter and heat the lithium carbonate solution to 60 C.
Transfer exactly 2 Gm. of uric acid to a 1-liter volumetric flask with the aid
of a funnel. Warm the flask in water at 60 C. Add the warm lithium carbonate
solution to the volumetric flask, washing down any uric acid remaining on the
funnel. Shake to dissolve the uric acid. If necessary, warm the flask in water at
60 C. (The lithium carbonate solution may not be perfectly clear even after
filtering. Do not mistake this turbidity for undissolved uric acid and keep
warming and shaking the flask too long. All the uric acid should be dissolved in
5 minutes.)
After solution, cool the flask under cold running water. Without undue delay,
add 20 ml. of 37 per cent formaldehyde solution and fill the flask about half
full with distilled water. Add 25 ml. of 1 N sulfuric acid (0.7 ml. of concentrated,
reagent sulfuric acid, sp.gr. 1.84, diluted to 25 ml. with distilled water) from a
pipet, slowly and with shaking. Dilute the solution to 1 liter, mix thoroughly
and transfer to a clean, brown, tightly-stoppered bottle. Store away from light.
One milliliter of this solution contains 2.0 mg. of uric acid. The solution can be
used indefinitely.
7. Standard uric acid solution. Transfer 1 ml. of the stock uric acid solution
(Reagent 6) to a 500-ml. volumetric flask and dilute to 500 ml. with distilled
water. Mix thoroughly. One milliliter of this solution contains 0.004 mg. of
uric acid. Keep in the refrigerator, but warm a portion of the solution to room
temperature before using. The solution may be used for 1 week only. Label the
bottle with the date of preparation.
Procedure for Blood or Serum
Prepare a Folin-Wu filtrate, (see Technical note 2), adding the acid very
slowly and allowing the coagulum to stand 15 to 20 minutes before filtering.
Transfer 2 ml. of the filtrate to a cuvet, test tube or small flask. In a similar
container, place 2 ml. of the standard uric acid solution. To each tube add 2
ml. of cyanide solution from a buret. (This reagent is highly poisonous and must
never be pipetted.) Mix by lateral shaking using paraffined corks. Add 2 ml. of
the urea solution to each tube and mix. Add 1 ml. of the uric acid reagent and
mix thoroughly. With multiple tests, allow about 30 seconds (or more if required)
between additions of the uric acid reagent to successive tubes. Allow the tubes
to stand at room temperature for 50 minutes. Add 3 ml. of distilled water and
MAY 1954
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mix thoroughly. Compare in a visual photometer, setting the standard at 20
mm. (see Technical note 3) and reading successive determinations at the same
time interval over which the uric acid reagent was added.
Calculation for blood or serum. If 2 ml. of Folin-Wu filtrate was taken for
analysis, the calculation is as follows:
Reading of standard
Reading of unknown
'
_ mg. of uric acid per 100 ml. of blood or serum.
(Calculate to 1 decimal -place only.)
Procedure for Urine
Collect a 24-hour specimen of urine (see Technical note 4) and measure the
volume. If any appreciable amount of protein is present, remove as directed in
Method 1, under Procedure for Urine. Transfer 1 ml. of urine to a 100-ml.
volumetric flask and dilute to 100 ml. with distilled water. Mix thoroughly.
Transfer 2 ml. of diluted urine to a cuvet, test tube or small flask, and 1 ml.
of diluted urine and 1 ml. of distilled water to another. In a similar container
place 2 ml. of standard uric acid solution, and proceed as given directly above
under Procedure for Blood or Serum.
Calculation for urine. If the urine was diluted 1 to 100 as given above, the
calculation is as follows:
Reading of standard
Reading of unknown
0.8
ml. of diluted urine used for analysis {usually 1 or 2 ml.)
= mg. of uric acid per ml. of urine. (Calculate to 1 decimal place only)
Mg. per ml. X 24-hour volume in ml. _ Gm. of uric acid excreted per day.
