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Amino acid metabolism in lactic acidosis
Amino Acid Metabolism in Lactic Acidosis
E. B. MARLISS, M.D.* T. T. AOKI, M.D. C. J. TOEWS, M.D.? Boston, Massachusetts P. FELIG, M.D. New Haven, Connecticut J. J. CONNON, M.D.S J. KYNER, M.D.g W. E. HUCKABEE, M.D. G. F. CAHILL, Jr., M.D. Boston, Massachusetts
From the Elliott P. Joslin Research Laboratory and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, the Evans Memorial Department of Clinical Research, Boston University School of Medicine, Boston, Massachusetts 02115, and the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510. This study was supported in part by U.S. Public Health Service Grants AM-09584, AM05077, AM-13526 and HE-06297. It was presented in part at the 30th Annual Meeting of the American Diabetes Association, St. Louis, Missouri, June 13, 14, 1970 [l]. Requests for reprints should be addressed to Dr. G. F. Cahill, Jr., Harvard University Medical School, Department of Medicisne, 170 Pilgrim Road, Boston, Massachusetts 02215. Manuscript received December 24, 1970. * Present address: lnstitut de Biochimie Clinique, Sentier de la Roseraie, Geneva, Switzerland. t Present address: 2083 Highview Drive, Cedar Springs Road, Burlington, Ontario, Canada. 3 Present address: University of Belfast Medical School, Belfast, Ireland. § Present ad,dress: University of Kansas Medical Center, Kansas City, Kansas.
474
individual plasma amino acid levels have been determined in six patients with severe lactic acidosis. A consistently distorted pattern was observed whether the disorder was due to shock or was of the idiopathic type. A sixfold elevation in mean alanine concentration was observed, and a significant correlation of alanine with pyruvate concentrations was demonstrated. Since alanine is normally released from muscle and stoichiometrically extracted by liver, normal formation of alanine from pyruvate is suggested by the findings. However, uptake and metabolism of alanine by isolated, perfused rat liver was significantly inhibited by perfusate concentrations of lactate and pyruvate equivalent to those observed in the patients. Thus the high levels of alanine in lactic acidosis are viewed as consistent with normal or increased peripheral release combined with impaired hepatic disposal. The levels of fifteen of the nineteen amino acids measured were elevated. Of the remaining four, three were intermediates of the urea cycle. The syndrome of metabolic
acidosis with massive accumulation of lactate and an elevated ratio of blood lactate to pyruvate concentrations was described by Huckabee in 1961 as lactic acidosis [2]. Blood lactate concentrations are regulated by synthesis and release by some tissues, including muscle, and uptake by others, predominately liver in a resting steady state. Thus accumulation must result from imbalance between these dynamic processes, that is, increased synthesis, decreased disposal or both [3]. Lactate can be synthesized only from pyruvate, therefore increased tissue lactate release could result only from increased pyruvate availability or increased conversion of available pyruvate. The corollary considerations apply for a ‘decrease in disposal, which can proceed only by reconversion to pyruvate. Thus decreased capacity for pyruvate metabolism via other pathways or decreased conversion of lactate to pyruvate could impede uptake and disposal. Since patients with lactic acidosis have exceedingly high blood pyruvate levels in addition to altered lactate to pyruvate ratios [2], a factor of increased cellular pyruvate concentrations probably contributes to the pathophysiology. The conversion of pyruvate to lactate, in addition to provision of oxidized nicotinamide adenine dinucleotide (NAD), may be viewed as a means of making available 3-carbon moieties not disposed of by other metabolic pathways, which are then
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AMINO
amounts, probably because its central position in many metabolic sequences results in much lower concentrations in cells and hence in blood. One such alternate fate of pyruvate is transamination to alanine. Felig et al. [43 recently demonstrated that pyruvate may be transported from peripheral tissues to the liver as alanine in physiologically significant amounts, as previously inferred by Mallette, Exton and Park [5]. Further, a linear relationship between blood pyruvate and alanine concentrations has been demonstrated both at rest and during exercise in normal subjects [6]. In light of these observations, and of the known stoichiometric relationship between ‘muscle release and hepatic uptake of most amino acids in normal man [4,7], individual plasma amino acid levels have been determined in six patients with documented lactic acidosis. In addition, to test whether the accumulation of lactate and pyruvate I
MARLISS
ET AL.
could interfere with hepatic amino acid disposal, the technic of the isolated, perfused rat liver has been employed using perfusate concentrations of lactate and pyruvate comparable to those demonstrated in the patients described.
recycled to the liver for new glucose syntheses (the afferent limb of the Cori cycle). Pyruvate itself is not normally transported in as large
TABLE
ACIDS IN LACTIC ACIDOSIS -
MATERIALS
AND METHODS
Patients. The patients were hospitalized with marked metabolic acidosis; all had blood lactate concentrations of greater than 15 mmoles/L, pyruvate levels greater than 0.28 mmoles/L and elevated lactate to pyruvate ratios. Four were diabetic: two of these (I.G. and M.L.) were suffering from concurrent ketoacidosis and are described in detail elsewhere [8]. One other (M.M.) was receiving therapy with phenethylbiguanide. Three patients were clinically in a state of circulatory failure (hypotension, peripheral vasoconstriction) and were considered to have “secondary lactic acidosis.” The remaining three showed no signs of circulatory collapse or hypoxemia and were considered to be representative of primary or idiopathic lactic acidosis [2]. Further clinical data are summarized in Table I. The course in all patients followed the usual one for severe lactic
Clinical Data in Patients with Lactic Acidosis
Patient M.L.
J.H.
77 F Idiopathic Diabetes, diarrhea
70 M Idiopathic Anemia, pneumonia, weight loss
I.G.
AgeW) Sex
Type of lactic acidosis Associated
disorders
72 F Idiopathic Diabetes, gastroenteritis
N.G.
M.M. 55 M Secondary Diabetes, myocardial infarction
64 M Secondary Diabetes, gastroenteritis, essential
J.J. 35 M Secondary Acute hemorrhagic pancreatitk
hypertension Vital signs Blood pressure (mm Hg) Pulse rate (per min) Respiratory rate (per min) Temperature (“F) Laboratory data Arterial blood ph PC% (mm Hg) HCQ (mEq/L) POZ (mm
Hgf
Serum Sodium (mEq/L) Chloride (mEq/L) Anion gap (mEq/L) Blood urea nitrogen (mg/lOO ml) Blood glucose (mg/lOO ml) Arterial blood Lactate (mmoles/L) Pyruvate (mmoles/L) Lactate: pyruvate Plasma alanine &moles/L) Alanine: pyruvate
Volume 52, April 1972
110/70 114 24 101.2
7.18 16 6 110
160/70 120 20 98.5
7.20 21 8 97
100/75 100 30 98.4
80/50 42 30 97.0
80/50 100 36 101.2
7.18 10 4 90
7.18 23 8 99
13 3 140
6.98
80/60 56 24 91.0
7.00 28 7 144
139 95 38 57
142
144 113 27 61
147 94 45 ...
