Determinations of DPN (TPN) Thiamine, Amino Acids and Amino Sugars in Blood of Animals with Experimental
Myocardial Infarction
By 0 . CABRERA, M.D., A. BEUREX, M.D. AND R. J. BJXG, M.D.
An investigation was made of the possible release from heart muscle of substances of small molecular weight following experimental myocardial infarction. These resulting chemical changes in coronary vein blood may be due to metabolic alterations of the cell which wouM either precede cell death or take place in cells with reversible metabolic changes. The coro- nary arteriovenous differences of pyridine nucleotides, thiamine and its phosphorylated deriva- tives, amino acids and amino sugars were investigated. No changes from the control were found. The negative results may have been caused by the rapid turnover of these substances in blood.
P
REVIOUS reports from this laboratory have described circulatory, pathologic and metabolic findings in humans and dogs fol- lowing myocardial infarction. Among these changes were early alterations in the myocar- dial usage of pyruvate and lactate, followed by an increase in plasma activity of certain enzymes, which appear to enter the circulation from necrotic heart muscle cells. Enzyme ac- tivity in blood begins to rise at the onset of coagulative necrosis and reaches its peak 24 hours after the injury of the heart, when cell necrosis is at its height. This paper deals with an investigation of the possible release from the cells of substances of smaller molecular weight. It was thought that these chemical changes may not be related to cell necrosis but might represent metabolic alterations of the cell which would either precede cell death or take place in cells with reversible metabolic changes. To accomplish this, the coronary arteriovenous differences across the heart of the following selected substances were studied:From the Department of Medicine, Washington University School of Medicine and the Washington University Medical Service, Veterans Administration Hospital, St. Louis, Mo.
This work was supported by U. S. Public Health Service grant no. H-2678, The Life Insurance Medi- cal Research Fund, The American Heart Association, Burroughs Wellcome, and the Tobacco Industry Re- search Fund.
Received for publication August 5, 1957.
pyridine nucleotides, thiamine and its phos- phorylated derivatives, amino acids and amino sugars. Although the results reported in this paper are entirely negative, it was felt that they were of sufficient general interest to war- rant publication.
METHODS
Experimental myocardial infarction was produc- ed in mongrel dogs according to methods previ- ously described.1 The chest of the animals was not opened. A no. 7 birdseye catheter was introduced via the external jugular vein into the coronary sinus and an Agress catheter was placed into the carotid artery.1 Arterial blood was collected from a catheter inserted into a femoral artery. During and im- mediately following the injection of the spheres, blood pressure was optically recorded from the femoral artery by means of a strain gage. A sus- tained fall in pressure of about 50 mm. Hg was taken as presumptive evidence of successful emboli- zation of the coronary arteries. All experiments were performed under Nembutal anesthesia (30 mg./Kg.). Samples were taken 5, 10, 15 and 30 min. and 1, 2, 12 and 24 hours following emboli- zation of the coronary arteries. At the end of the experimental period, the dogs were killed and the hearts were examined for gross evidence of myocardial infarction.
Determination of DPN (TPN). The fluorometric method for pyridine nucleotides of Lowry and co- workers* was used. This method is based on the fluorescence emitted by oxidized pyridine nucleo- tides when dissolved in strong alkaline solutions.
The method as applied to the determination of DPX also measures TPN. The general procedure was as follows: 10 ml. blood samples were with-
Circulation Rritearch, Volume VI, January \OTt3
16 CABRERA, BEUREN AND BIXG drawn into clean syringes wetted with heparin
solution, and transferred immediately into centri- fuge tubes containing sufficient nicotinamide to make a final concentration of .03 M of nicotina- mide. The mixture was then centrifuged and the plasma decanted. Trichloroacetic acid (TCA) was then added in the proportions of 1 volume of plasma to 2 volumes of TCA to make a final con- centration of 5 per cent TCA. A protein free supernatant was then obtained by centrifngation of the precipitate. Within 10 minutes, 10 nl. ali- quots of the clear supernatants were transferred to 3 ml. fluorometer tubes containing 100 |il. of 7N-NaOH and mixed promptly. After one hour standing at room temperature, 1 ml. of water was added to each fluorometer tube and the samples were read.
One of the difficulties in determining plasma concentration of DPX (TPX) is the presence of DPN-ase in blood which hydrolyses these nucleo- tides. It was found, however, that collections of blood in the presence of .03 M nicotinamide, which inhibits the DPN-ase, results in satisfactory re-
coveries (95-100 per cent) of added DPN.
The fluorometer used was the model A of the Farrand Optical Company with primary filter Corning no. 5850 and secondary filters Corning nos. 4308, 5562 and 3387. Quinine sulfate solu- tions in 0.1 X H»SO< served as working fluor- ometer standard. Two DPN standards containing respectively 5 X 10"" and 1 X 1O'10 M and appro- priate blanks were given identical treatment. All determinations were done in duplicate.
