Pathways of reducing equivalents in hepatocytes from starved, ethanol-induced, and hyperthyroid rats during ethanol and xylitol metabolism

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 211, No. 2, October 15, pp. 697-708, 1981

Pathways

of Reducing Equivalents in Hepatocytes from Starved, Ethanol-Induced, and Hyperthyroid Rats during Ethanol and Xylitol Metabolism CONSTANCE VIND

AND

NIELS GRUNNET

Department of Biochemistry A, University of Copenhagen, DK 2200 Copenhagen N, Denmark Received January

8, 1981

The metabolism of [2-SH]xylitol was studied in the absence or presence of ethanol in cells from 24-h-starved rats, either untreated, Ts treated, or ethanol induced. The relative yields of tritium in water, glucose, and lactate were determined. A simple metabolic model is proposed, from which the specific radioactivity of cytosolic NADH, flux through lactate dehydrogenase, and the relative yield of tritium in water and glucose can be calculated. The model assumes one cytosolic NAD pool and metabolic and isotopic steady state. The measured incorporation of tritium into glucose indicate that the 4A- and 4Bhydrogens of NADH are not fully equilibrated. The specific radioactivity of the CZ-hydrogen of lactate does not parallel that of cytosolic NADH as calculated from the incorporation of tritium into lactate and the total production of reducing equivalents in the cystosol. The flux through lactate dehydrogenase was calculated from the specific radioactivities of NADH and lactate to be l-6 amol/min g wet wt. In cells from Ta-treated and ethanol-induced rats, the relative yield of tritium in water was higher when xylitol was metabolized in the presence of ethanol than in its absence. Furthermore, the specific radioactivity of cytosolic NADH was not decreased as much as expected by ethanol addition. These results are taken as evidence for the presence of a microsomal ethanol oxidation system using NADH as an electron donor in Ta-treated and ethanol-induced animals.

Xylitol is rapidly metabolized in hepatocytes by a cytosolic NAD-linked xylitol dehydrogenase (xylitol: NAD oxidoreductase, EC 1.1.1.9) (1) with glucose as the main carbon end product. Glucose formation from D-Xy]U]OSt?, the product of NAD-xylitol dehydrogenase, involves phosphorylation of the pentose by ATP to D-xylulose &phosphate, which is converted to fructose 6-phosphate and triose phosphates by the transaldolase and transketolase reactions of the pentose phosphate cycle (2). Gluconeogenesis from xylitol is (unlike, e.g., gluconeogenesis from lactate) associated with the net generation of NADH in the cytosol. Reoxidation of cytosolic NADH, therefore, is an obligatory step for xylitol metabolism as well as for ethanol oxidation. Three different reaction mechanisms

for oxidation of ethanol to acetaldehyde have been described in liver tissue. These are the pathway catalyzed by the NADdependent alcohol dehydrogenase, the pathway involving hydrogen peroxide and catalase, and the microsomal ethanol-oxidizing system (MEOS)’ involving NADPH, cytochrome P-450 (3), and possibly NADH (4-6). The quantitatively most important pathway, catalyzed by alcohol dehydrogenase, is localized in the cytoplasmic compartment of the cell, while the system dependent on cytochrome P-450 is microsome bound. Xylitol oxidation is similar to ethanol metabolism as regards the rate-limiting step (7) and the equilibrium constants of xylitol dehydrogenase and alcohol dehy’ Abbreviations used: MEOS, microsomal oxidizing system; EtOH, ethanol.

697

ethanol-

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0 1981 by Academic Press. Inc. of reproduction in any form reserved.

698

VIND AND GRUNNET

drogenase are similar (8,9). However, xylitol is a donor of reducing equivalents exclusively to cytosolic NAD+, whereas studies of ethanol metabolism are complicated by the formation of acetaldehyde, which is a very efficient donor of reducing equivalents directly to mitochondrial NAD+. Thus, results obtained by the use of [23H]xylitol provide basic information on the intracellular pathways of reducing equivalents, which is of relevance also to problems of ethanol utilization by the liver (10). Studies of the metabolism of [23H]xylitol in the presence of ethanol may provide information of the interaction between the two dehydrogenases. In the present paper the metabolism of [2-3H]xylitol is studied in isolated hepatocytes from untreated, T3-treated, and chronic ethanol-fed rats. T3-treated and ethanol-induced rats were used in an attempt to increase the utilization of reducing equivalents. Israel et al. (11) have reported that some similarities may exist between livers of alcohol-treated animals and livers from hyperthyroid animals especially since hepatic uptake of thyroxine was found to be increased following chronic alcohol consumption. Moreno et al. (12) suggest that in the “hyperthyroid hepatic state” created by chronic alcohol consumption some of the changes are due to an induction of MEOS activity. The results obtained with [2-3H]xylitol as the labeled substrate are used to estimate the flux of reducing equivalents through lactate dehydrogenase and the specific radioactivity of NADH and the influence of some dietary and hormonal treatments on these parameters. From these data we calculate the expected relative yield of tritium in water and glucose and compare them with our experimental results. Furthermore, indication for the involvment of the microsomal ethanol-oxidizing system in the reoxidation of NADH during ethanol metabolism is obtained. MATERIALS Enzymes and coenzymes were from Boehringer, Mannheim GmbH (Mannheim, W. Germany). [l,l-