139 88 48 103
123
103 31 138
298
311
95
50
144
88
24.2 0.51 48 1,188
15.0 0.28 52 1,336
2.3
4.8
22.6 0.30 76 426 1.3
75 41 21
24.0 0.48 50 1,961
29.0 0.77 38 5,390
27.0 0.33 82 866
4.1
7.0
2.6
475
AMINO
ACIDS IN LACTIC ACIDOSIS -
MARLISS
FT AL.
acidosis [3], and they died between one and three days from the time the samples were taken. Normal values for plasma amino acids were obtained under basal conditions in ten postabsorptive nonobese subjects with normal intravenous glucose tolerance (K >1.2 per cent as absolute per cent disappearance per minute, after the administration of 0.5 gmlkg). Similar conditions applied to eight different subjects in whom normal values were obtained for arterial blood lactate (0.910 * 0.155 mmoles/L, mean + SEM), pyruvate (0.075 f 0.010 mmoles/L) and lactate to pyruvate ratios (12.7 2 1.5). Rat Liver Perfusion Studies. Livers from male Sprague-Dawley rats (200 to 250 gm) fasted overnight (P<.OS.
ALA
PA)
ML
LVS LEU SLY
UNPAIREO
TAU TM)
1)
ILE
mrantSEN 0
LactlC ACLAW (n41
I
Normdr
(n*IOl
HbS TYR O+lt$hEl BUT
PME
Figure 1. Plasma amino acid levels that are significantly elevated in six patients with lactic acidosis. Values for normal subjects are shown for comparison. Mean & standard error of mean (SEM).
were perfused for sixty minutes in a modified Miller apparatus [9] with Krebs-Ringer bicarbonate buffer at pH 7.4 having the same amino acid concentration as found in the plasma of the patients with lactic acidosis. The amino acids were obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio) and Sigma Chemical Co. (St. Louis, MO.). Eight livers were perfused with a medium containing high lactate and pyruvate concentrations which were maintained by their constant infusion into the perfusate. Eight control livers were perfused without the addition of lactate and pyruvate to the perfusate. Oxygenation was maintained with 95 per cent oxygen and 5 per cent carbon dioxide in both groups. The conversion of L-alanine-U-l% (New England Nuclear Corp., Boston, Mass.) added initially to the medium, to *%-oxygen and 14Cglucose, and the perfusion technic, materials used and enzymatic fluorometric methods for the assay of metabolites are as previously described [lo]. Individual amino acid levels were deterMethods. mined by automated ion exchange chromatography using a Beckman 120C amino acid analyzer (Beckman Instrument Co., Palo Alto, Calif.), modified to provide simultaneous recording from two columns [ll]. Plasma and perfusate samples were deproteinized with sulfosalicylic acid as described previously [7]. In three instances deproteinization of plasma samples was performed after storage at 20°C and therefore values for half cystine are not reported. The sulfosalicylic acid filtrates were immediately frozen and stored at -20°C until analysis. Glutamine, asparagine and glutamate levels were not reliably determined by this method. Plasma glutamine was determined in three of the subjects within three days of sampling by an enzymatic method employing glutaminase [12]. Arterial blood was immediately deproteinized for determination of lactate and pyruvate content by either enzymatic fluorometric methods on perchloric acid fil-
meon? SEM
160NS
0
Loctlc
n
Normols
Actdos~s
(n=6)
h=IO)
140 ALANINE pHlL
IZO@IL tooNS
PC.OS
NS
80. 604020-
hil.ORN
Plasma amino Figure 2. decreased in six patients sented as in Figure 1.
476
A
Cl1
acid levels that are normal or with lactic acidosis. Data pre-
0’
*
4
*
1002003co400soo600700mo900 BLOOD PYRUVATE CM/L
Figure 3. Correlation of simultaneous arterial blood pyruvate and venous plasma alanine concentrations in lactic acidosis.
The
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AMINO
trates [lo] chloroacetic parameters
or by modified chemical methods on triacid filtrates [13] or both. Acid-base and standard clinical blood chemistry analy-
ses were performed as previously reported [14]. Statistical analyses were performed according method of Snedecor [15].
to the
RESULTS Patients with Lactic Acidosis. Markedly elevated levels of lactate and pyruvate and an increased lactate to pyruvate ratio were observed in all patients, as compared with the normal subjects (Table I). Plasma individual amino acids showed a consistently distorted pattern. Striking elevations of three to six times normal were observed for alanine, proline, ;I-amino-N-butyric acid, lysine, methionine, isoleucine, leucine and taurine (p ~0.02, unpaired t test). Less marked but sig nificant elevations were noted for threonine, glytine, valine, phenylalanine, tyrosine and histidine (p ~0.05). These values, with normal levels for comparison, are depicted in Figure 1. Glutamine levels in the three subjects in whom they were determined were moderately elevated (840 4 20 pmoles/L, normal 584 fi 43 pmoles/L). The levels of the remaining amino acids are shown in Figure 2. No alteration was observed in the levels of citrulline, ornithine or serine. The only amino acid which showed a significant decrease was arginine (p cO.05). Alanine concentrations showed the greatest increase (1,966 2 891 mmoles/L + SEM). A sig nificant correlation of alanine and pyruvate concentrations is illustrated in Figure 3 with r = 0.93
ACIDS IN LACTIC ACIDOSIS-MARLISS
ET AL.
bon dioxide and glucose was not simply due to dilution of radioactivity in a much larger intracellular pyruvate pool. Figure 5 shows the amino acid concentrations of the perfusate at the beginning and at sixty minutes, the latter for both the perfusate with and without high lactate and pyruvate concentrations. Although most amino acids were taken up to some degree by the livers perfused with either solution, a difference attributable to the presence of lactate and pyruvate in the medium was apparent only for alanine, proline, tyrosine, methionine and phenylalanine. The magnitude of the inhibition of uptake was greatest
YINIJTES
r
(P
Rat Liver Perfusion Studies. The appearance of labelled carbons in carbon dioxide and in glucose is shown in Figure 4. With lactate and pyruvate in the perfusion medium, the activity of l*C appearing in carbon dioxide from L-alanine-l*C was markedly decreased as compared to that obtained with the perfusate containing no lactate or pyruvate. The differences were highly significant at both thirty and sixty minutes (p
Volume 52, April 1972
30
MINUTES Figure 4. Effect of high perfusate concentrations of lactate and pyruvate upon conversion L-alanineUIT to “CO, (top) and glucose (bottom) in isolated rat livers. Mean r+ SEM.
477
AMINO ACIDS IN LACTIC ACIDOSIS -
MARL&S
ET AL
INITIAL
PERFUSATE
60
MIN..
HIGH
60
MIN..
CONTROL
CONCENTRATION
LACTATE
+ PVRUVATE
In = 5)
I600 tn = 5)
(HwniSEY)
Figure 5. Effect of high perfusate concentrations of lactate and pyru-
600 400 ‘ml
lM)s
l.001
200 0 ALA
PRO
VAL
LVS
LEU
GLV
TAU
THR
ILE
HIS
TVR
MET
for alanine, which showed a fourfold difference in concentration (265 & 46 Pmoles/L in the livers perfused without lactate and pyruvate in the perfusate and 959 2 79 Pmoles/L in the livers perfused with high lactate and pyruvate concentrations in the perfusate). The smallest proportional uptakes were for valine, leucine and isoleucine (less than 20 per cent). Citrulline and ornithine were taken up to the extent of about 30 per cent, arginine uptake was greater than 60 per cent. The remaining amino acids were taken up by 40 to 60 per cent, except for histidine which was taken up by 80 per cent. Lactate concentrations of the perfusate were maintained at 25 to 27 mmoles/L and pyruvate concentrations at 0.43 to 0.97 mmoles/L by the rates of infusion employed, resulting in lactate to pyruvate ratios of 30 to 70. In the control group, the lactate concentration in the perfusate throughout perfusion was 0.6 to 1.5 mmoles/L and pyruvate concentration less than 0.05 mmoles/L. Glucose production was significantly greater in the livers perfused with the medium containing high lactate and pyruvate concentrations at all periods: at thirty and sixty minutes, control values were 9 and 15 Pmoles/gm, whereas those perfused with high lactate-pyruvate solutions were 31 and 50 rmoles/gm, respectively. COMMENTS Of the subjects reported, three represent lactic acidosis secondary to circulatory failure (type 2A of Huckabee [13]). The other three may be considered to have suffered from idiopathic lactic acidosis (type 2B) since no evidence of inadequate
478
PHE
SER
ORN
ARC
CIT
vate upon uptake of amino acids by isolated rat livers. Initial amino acid concentrations were comparable to those in the lactic acidosis patients. See text for discussion. Mean + SEM.