Determination of Total Thiamine. Burcb and co- workers' found in normal human subjects a thin- niine content in whole blood of 5 (j,g. per cent with an average of .8 \ig. per cent for plasma. The values for dog's plasma in the present experiments were considerably higher with a range of from 2-5 [j.g. per cent of plasma. This is probably due to the fact that the original method was not followed in which thioehrome is estimated as the differences of the fluorescences before and after irradiation of the fluorometer tubes with ultraviolet light. Con- sequently, absolute values as determined here, are too high. It was felt, however, that this source of error would not invalidate the results since the degree of fluorescence due to extraneous material was probably the same in coronary arterial and coronary venous blood.
The total thiamine content of plasma specimens was determined by following the fluorometric thio- ehrome method of Burch and co-workers.' Plasma samples were obtained by centrifugation of the heparinized blood specimens immediately after their withdrawal. One millUiter aliquots of plasma were delivered into test tubes containing 2 ml. of 15 per cent TCA and mixed vigorously. After 30 to 60 min. at room temperature, the specimens were
centrifuged and 0.4 ml. aliquots were used for determination of total thiamine according to the procedure of Burch. Appropriate blanks and stand- ards containing 10 and 20 millimicrograms of thia- mine were treated in an identical manner. All samples were run in duplicate and read in the Farrand fluorometer with primary filter Corning no. 5860 and secondary filters nos. 3384 and 4303.
Chromatoyraphic Analysis of Amino Acids and Amino Sugars. The filter paper partition chrom- atography technic of Dent" was used for determi- nation of amino acids. Plasma samples were ob- tained as described for the determination of thia- mine. The specimens were then transferred to dialysis bags made from Visking tubing and ultra- filtrates obtained by housing the dialysis bags in appropriate boiling tubes placed in crushed ice.
The dialysis bags remained under reduced pressure overnight. By the use of a manifold, 12 individual ultrafiltrations could be carried out simultaneously.
The following day, 5 ml. aliquots of the ultrafil- trates were desalted by electrodialysis in a Reco desalter. Amounts of desalted ultraflltrates equiva- lent to 0.25 ml. of plasma were then simultaneously evaporated to dryness under a current of warm air;
the residue was then transferred to squares of filter paper (Whatman no. 4, 20 X 20 cm.). A metal frame for the simultaneous chroniatographic development of 12 such squares described by Datta Hiid co-workers" was used. The ascending type of chromatography was employed. The first solvent consisted of phenol-HN> and water (4 parts of phenol to 1 part of water) ; for the second solvent, a collidine-lutidine-water mixture (1:1:1) was used. The amino acid spots were detected by spray- ing the papers with a 0.1 per cent solution of nin- hydrin in 95 per cent ethanol, and leaving them to stand at room temperature.
For the chroniatographic analysis of the amino sugars, the filter paper partition chromatography technic of Partridge7 was employed. This method employs one dimensional chromatography, using phenol saturated with water as the solvent pair and a modified Morgan and Elson8 reagent for separate consecutive detection of hexosamines and N-acetyl hexosamines.
REKULTB AND COMMENTS
DPN (TPN). Blood concentrations of DPN (TPX) were determined in 7 dogs with defi- nite myocardial infarction. Statistical analy- sis of the coronary arteriovenous differences indicates that there was no significant differ- ence between the control observations and data collected at various intervals up to 2 hours after the onset of infarction (table 1).
The range of experimental A'alues in plasma
DETERMINATION'S POLLOAVING MYOCARDIAL INFARCTION
TABLE 1.—Plasma DPK (TPN) Levels ( Arteriovenous Difference, mH '/<) Following Experimental ifyo-
cardial Infarction.
Mean S. E.
X V
Control
- 0
± 0 6
>
10 25 7
0—5 min.
-0.39
±0.32 7
> . 2
Differences from contro 6-10
- 0
± 0 3
>
min.
53 61 4
11-20 min.
-0.45
±0.38 6
> . 2
1-2 hour*
- 0
± 0 7
>
23 28 4
fell between 15 X 10^" and 50 X 10"6 M/L.
In anesthetized dogs, before the injection of the spheres, concentrations of nucleotides in coronary vein blood exceeded that in coronary arterial blood by 1 X 10""6 M/L.; this value was not statistically significant (table 1).
Glock9 has shown that the DPN and TPN con- tent of vertebrate heart is approximately 5 X lO""1 M/Kg. of wet tissue. If it is assumed that the same concentration is present in the heart of dogs, then the release of these co-en- zymes from 1 Gm. of infarcted tissue in 5 min.
at a coronary flow of 80 ml./min. would result in concentration in the coronary sinus blood of approximately 1 X 10~" M/L. This would fall within the errors of the method.
Thiamine. Determinations of thiamine were carried out in 11 dogs with verified myocardial infarction (table 2). Following embolization of the coronary arteries, the concentration of thiamine in coronary vein blood exceeded that in arterial blood. However, these differences were not significant statistically. If one as- sumes that the total thiamine content of the heart is about 4 [Ag./Gm. of tissue and that 1 Gm. of infarcted tissue releases all its thiamine content in 5 min. at a coronary flow of 80 ml./
min., then the amount of total thiamine recov- ered in the plasma would be of the order of 0.1 (Jig. per cent. This figure is barely within the error of the method. Furthermore, the possibility cannot be ruled out that thiamine pyrophosphate (cocarboxylase) is immediately bound by active proteins and is removed from the solutions with the TCA precipitate. It is known that eocarboxylase dissociates from its protein partner in alkaline solutions.