1000
(Calculated to 1 decimal place only)
Normal findings. The normal concentration of uric acid in a person on an
average diet and after a 12- to 14-hour fast, varies from 1.9 to 6.7 mg. per 100
ml. of serum or plasma. The normal adult on an average diet excretes from 0.5
to 0.7 Gm. of uric acid per day. On a purine-free diet, the excretion varies
from 0.3 to 0.5 Gm. per day. On a high-purine diet, the excretion may rise to
1 Gm.
METHOD 10. TJKIC ACID
Objective Photometer and Spectrophotometer Procedure
Principle and reagents. Refer to Method 9, above.
Filler. Red.
Wavelength. 700 im*.
Calibration curve. To tubes or flasks, transfer the following amounts of standard uric acid solution: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 ml. Dilute the contents
of each tube to 5 ml. with distilled water. To each tube add 2 ml. of the cyanide
solution from a buret. (This reagent is highly poisonous and must never be pipetted.)
Mix by lateral shaking, using a paraffined cork. Add 2 ml. of the urea solution
to each tube and mix. Add 1 ml. of the uric acid reagent and mix thoroughly,
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allowing about 30 seconds (or more if required) between additions to successive
tubes. Allow to stand at room temperature for 50 minutes and then read in the
photometer or spectrophotometer, using the blank or " 0 " tube as the reference
liquid (see Technical note 5) and reading successive tubes at the same time
interval over which the uric acid reagent was added.
Prepare a calibration curve on coordinate paper, using the following figures
to correspond to the amounts of standard solution used above: 0, 2, 4, 6, 8, 10,
12 and 14 mg. of uric acid per 100 ml. of serum or plasma, and plotting against
the corresponding readings in the photometer or spectrophotometer. (See
Technical note 6.) Prepare a new curve with each new lot of uric acid reagent.
Procedure for Blood or Serum
Follow the procedure as given above under Method 9, Procedure for Blood
or Serum, except use 1 ml. of nitrate and 1 ml. of distilled water (instead of the
2 ml. of filtrate prescribed) and do not prepare the standard solution. Read in
the photometer or spectrophotometer using as the reference liquid a blank
containing 5 ml. of water to which are added the cyanide, urea and uric acid
reagent. (See Technical notes 5, 7 and 8.)
Calcidation for serum or plasma. Read the value directly from the calibration
curve in terms of mg. of uric acid per 100 ml. of serum or plasma. (Calcidate to
1 decimal -place only.)
Procedure for Urine
Collect a 24-hour specimen of urine (see Technical note 4) and measure the
volume. If any appreciable amount of protein is present, remove as directed in
Method 1, under Procedure for Urine. Transfer 1 ml. of urine to a 200-ml.
volumetric flask and dilute to 200 ml. with distilled water. Mix thoroughly.
Transfer 2 ml. of diluted urine to a cuvet, test tube or small flask, and 1 ml. of
diluted urine and 1 ml. of distilled water to another. Follow the procedure as
given above under Method 9, Procedure for Blood or Serum, but do not prepare
the standard solution. Read in the photometer or spectrophotometer, using as
the reference liquid a blank containing 5 ml. of water to which are added the
cyanide, urea and uric acid reagent. (See Technical notes 5, 7 and 8.)
Calcidation for urine. Use the same curve as prepared for serum or plasma. If
the urine was diluted 1 to 200 as given above, calculate as follows:
Value from the calibration curve in
terms of mg. of uric acid per 100 ml. _ mg. of uric acid per ml. of urine.
5 X ml. of diluted urine used for
{Calcidate to 1 decimal place only.)
analysis (usually 1 or 2 ml.)
Mg. per ml. X 24-hour volume in ml. _ Gm. of uric acid excreted per day.
1000
(Calculate to 1 decimal place only)
Normal findings. Refer to Method 9, above.
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