tissue perfusion or hypoxemia was present at the time the samples were taken. Despite this difference, the acid-base, lactate and pyruvate, and amino acid parameters were comparable. Thus the data demonstrate further similarities between the two disorders, but specific conclusions regarding the etiology of idiopathic lactic acidosis are not warranted. The data are consistent with the hypothesis of Huckabee of inapparent and inefficient redistribution of blood flow [16], and would suggest an important component of hepatic incapacity to maintain metabolic homeostasis. Indeed, net hepatic release of lactate [17] and of pyruvate and amino acids is not excluded. Five of the patients described showed marked metabolic acidosis, with appropriate respiratory compensation, but one (J.J.) demonstrated inadequate compensation and thus had combined metabolic and respiratory components [18]. All had markedly elevated blood lactate and pyruvate levels and lactate to pyruvate ratios (Table I), reflecting an altered cellular oxidation-reduction state [ 191. The plasma alanine level has been shown to be regulated in the postabsorptive state primarily by the rates of release from muscle and uptake by the liver, for which a near stoichiometric relationship exists [4,7]. Alanine is quantitatively important as a substrate for hepatic gluconeogenesis, and glucose so synthesized may be recycled to the periphery, in an “alanine cycle” analogous to the Cori cycle [20]. These two cycles are closely interrelated, via the reversible transamination step between pyruvate and alanine, catalyzed by alanine aminotransferase (EC 2.6.1.2).
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AMINO ACIDS
It has recently been shown that for the effluent from limb muscle, a linear correlation exists between alanine and pyruvate concentrations at rest and during exercise in normal subjects [6]. Hence, the demonstration of a sirmilar correlation between alanine and pyruvate in the current study (Figure 3) suggests that formation of alanine from pyruvate remains intact in lactic acidosis. The disproportionate elevation in alanine levels with respect to the other amino acids could be a consequence of increased intracellular pyruvate levels, which may be inferred from the markedly elevated blood pyruvate concentration. A shift of cellular redox potential toward the reduced state would be expected to diminish pyruvate levels in the absence of altered pyruvate synthesis (from glycolysis or transamination of alanine) or disposal (via the tricarboxylic acid cycle). Thus the elevated levels must reflect an alteration in pyruvate flux through such pathways. Tissue hypoxia in the three patients with circulatory failure could account for this finding, but such an explanation is lacking in the remaining patients. Although the alanine to pyruvate ratio in the patients with lactic acidosis (Table I) fell within the normal range* in two, it was decreased in one and elevated in three. Those with the elevated alanine to pyruvate ratios had the highest alanine concentrations. A further factor may have contributed to the increase in alanine in these instances, namely, the possibility of reduced redox state of all cellular compartments. The lactate to pyruvate ratio reflects only the redox potential of the cytosol, since lactate dehydrogenase is cytoplasmic. The following derivation raises the likelihood that the mitochondrial redox state may be similarly disturbed in lactic acidosis. The alanine to pyruvate ratio is indirectly a “redox couple” as indicated from the reactions catalyzed by glutamate dehydrogenase (GDH, EC 1.4.1.3., equation 1) and alanine aminotransferase (T., equation 2). Glutamate
+ NAD = a-ketoglutarate
+
NADH
+
NH,+
(1)
Kc,,= km, = [glutamate] [or-ketoglutarate]
[NAD] [NADH]
[NH,+]
(la)
* From the unpublished data of P. Felig and J. Wahren: normal alanine to pyruvate ratio in blood is 3.3 2 0.1 (mean -e SEM), range 2.5 to 3.8, as determined in eighteen nmormalsubjects.
Volume 52, April 1972
Glutamate
+
IN LACTIC ACIDOSIS -
pyruvate
MARLISS
ET AL.
Z+
a-ketoglutarate
+
alanine
By substituting the value for [glutamate]/[a-ketoglutarate] from equation 2a into equation la, following is obtained: K,
[N,ADH]
* Cm, -
[NH,+]
WADI
=
lIalaninel cpyruvatet~
(2)
the
(3)
Thus the ratio of alanine to pyruvate concentration reflects the redox couple of the glutamate dehydrogenase reaction if (NH4+) remains constant. An equilibrium constant for this “alanine dehydrogenase” system has been determined for rat liver [21]. This system might be expected to reflect prijmarily mitochondrial redox state, since glutamate dehydrogenase is a mitochondrial enzyme [22]. In the two patients in whom diabetic ketoacidosis and lactic acidosis occurred concurrently an elevated beta-hydroxybutyrate to acetoacetate ratio has been reported previously [8]. This ratio also reflects mitochondrial redox state [22]. The elevation of the redox ratios of these two mitochondrial systems in blood is consistent with the findings of Brosnan, Krebs and Williamson [21] who demonstrated a similar increase, but with maintenance of the equilibrium between the two systems, in ischemic rat liver. The appearance of elevated alanine to pyruvate ratios in only half the patients, not correlated with the magnitude of elevation in lactate to pyruvate ratio, and occurring in only one of the two patients with ketoacidosis, is unexplained. Nonetheless, these two preliminary lines of evidence support the contention that mitochondrial as well as cytoplasmic redox state may be reflected by the ratios of these metabolites in blood. Inasmuch as an elevation was noted in the concentration of fourteen amino acids in addition to alanine, including eight which are essential, factors other than increased amino acid formation and release must be invoked to account for the hyperaminoacidemia of lactic acidosis. Since hepatic uptake is a primary route of alanine disposal, the possibility of an hepatic component in causing the high alanine levels was assessed by use of the isolated, perfused liver technic. It was shown that the elevated concentra-
479
AMINO
ACIDS IN LACTIC ACIDOSIS-
MARLISS ET AL.