Amino Acids and Amino Sugars. It is well known that changes in concentration of trans-
TABLE 2.—Plasma Thiamine Levels (Arteriovenous Difference, [1*7. 9}-) Following Experimental Hyo-
canlial Infarction
Mean S. E.
X V
Control
0.19
±4.34 7
Differences from control 0-5 min. 6-10 min. 11-20 min. 1-2 hours
-5.34
±4.72 11
>.2
0.25
±4.70 8
-1.63
±3.02 C
2.14
±5.32 5
>7
aminases appear in the heart muscle after myocardial infarction; this change, together with the suppression of anabolism which oc- curs as an early manifestation of tissue ische- mia, might lead to an increase in concentration of the intracellular amino acid pool. It seemed conceivable that such an increase would lead to a release of ainino acids from the cell and result in increased concentration of amino acids in coronary vein blood. Technics are already available for screening purposes, such as filter paper chromatography; thus, changes in amino acid pattern of plasma may be recog- nized by using known amounts of standards of pure amino acids and comparing their chromatographic spots with the unknown sample.
Studies were performed on 10 animals with proven myocardial infarction. The normal amino acid pattern of dog's blood as described by Dent1" was found in control experiments.
No obvious changes were observed after the onset of infarction. There was no change in the relative amount of amino acid normally present, nor was there any appearance of ab- normal chromatographic pattern, suggesting the appearance of amino acids not previously present. AVhile this work was in progress, a report by Chan in and co-workers11 appeared which described changes in amino acid pattern in heart tissue several hours after onset of in- farction.
Because one of the earliest metabolic mani- festations of cytolysis following tissue ische- mia is the breakdown of mucopolysaccharides, the appearance of free hexosamines in the coronary vein blood was investigated. These substances are ninhydrin reactive; conse-
18 CABRERA, BEUREN AND BING quently their presence might have been de-
tected in the chromatographic studies on arn- ino acids described above. However, since this was not the case, the specific technic for their detection devised by Partridge7 was employed.
An aliquot specimen equivalent to 1.0 ml. of plasma was evaporated under a current of warm air to a convenient volume and run on the chromatograms. Desalted ultrafiltrates previously prepared for the determinations of amino acids and kept at a temperature of
—10 C. were used. No change in the coronary arteriovenous differences of free hexosamines were observed after coronary embolization.
Changes of hexosamines bound to proteins have been described in several human diseases, including myocardial infarction.12 With the technic used in this preliminary report, only free hexosamines or N-aeetyl hexosamines can be detected. It is possible that an analysis of hydrolysates of these specimens may show different results.
Results reported in this paper demonstrate no change in the coronary arteriovenous dif- ference of DPN (TPN) and total thiamine following arterial embolization of the coronary arteries in the dog. Results obtained on changes in amino acid pattern of coronary artery and vein blood or coronary arterio- venous difference of hexosamines are equally negative. The likelihood exists that these neg- ative results were due to the fact that, al- though the methods of analysis were extremely sensitive, the analytic error was probably still too great. In addition, substances of small molecular weight, as determined in this report, are metabolized and excreted at a higher rate than large protein molecules. Their accumu- lation in blood would be prevented, therefore, in contrast to the protein enzymes which are readily detected following myocardial infarc- tion.
SUMMARY
Coronary arteriovenous differences of DPX (TPN), total thiamine, amino acids, and am- ino sugars were determined in dogs following experimental myocardial infarction. Samples were drawn 5, 10, 15, 30 min. and 1, 2, 12 and
24 hours following embolization of the coro- nary arteries.
No change in the coronary arteriovenous differences of these substances were found.
The likelihood exists that these negative re- sults were due to the fact that, although the methods of analysis were extremely sensitive, the analytic error was probably still too great;
it is also possible that the substances analyzed were so rapidly metabolized that their accu- mulation in blood could not be detected.
SUMMARIO IN I N T E R L E N Q U A
Differentias del contento de nucleotido di- phosphopyridina (triphosphopyridina), thia- mina total, amino-acidos, e amino-sucros in arterias coronari e in venas esseva determinate in canes post effectuation experimental de in- farcimento myocardial. Specimens de sanguine esseva prendite 5, 10, 15, e 30 minutas e 1, 2, 12, e 24 horas post le embolisation del arterias coronari.
Nulle alterationes del differentias de iste substantias inter arterias coronari e venas es- seva constatate. II non es improbabile que le negativitate del resultatos debe esser attribuite al facto que le errores analytic esseva troppo grande, ben que le methodos usate in le ana- lyse esseva extrememente sensibile. II es etiam possibile que le metabolisation del substantias in question esseva si rapide que lor accumu- lation in le sanguine non poteva esser detegite.
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