tions of lactate and pyruvate could inhibit alanine uptake and its metabolism to glucose and carbon dioxide. Whether either lactate or pyruvate in high concentrations could individually induce the same response was not examined, and therefore precise definition of the mechanism of the effect observed is not possible. However, these studies do permit the extrapolation that part of the increase in alanine concentration in vivo could be on the basis of decreased hepatic utilization secondary to the marked increase in lactate and pyruvate concentrations. These would act presumably via increased cellular pyruvate ‘concentrations interfering with alanine conversion to pyruvate. The same might apply for proline, tyrosine, methionine and phenylalanine, whose uptake by the perfused liver also appeared to be inhibited by the presence of high lactate and pyruvate concentrations, although the mechanism is less apparent in these instances. The association of abnormal amino acid metabolism with other clinical disorders involving lactate and pyruvate has recently become apparent. Hyperalaninemia and alaninuria have been reported in an infant during recurrent episodes of lactic and pyruvic acidosis of unknown cause, which responded to thiamine therapy [23]. Similarly, inappropriately elevated alanine levels accompanied the high lactate and pyruvate levels after twenty-four hours of fasting in an adult with chronic lactic acidosis described by Sussman et al. [24]. This same patient had slightly elevated fasting ratios of beta-hydroxybutyrate to acetoacetate by comparison to normal subjects [25], and the alanine to pyruvate ratios were also elevated. The decrease in plasma arginine concentration and the absence of an increase in ornithine and citrulline in the present study suggest that hepatic urea cycle activity did not increase consonant with the increased demand for amino acid catabolism. Plasma concentrations of the latter two of these urea cycle intermediates are probably determined by hepatic release and peripheral disposal rather than the reverse, as obtains for most of the remaining amino acids [4,7]. Such an alteration in urea cycle activity could result in part from inhibition of alanine uptake by the high concentrations
of lactate and pyruvate, as already discussed. Alternatively, diminished splanchnic perfusion with decreased presentation of amino acids to the liver could also account for a generalized hyperaminoacidemia in association with a relative or absolute decline in the level of urea cycle intermediates. In either case, current data suggest that alterations in hepatic disposal of amino acids whether due to primmary inhibition of hepatic amino acid metabolism or consequent to decreased splanchnic perfusion, contribute to their accumulation in plasma. An alternative or contributory explanation for the relatively lower urea cycle amino acids lies again in the altered redox state, in which a decrease in aspartate concentration might occur consequent upon altered malate-oxaloacetate ratios. Since the cellular aspartate concentrations approach the K, of the argininosuccinate synthetase step [26], any decrease in aspartate could be Iilmiting for arginine synthesis in the urea cycle, and thus the operation of the cycle affected. The in vitro studies of ischemic rat liver have demon-, strated such a decrease in aspat-tate concentration
WI.
Finally, the levels of serine may be accounted for by its unique behavior with respect to muscle and to the hepatic gluconeogenic pathway. Felig and Wahren [27] have recently demonstrated a net uptake by leg muscle. Serine has also been demonstrated to be released by kidney into the renal vein [7,28]. It is unique among the glucogenie amino acids in that its carbon skeleton has recently been shown to enter the gluconeogenic pathway above pyruvate [29], and thus ‘may not be susceptible to the same inhibitory influences as the remaining amino acids. ACKNOWLEDGMENT We express our gratitude to the following for their excellent technical assistance in the performance of these studies: Misses Patricia Barlas, Adele Rymut, Mrs. Dzidra Rumba and Mr. Lynn Lowe. Dr. John L. Ohman, Jr., gave invaluable assistance in the study of two of the subjects.
REFERENCES 1.
2.
3. 4.
480
Marliss EB, Aoki TT, Felig P, Huckabee
WE, Cahill GF Jr: Altered amino acid metabolism in lactic acidosis. Diabetes 19: 355, 1970. Huckabee WE: Abnormal resting blood lactate. II. Lactic acidosis. Amer J Med 30: 840, 1961. Oliva PB: Lactic acidosis. Amer J Med 48: 309, 1970. Felig P, Pozefsky T, Marliss E, Cahill GF Jr: Alanine:
5.
6.
key role in gluconeogenesis. Science 167: 1003, 1970. Mallette LE, Exton JH, Park CR: Control of gluconeogenesis from amino acids in the perfused rat liver. J Biol Chem 244: 5713, 1969. Felig P, Wahren J: Evidence for a glucose-alanine cycle: amino acid metabolism during muscular exercise. J Clin Invest 49: 282, 1970.
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Felig P, Owen OE, Wahren J, Cahill GF Jr: Amino acid metabolism during prolonged starvation. J Clin Invest 48: 584, 1969. Marliss EB, Ohman JL Jr, Aoki lT, Kozak GP: Altered redox state obscuring ketoacidosis in diabetics with lactic acidosis. New Eng J Med 283: 978, 1970. Miller LL: Some direct actions of insulin, glucagon and hydrocortisone on the isolated, perfused rat liver. Recent Proer Horm,one Res 17: 539. 1961. Ruderman NE, Tolws CJ, Lowy C, Vreelanb I, Shafrir E: l’nhibition of hepatic gluconeogenesis and fatty acid oxidation by pent-Cenoic acid. Amer J Physiol 291: 51, 1970. Striver CR, Davies E, Lamm P: Accelerated selective short colum,n chromatography of neutral and acidic amino acids on a Beckman-Spinco analyzer modified for simultaneous analysis of two samoles. Clin Biochem 1: 179. 1968. Pagfiara AS, Goodman AD: Plasma glutamate in gout: possible role in pathogenesis of hyperuricemia. New Eng J Med 281: 757,1969. Huckabee WE: Abnormal resting blood lactate. I. The significance of hyperlactatemia in hospitalized patients. Amer J Med 30: 833, 1961. Ohman JL, Marliss EB, Aoki TT, Munichoodappa CS, Khanna VV, Kozak GP: The cerebrospinal fluid in diabetic ketoacidosis. New Eng J Med 284: 283, 1970. Snedecor GW: Statistical Methods, 5th ed, Ames, Iowa State College Press, 1956. Huckabee WC: Lactic acidosis. Amer J Cardiol 12: 663, 1963. Berry HN, Schener J: Splanchnic lactic acid metab‘olism in hyperve’ntilation, metabolic acidosis, and shock. Metabolism 16: 537, 1967. Stinebaugh BJ, Austin WH: Acid-base balance. Common sense approach. Arch Intern Med (Chi-
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52, April 1972
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cage) 119: 182, 1967. Huckabee WE: Relationships of pyruvate and lactate during anaerobic metabolism. I-III. J Clin Invest 37: 244, 1958. Cori CF, Cori GT: Glycogen formation in the liver from d- and l-lactic acid. J Biol Chem 81: 389, 1929. Brosnan JT, Krebs HA, Williamson DH: Effects of ischemia on metabolite concentrations in rat liver. Biochem J 117: 91, 1970. Krebs HA: The redox state of ‘nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Advances Enzym Regulat 5: 409, 1967. Gagnon-Brunette M, Hazel B, Striver CR: Thiamine dependent neonatal lactic acidosis. Clin Res 17: 659, 1969. Sussman KE, Alfrey A, Kirsch WM, Zweig P, Felig P, Messner F: Chronic lactic acidosis in an adult. Amer J Med 48: 104, 1970. Cahill GF Jr, Herrera MG, Morean AP, Soeldner JS, Steinke J, Levy PL, Reichard GA Jr, Kipnis DM: Hormone fuel interrelationsh,ip during fasting. J Clin Invest 45: 1751, 1966. Ratner S: Nitrogen transfer from aspartic acid, The Enzymes, 2nd ed, vol 6 (Boyer PD, Lardy H, Myrback K, eds), New York and London, Academic Press, 1960, p 495. Felig P, Wahren J: Interrelationship between amino acid and carbohydrate metabolism during exercise. The glucose-alanine cycle, Muscle Metabolism During Exercise (Pornow B, Sahin B, eds), New York, Plenum Press, 1971, p 143. Pitts RF, Damian AC, MacLeod MB: Synthesis of serine by rat kidney in vivo and in vitro. Amer J Physiol 219: 584, 1970. Ven’eziale CM, Gabrielli F, Kneer N, Lardy HA: Gluconeogenesis from L-serine tin isolated perfused rat livers. Fed Proc 28: 411, 1969.
481
E. B. MARLISS, M.D.* T. T. AOKI, M.D. C. J. TOEWS, M.D.? Boston, Massachusetts P. FELIG, M.D. New Haven, Connecticut J. J. CONNON, M.D.S J. KYNER, M.D.g W. E. HUCKABEE, M.D. G. F. CAHILL, Jr., M.D. Boston, Massachusetts
From the Elliott P. Joslin Research Laboratory and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, the Evans Memorial Department of Clinical Research, Boston University School of Medicine, Boston, Massachusetts 02115, and the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510. This study was supported in part by U.S. Public Health Service Grants AM-09584, AM05077, AM-13526 and HE-06297. It was presented in part at the 30th Annual Meeting of the American Diabetes Association, St. Louis, Missouri, June 13, 14, 1970 [l]. Requests for reprints should be addressed to Dr. G. F. Cahill, Jr., Harvard University Medical School, Department of Medicisne, 170 Pilgrim Road, Boston, Massachusetts 02215. Manuscript received December 24, 1970. * Present address: lnstitut de Biochimie Clinique, Sentier de la Roseraie, Geneva, Switzerland. t Present address: 2083 Highview Drive, Cedar Springs Road, Burlington, Ontario, Canada. 3 Present address: University of Belfast Medical School, Belfast, Ireland. § Present ad,dress: University of Kansas Medical Center, Kansas City, Kansas.
474
individual plasma amino acid levels have been determined in six patients with severe lactic acidosis. A consistently distorted pattern was observed whether the disorder was due to shock or was of the idiopathic type. A sixfold elevation in mean alanine concentration was observed, and a significant correlation of alanine with pyruvate concentrations was demonstrated. Since alanine is normally released from muscle and stoichiometrically extracted by liver, normal formation of alanine from pyruvate is suggested by the findings. However, uptake and metabolism of alanine by isolated, perfused rat liver was significantly inhibited by perfusate concentrations of lactate and pyruvate equivalent to those observed in the patients. Thus the high levels of alanine in lactic acidosis are viewed as consistent with normal or increased peripheral release combined with impaired hepatic disposal. The levels of fifteen of the nineteen amino acids measured were elevated. Of the remaining four, three were intermediates of the urea cycle. The syndrome of metabolic
acidosis with massive accumulation of lactate and an elevated ratio of blood lactate to pyruvate concentrations was described by Huckabee in 1961 as lactic acidosis [2]. Blood lactate concentrations are regulated by synthesis and release by some tissues, including muscle, and uptake by others, predominately liver in a resting steady state. Thus accumulation must result from imbalance between these dynamic processes, that is, increased synthesis, decreased disposal or both [3]. Lactate can be synthesized only from pyruvate, therefore increased tissue lactate release could result only from increased pyruvate availability or increased conversion of available pyruvate. The corollary considerations apply for a ‘decrease in disposal, which can proceed only by reconversion to pyruvate. Thus decreased capacity for pyruvate metabolism via other pathways or decreased conversion of lactate to pyruvate could impede uptake and disposal. Since patients with lactic acidosis have exceedingly high blood pyruvate levels in addition to altered lactate to pyruvate ratios [2], a factor of increased cellular pyruvate concentrations probably contributes to the pathophysiology. The conversion of pyruvate to lactate, in addition to provision of oxidized nicotinamide adenine dinucleotide (NAD), may be viewed as a means of making available 3-carbon moieties not disposed of by other metabolic pathways, which are then
The American
Journal of Medlclne
AMINO
amounts, probably because its central position in many metabolic sequences results in much lower concentrations in cells and hence in blood. One such alternate fate of pyruvate is transamination to alanine. Felig et al. [43 recently demonstrated that pyruvate may be transported from peripheral tissues to the liver as alanine in physiologically significant amounts, as previously inferred by Mallette, Exton and Park [5]. Further, a linear relationship between blood pyruvate and alanine concentrations has been demonstrated both at rest and during exercise in normal subjects [6]. In light of these observations, and of the known stoichiometric relationship between ‘muscle release and hepatic uptake of most amino acids in normal man [4,7], individual plasma amino acid levels have been determined in six patients with documented lactic acidosis. In addition, to test whether the accumulation of lactate and pyruvate I
MARLISS
ET AL.
could interfere with hepatic amino acid disposal, the technic of the isolated, perfused rat liver has been employed using perfusate concentrations of lactate and pyruvate comparable to those demonstrated in the patients described.
recycled to the liver for new glucose syntheses (the afferent limb of the Cori cycle). Pyruvate itself is not normally transported in as large
TABLE
ACIDS IN LACTIC ACIDOSIS -
MATERIALS
AND METHODS
Patients. The patients were hospitalized with marked metabolic acidosis; all had blood lactate concentrations of greater than 15 mmoles/L, pyruvate levels greater than 0.28 mmoles/L and elevated lactate to pyruvate ratios. Four were diabetic: two of these (I.G. and M.L.) were suffering from concurrent ketoacidosis and are described in detail elsewhere [8]. One other (M.M.) was receiving therapy with phenethylbiguanide. Three patients were clinically in a state of circulatory failure (hypotension, peripheral vasoconstriction) and were considered to have “secondary lactic acidosis.” The remaining three showed no signs of circulatory collapse or hypoxemia and were considered to be representative of primary or idiopathic lactic acidosis [2]. Further clinical data are summarized in Table I. The course in all patients followed the usual one for severe lactic
Clinical Data in Patients with Lactic Acidosis
Patient M.L.
J.H.
77 F Idiopathic Diabetes, diarrhea
70 M Idiopathic Anemia, pneumonia, weight loss
I.G.
AgeW) Sex
Type of lactic acidosis Associated
disorders
72 F Idiopathic Diabetes, gastroenteritis
N.G.
M.M. 55 M Secondary Diabetes, myocardial infarction
64 M Secondary Diabetes, gastroenteritis, essential
J.J. 35 M Secondary Acute hemorrhagic pancreatitk
hypertension Vital signs Blood pressure (mm Hg) Pulse rate (per min) Respiratory rate (per min) Temperature (“F) Laboratory data Arterial blood ph PC% (mm Hg) HCQ (mEq/L) POZ (mm
Hgf
Serum Sodium (mEq/L) Chloride (mEq/L) Anion gap (mEq/L) Blood urea nitrogen (mg/lOO ml) Blood glucose (mg/lOO ml) Arterial blood Lactate (mmoles/L) Pyruvate (mmoles/L) Lactate: pyruvate Plasma alanine &moles/L) Alanine: pyruvate
Volume 52, April 1972
110/70 114 24 101.2
7.18 16 6 110
160/70 120 20 98.5
7.20 21 8 97
100/75 100 30 98.4
80/50 42 30 97.0
80/50 100 36 101.2
7.18 10 4 90
7.18 23 8 99
13 3 140
6.98
80/60 56 24 91.0
7.00 28 7 144
139 95 38 57
142
144 113 27 61
147 94 45 ...
139 88 48 103
123
103 31 138
298
311
95
50
144
88
24.2 0.51 48 1,188
15.0 0.28 52 1,336
2.3
4.8
22.6 0.30 76 426 1.3
75 41 21
24.0 0.48 50 1,961
29.0 0.77 38 5,390
27.0 0.33 82 866
4.1
7.0
2.6
475
AMINO
ACIDS IN LACTIC ACIDOSIS -
MARLISS
FT AL.
acidosis [3], and they died between one and three days from the time the samples were taken. Normal values for plasma amino acids were obtained under basal conditions in ten postabsorptive nonobese subjects with normal intravenous glucose tolerance (K >1.2 per cent as absolute per cent disappearance per minute, after the administration of 0.5 gmlkg). Similar conditions applied to eight different subjects in whom normal values were obtained for arterial blood lactate (0.910 * 0.155 mmoles/L, mean + SEM), pyruvate (0.075 f 0.010 mmoles/L) and lactate to pyruvate ratios (12.7 2 1.5). Rat Liver Perfusion Studies. Livers from male Sprague-Dawley rats (200 to 250 gm) fasted overnight (P<.OS.
ALA
PA)
ML
LVS LEU SLY
UNPAIREO
TAU TM)
1)
ILE
mrantSEN 0
LactlC ACLAW (n41
I
Normdr
(n*IOl
HbS TYR O+lt$hEl BUT
PME
Figure 1. Plasma amino acid levels that are significantly elevated in six patients with lactic acidosis. Values for normal subjects are shown for comparison. Mean & standard error of mean (SEM).
were perfused for sixty minutes in a modified Miller apparatus [9] with Krebs-Ringer bicarbonate buffer at pH 7.4 having the same amino acid concentration as found in the plasma of the patients with lactic acidosis. The amino acids were obtained from Nutritional Biochemicals Corp. (Cleveland, Ohio) and Sigma Chemical Co. (St. Louis, MO.). Eight livers were perfused with a medium containing high lactate and pyruvate concentrations which were maintained by their constant infusion into the perfusate. Eight control livers were perfused without the addition of lactate and pyruvate to the perfusate. Oxygenation was maintained with 95 per cent oxygen and 5 per cent carbon dioxide in both groups. The conversion of L-alanine-U-l% (New England Nuclear Corp., Boston, Mass.) added initially to the medium, to *%-oxygen and 14Cglucose, and the perfusion technic, materials used and enzymatic fluorometric methods for the assay of metabolites are as previously described [lo]. Individual amino acid levels were deterMethods. mined by automated ion exchange chromatography using a Beckman 120C amino acid analyzer (Beckman Instrument Co., Palo Alto, Calif.), modified to provide simultaneous recording from two columns [ll]. Plasma and perfusate samples were deproteinized with sulfosalicylic acid as described previously [7]. In three instances deproteinization of plasma samples was performed after storage at 20°C and therefore values for half cystine are not reported. The sulfosalicylic acid filtrates were immediately frozen and stored at -20°C until analysis. Glutamine, asparagine and glutamate levels were not reliably determined by this method. Plasma glutamine was determined in three of the subjects within three days of sampling by an enzymatic method employing glutaminase [12]. Arterial blood was immediately deproteinized for determination of lactate and pyruvate content by either enzymatic fluorometric methods on perchloric acid fil-
meon? SEM
160NS
0
Loctlc
n
Normols
Actdos~s
(n=6)
h=IO)
140 ALANINE pHlL
IZO@IL tooNS
PC.OS
NS
80. 604020-
hil.ORN
Plasma amino Figure 2. decreased in six patients sented as in Figure 1.
476
A
Cl1
acid levels that are normal or with lactic acidosis. Data pre-
0’
*
4
*
1002003co400soo600700mo900 BLOOD PYRUVATE CM/L
Figure 3. Correlation of simultaneous arterial blood pyruvate and venous plasma alanine concentrations in lactic acidosis.
The
American
Journal of Medicine
AMINO
trates [lo] chloroacetic parameters
or by modified chemical methods on triacid filtrates [13] or both. Acid-base and standard clinical blood chemistry analy-
ses were performed as previously reported [14]. Statistical analyses were performed according method of Snedecor [15].
to the
RESULTS Patients with Lactic Acidosis. Markedly elevated levels of lactate and pyruvate and an increased lactate to pyruvate ratio were observed in all patients, as compared with the normal subjects (Table I). Plasma individual amino acids showed a consistently distorted pattern. Striking elevations of three to six times normal were observed for alanine, proline, ;I-amino-N-butyric acid, lysine, methionine, isoleucine, leucine and taurine (p ~0.02, unpaired t test). Less marked but sig nificant elevations were noted for threonine, glytine, valine, phenylalanine, tyrosine and histidine (p ~0.05). These values, with normal levels for comparison, are depicted in Figure 1. Glutamine levels in the three subjects in whom they were determined were moderately elevated (840 4 20 pmoles/L, normal 584 fi 43 pmoles/L). The levels of the remaining amino acids are shown in Figure 2. No alteration was observed in the levels of citrulline, ornithine or serine. The only amino acid which showed a significant decrease was arginine (p cO.05). Alanine concentrations showed the greatest increase (1,966 2 891 mmoles/L + SEM). A sig nificant correlation of alanine and pyruvate concentrations is illustrated in Figure 3 with r = 0.93
ACIDS IN LACTIC ACIDOSIS-MARLISS
ET AL.
bon dioxide and glucose was not simply due to dilution of radioactivity in a much larger intracellular pyruvate pool. Figure 5 shows the amino acid concentrations of the perfusate at the beginning and at sixty minutes, the latter for both the perfusate with and without high lactate and pyruvate concentrations. Although most amino acids were taken up to some degree by the livers perfused with either solution, a difference attributable to the presence of lactate and pyruvate in the medium was apparent only for alanine, proline, tyrosine, methionine and phenylalanine. The magnitude of the inhibition of uptake was greatest
YINIJTES
r
(P
Rat Liver Perfusion Studies. The appearance of labelled carbons in carbon dioxide and in glucose is shown in Figure 4. With lactate and pyruvate in the perfusion medium, the activity of l*C appearing in carbon dioxide from L-alanine-l*C was markedly decreased as compared to that obtained with the perfusate containing no lactate or pyruvate. The differences were highly significant at both thirty and sixty minutes (p
Volume 52, April 1972
30
MINUTES Figure 4. Effect of high perfusate concentrations of lactate and pyruvate upon conversion L-alanineUIT to “CO, (top) and glucose (bottom) in isolated rat livers. Mean r+ SEM.
477
AMINO ACIDS IN LACTIC ACIDOSIS -
MARL&S
ET AL
INITIAL
PERFUSATE
60
MIN..
HIGH
60
MIN..
CONTROL
CONCENTRATION
LACTATE
+ PVRUVATE
In = 5)
I600 tn = 5)
(HwniSEY)
Figure 5. Effect of high perfusate concentrations of lactate and pyru-
600 400 ‘ml
lM)s
l.001
200 0 ALA
PRO
VAL
LVS
LEU
GLV
TAU
THR
ILE
HIS
TVR
MET
for alanine, which showed a fourfold difference in concentration (265 & 46 Pmoles/L in the livers perfused without lactate and pyruvate in the perfusate and 959 2 79 Pmoles/L in the livers perfused with high lactate and pyruvate concentrations in the perfusate). The smallest proportional uptakes were for valine, leucine and isoleucine (less than 20 per cent). Citrulline and ornithine were taken up to the extent of about 30 per cent, arginine uptake was greater than 60 per cent. The remaining amino acids were taken up by 40 to 60 per cent, except for histidine which was taken up by 80 per cent. Lactate concentrations of the perfusate were maintained at 25 to 27 mmoles/L and pyruvate concentrations at 0.43 to 0.97 mmoles/L by the rates of infusion employed, resulting in lactate to pyruvate ratios of 30 to 70. In the control group, the lactate concentration in the perfusate throughout perfusion was 0.6 to 1.5 mmoles/L and pyruvate concentration less than 0.05 mmoles/L. Glucose production was significantly greater in the livers perfused with the medium containing high lactate and pyruvate concentrations at all periods: at thirty and sixty minutes, control values were 9 and 15 Pmoles/gm, whereas those perfused with high lactate-pyruvate solutions were 31 and 50 rmoles/gm, respectively. COMMENTS Of the subjects reported, three represent lactic acidosis secondary to circulatory failure (type 2A of Huckabee [13]). The other three may be considered to have suffered from idiopathic lactic acidosis (type 2B) since no evidence of inadequate
478
PHE
SER
ORN
ARC
CIT
vate upon uptake of amino acids by isolated rat livers. Initial amino acid concentrations were comparable to those in the lactic acidosis patients. See text for discussion. Mean + SEM.
tissue perfusion or hypoxemia was present at the time the samples were taken. Despite this difference, the acid-base, lactate and pyruvate, and amino acid parameters were comparable. Thus the data demonstrate further similarities between the two disorders, but specific conclusions regarding the etiology of idiopathic lactic acidosis are not warranted. The data are consistent with the hypothesis of Huckabee of inapparent and inefficient redistribution of blood flow [16], and would suggest an important component of hepatic incapacity to maintain metabolic homeostasis. Indeed, net hepatic release of lactate [17] and of pyruvate and amino acids is not excluded. Five of the patients described showed marked metabolic acidosis, with appropriate respiratory compensation, but one (J.J.) demonstrated inadequate compensation and thus had combined metabolic and respiratory components [18]. All had markedly elevated blood lactate and pyruvate levels and lactate to pyruvate ratios (Table I), reflecting an altered cellular oxidation-reduction state [ 191. The plasma alanine level has been shown to be regulated in the postabsorptive state primarily by the rates of release from muscle and uptake by the liver, for which a near stoichiometric relationship exists [4,7]. Alanine is quantitatively important as a substrate for hepatic gluconeogenesis, and glucose so synthesized may be recycled to the periphery, in an “alanine cycle” analogous to the Cori cycle [20]. These two cycles are closely interrelated, via the reversible transamination step between pyruvate and alanine, catalyzed by alanine aminotransferase (EC 2.6.1.2).
The American
Joumel
of Medicine
AMINO ACIDS
It has recently been shown that for the effluent from limb muscle, a linear correlation exists between alanine and pyruvate concentrations at rest and during exercise in normal subjects [6]. Hence, the demonstration of a sirmilar correlation between alanine and pyruvate in the current study (Figure 3) suggests that formation of alanine from pyruvate remains intact in lactic acidosis. The disproportionate elevation in alanine levels with respect to the other amino acids could be a consequence of increased intracellular pyruvate levels, which may be inferred from the markedly elevated blood pyruvate concentration. A shift of cellular redox potential toward the reduced state would be expected to diminish pyruvate levels in the absence of altered pyruvate synthesis (from glycolysis or transamination of alanine) or disposal (via the tricarboxylic acid cycle). Thus the elevated levels must reflect an alteration in pyruvate flux through such pathways. Tissue hypoxia in the three patients with circulatory failure could account for this finding, but such an explanation is lacking in the remaining patients. Although the alanine to pyruvate ratio in the patients with lactic acidosis (Table I) fell within the normal range* in two, it was decreased in one and elevated in three. Those with the elevated alanine to pyruvate ratios had the highest alanine concentrations. A further factor may have contributed to the increase in alanine in these instances, namely, the possibility of reduced redox state of all cellular compartments. The lactate to pyruvate ratio reflects only the redox potential of the cytosol, since lactate dehydrogenase is cytoplasmic. The following derivation raises the likelihood that the mitochondrial redox state may be similarly disturbed in lactic acidosis. The alanine to pyruvate ratio is indirectly a “redox couple” as indicated from the reactions catalyzed by glutamate dehydrogenase (GDH, EC 1.4.1.3., equation 1) and alanine aminotransferase (T., equation 2). Glutamate
+ NAD = a-ketoglutarate
+
NADH
+
NH,+
(1)
Kc,,= km, = [glutamate] [or-ketoglutarate]
[NAD] [NADH]
[NH,+]
(la)
* From the unpublished data of P. Felig and J. Wahren: normal alanine to pyruvate ratio in blood is 3.3 2 0.1 (mean -e SEM), range 2.5 to 3.8, as determined in eighteen nmormalsubjects.
Volume 52, April 1972
Glutamate
+
IN LACTIC ACIDOSIS -
pyruvate
MARLISS
ET AL.
Z+
a-ketoglutarate
+
alanine
By substituting the value for [glutamate]/[a-ketoglutarate] from equation 2a into equation la, following is obtained: K,
[N,ADH]
* Cm, -
[NH,+]
WADI
=
lIalaninel cpyruvatet~
(2)
the
(3)
Thus the ratio of alanine to pyruvate concentration reflects the redox couple of the glutamate dehydrogenase reaction if (NH4+) remains constant. An equilibrium constant for this “alanine dehydrogenase” system has been determined for rat liver [21]. This system might be expected to reflect prijmarily mitochondrial redox state, since glutamate dehydrogenase is a mitochondrial enzyme [22]. In the two patients in whom diabetic ketoacidosis and lactic acidosis occurred concurrently an elevated beta-hydroxybutyrate to acetoacetate ratio has been reported previously [8]. This ratio also reflects mitochondrial redox state [22]. The elevation of the redox ratios of these two mitochondrial systems in blood is consistent with the findings of Brosnan, Krebs and Williamson [21] who demonstrated a similar increase, but with maintenance of the equilibrium between the two systems, in ischemic rat liver. The appearance of elevated alanine to pyruvate ratios in only half the patients, not correlated with the magnitude of elevation in lactate to pyruvate ratio, and occurring in only one of the two patients with ketoacidosis, is unexplained. Nonetheless, these two preliminary lines of evidence support the contention that mitochondrial as well as cytoplasmic redox state may be reflected by the ratios of these metabolites in blood. Inasmuch as an elevation was noted in the concentration of fourteen amino acids in addition to alanine, including eight which are essential, factors other than increased amino acid formation and release must be invoked to account for the hyperaminoacidemia of lactic acidosis. Since hepatic uptake is a primary route of alanine disposal, the possibility of an hepatic component in causing the high alanine levels was assessed by use of the isolated, perfused liver technic. It was shown that the elevated concentra-
479
AMINO
ACIDS IN LACTIC ACIDOSIS-
MARLISS ET AL.
tions of lactate and pyruvate could inhibit alanine uptake and its metabolism to glucose and carbon dioxide. Whether either lactate or pyruvate in high concentrations could individually induce the same response was not examined, and therefore precise definition of the mechanism of the effect observed is not possible. However, these studies do permit the extrapolation that part of the increase in alanine concentration in vivo could be on the basis of decreased hepatic utilization secondary to the marked increase in lactate and pyruvate concentrations. These would act presumably via increased cellular pyruvate ‘concentrations interfering with alanine conversion to pyruvate. The same might apply for proline, tyrosine, methionine and phenylalanine, whose uptake by the perfused liver also appeared to be inhibited by the presence of high lactate and pyruvate concentrations, although the mechanism is less apparent in these instances. The association of abnormal amino acid metabolism with other clinical disorders involving lactate and pyruvate has recently become apparent. Hyperalaninemia and alaninuria have been reported in an infant during recurrent episodes of lactic and pyruvic acidosis of unknown cause, which responded to thiamine therapy [23]. Similarly, inappropriately elevated alanine levels accompanied the high lactate and pyruvate levels after twenty-four hours of fasting in an adult with chronic lactic acidosis described by Sussman et al. [24]. This same patient had slightly elevated fasting ratios of beta-hydroxybutyrate to acetoacetate by comparison to normal subjects [25], and the alanine to pyruvate ratios were also elevated. The decrease in plasma arginine concentration and the absence of an increase in ornithine and citrulline in the present study suggest that hepatic urea cycle activity did not increase consonant with the increased demand for amino acid catabolism. Plasma concentrations of the latter two of these urea cycle intermediates are probably determined by hepatic release and peripheral disposal rather than the reverse, as obtains for most of the remaining amino acids [4,7]. Such an alteration in urea cycle activity could result in part from inhibition of alanine uptake by the high concentrations
of lactate and pyruvate, as already discussed. Alternatively, diminished splanchnic perfusion with decreased presentation of amino acids to the liver could also account for a generalized hyperaminoacidemia in association with a relative or absolute decline in the level of urea cycle intermediates. In either case, current data suggest that alterations in hepatic disposal of amino acids whether due to primmary inhibition of hepatic amino acid metabolism or consequent to decreased splanchnic perfusion, contribute to their accumulation in plasma. An alternative or contributory explanation for the relatively lower urea cycle amino acids lies again in the altered redox state, in which a decrease in aspartate concentration might occur consequent upon altered malate-oxaloacetate ratios. Since the cellular aspartate concentrations approach the K, of the argininosuccinate synthetase step [26], any decrease in aspartate could be Iilmiting for arginine synthesis in the urea cycle, and thus the operation of the cycle affected. The in vitro studies of ischemic rat liver have demon-, strated such a decrease in aspat-tate concentration
WI.
Finally, the levels of serine may be accounted for by its unique behavior with respect to muscle and to the hepatic gluconeogenic pathway. Felig and Wahren [27] have recently demonstrated a net uptake by leg muscle. Serine has also been demonstrated to be released by kidney into the renal vein [7,28]. It is unique among the glucogenie amino acids in that its carbon skeleton has recently been shown to enter the gluconeogenic pathway above pyruvate [29], and thus ‘may not be susceptible to the same inhibitory influences as the remaining amino acids. ACKNOWLEDGMENT We express our gratitude to the following for their excellent technical assistance in the performance of these studies: Misses Patricia Barlas, Adele Rymut, Mrs. Dzidra Rumba and Mr. Lynn Lowe. Dr. John L. Ohman, Jr., gave invaluable assistance in the study of two of the subjects.
REFERENCES 1.
2.
3. 4.
480
Marliss EB, Aoki TT, Felig P, Huckabee
WE, Cahill GF Jr: Altered amino acid metabolism in lactic acidosis. Diabetes 19: 355, 1970. Huckabee WE: Abnormal resting blood lactate. II. Lactic acidosis. Amer J Med 30: 840, 1961. Oliva PB: Lactic acidosis. Amer J Med 48: 309, 1970. Felig P, Pozefsky T, Marliss E, Cahill GF Jr: Alanine:
5.
6.
key role in gluconeogenesis. Science 167: 1003, 1970. Mallette LE, Exton JH, Park CR: Control of gluconeogenesis from amino acids in the perfused rat liver. J Biol Chem 244: 5713, 1969. Felig P, Wahren J: Evidence for a glucose-alanine cycle: amino acid metabolism during muscular exercise. J Clin Invest 49: 282, 1970.
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Journal
of Medicine
AMINO
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Felig P, Owen OE, Wahren J, Cahill GF Jr: Amino acid metabolism during prolonged starvation. J Clin Invest 48: 584, 1969. Marliss EB, Ohman JL Jr, Aoki lT, Kozak GP: Altered redox state obscuring ketoacidosis in diabetics with lactic acidosis. New Eng J Med 283: 978, 1970. Miller LL: Some direct actions of insulin, glucagon and hydrocortisone on the isolated, perfused rat liver. Recent Proer Horm,one Res 17: 539. 1961. Ruderman NE, Tolws CJ, Lowy C, Vreelanb I, Shafrir E: l’nhibition of hepatic gluconeogenesis and fatty acid oxidation by pent-Cenoic acid. Amer J Physiol 291: 51, 1970. Striver CR, Davies E, Lamm P: Accelerated selective short colum,n chromatography of neutral and acidic amino acids on a Beckman-Spinco analyzer modified for simultaneous analysis of two samoles. Clin Biochem 1: 179. 1968. Pagfiara AS, Goodman AD: Plasma glutamate in gout: possible role in pathogenesis of hyperuricemia. New Eng J Med 281: 757,1969. Huckabee WE: Abnormal resting blood lactate. I. The significance of hyperlactatemia in hospitalized patients. Amer J Med 30: 833, 1961. Ohman JL, Marliss EB, Aoki TT, Munichoodappa CS, Khanna VV, Kozak GP: The cerebrospinal fluid in diabetic ketoacidosis. New Eng J Med 284: 283, 1970. Snedecor GW: Statistical Methods, 5th ed, Ames, Iowa State College Press, 1956. Huckabee WC: Lactic acidosis. Amer J Cardiol 12: 663, 1963. Berry HN, Schener J: Splanchnic lactic acid metab‘olism in hyperve’ntilation, metabolic acidosis, and shock. Metabolism 16: 537, 1967. Stinebaugh BJ, Austin WH: Acid-base balance. Common sense approach. Arch Intern Med (Chi-
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cage) 119: 182, 1967. Huckabee WE: Relationships of pyruvate and lactate during anaerobic metabolism. I-III. J Clin Invest 37: 244, 1958. Cori CF, Cori GT: Glycogen formation in the liver from d- and l-lactic acid. J Biol Chem 81: 389, 1929. Brosnan JT, Krebs HA, Williamson DH: Effects of ischemia on metabolite concentrations in rat liver. Biochem J 117: 91, 1970. Krebs HA: The redox state of ‘nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Advances Enzym Regulat 5: 409, 1967. Gagnon-Brunette M, Hazel B, Striver CR: Thiamine dependent neonatal lactic acidosis. Clin Res 17: 659, 1969. Sussman KE, Alfrey A, Kirsch WM, Zweig P, Felig P, Messner F: Chronic lactic acidosis in an adult. Amer J Med 48: 104, 1970. Cahill GF Jr, Herrera MG, Morean AP, Soeldner JS, Steinke J, Levy PL, Reichard GA Jr, Kipnis DM: Hormone fuel interrelationsh,ip during fasting. J Clin Invest 45: 1751, 1966. Ratner S: Nitrogen transfer from aspartic acid, The Enzymes, 2nd ed, vol 6 (Boyer PD, Lardy H, Myrback K, eds), New York and London, Academic Press, 1960, p 495. Felig P, Wahren J: Interrelationship between amino acid and carbohydrate metabolism during exercise. The glucose-alanine cycle, Muscle Metabolism During Exercise (Pornow B, Sahin B, eds), New York, Plenum Press, 1971, p 143. Pitts RF, Damian AC, MacLeod MB: Synthesis of serine by rat kidney in vivo and in vitro. Amer J Physiol 219: 584, 1970. Ven’eziale CM, Gabrielli F, Kneer N, Lardy HA: Gluconeogenesis from L-serine tin isolated perfused rat livers. Fed Proc 28: 411, 1969.
481