‘HalEthanol was purchased from NEN Chemicals GmbH (Frankfurt/Main, W. Germany). D-Kylulose was kindly provided by Dr. B. Stig Enevoldsen (Carlsberg Research Center, Copenhagen, Denmark). Chemicals were of analytical grade. Serum albumin from Armour Pharmaceutical Company (Eastbourne, England) was treated with charcoal to remove fatty acids (13). Dowex I was from Fluka AG (Buchs, Schweiz), DEAE-Sephadex A-25 from Pharmacia Fine Chemicals (Uppsala, Sweden), and Instafluor from Packard (Groningen, The Netherlands). Liquid ethanol diet was from Bio-Serv, Inc. (Frenchtown, N. J.). METHODS Treatment ofrats. Female rats of the Wistar strain weighing 150-200 g, either untreated, Tr-treated, or EtOH-induced were used after 24 h starvation. The Ts-treated rats were obtained by intraperitoneal injection with L-3,3’,5-triiodothyronine (sodium salt from Sigma). An injection of 50 ~g/lOO g body wt dissolved in 0.5 ml alkali was given every second day, for a total of two or three doses. The last injection was given and the food was withdrawn 24 h prior to cell preparation (14). The ethanol-induced rats were fed a liquid ethanol diet (15) containing in terms of energy: 35% fat, 11% carbohydrate, 18% protein, and 36% ethanol. The rats were switched to the liquid diets when weighing 130-140 g and diets were administered for 4-5 weeks. Preparation ofhepatocytes. Cells were prepared as described (16). More than 90% of the cells excluded Trypan blue. Incubation conditions. The experiments were carried out in the presence of a physiological concentration of glucose and with the lactate-to-pyruvate ratio poised at a value of 10 by addition of lactate and pyruvate. Packed cells (0.1 ml; approx. 0.11 g wet wt) were incubated at 37°C in Krebs-Henseleit bicarbonate buffer containing 1% serum albumin and 7.5 mru glucose in a total volume of 2.25 ml. The gas phase was 95% Oa + 5% COr. The cells were incubated with 2.5 mM lactate and 0.25 mM pyruvate for 5 min prior to the ethanol and xylitol addition. After incubation with 6 mM [2-3H]xylitol (specific radioactivity ca. 100 cpm/nmol) with or without 7 mM ethanol for 0, 15, 30, and 45 min, the reaction was stopped by addition of perchloric acid to 0.7 M. The supernatant was neutralized by KOH containing triethanolamine. of the distribution of tritium from Determination [Z-‘Hjxglitol. The neutralized perchloric acid supernatant was used for isolation of water, glucose, lactate, and xylitol. Radioactivity in water was obtained as the difference between total radioactivity and the radioactivity in dry matter. In some of the experiments the ethanol was removed by distillation before

PATHWAYS

OF REDUCING

EQUIVALENTS

drying to determine the radioactivity of ethanol. We did not find any tritium in ethanol. Glucose and xylitol were isolated as the neutral fraction from a Dowex I (acetate form) ion-exchange column. Lactate and (3-hydroxybutyrate was eluted with 4 N acetic acid. The tritium content in &hydroxybutyrate was less than 1% of the tritium applied to the column and therefore neglected. Other anions were eluted with 2 N potassium formate, pH 3. By cation-exchange chromatography (Dowex 50) negligible amounts of radioactivity were found in amino acids. Glucose and xylitol were separated by phosphorylation of glucose with ATP and hexokinase and subsequent separation by Dowex I ion-exchange chromatography. Xylitol was collected as the neutral fraction and the phosphorylated glucose was eluated with 2 N potassium formate, pH 3. Samples were counted in a Packard 2425 scintillation spectrometer. 1nstafluor:Triton X-114 (21) was used as the scintillation liquid with a sample volume of 23%. The tritium from the oxidation of [2-8Hlxylitol was recovered in water, lactate, glucose, and the fraction of other anions. The total recovery were between 99 and 110% of the tritium used. The relative amount found in the fraction of other anions was less than 2% and has been neglected in our calculations. The yield of radioactivity was calculated relative to the total yield in lactate, glucose, and water. Preparation of[2-SHjX&ol. [2-‘H]Xylitol was prepared from [4A-3H]NADH and D-XyhhW. with sorbitol dehydrogenase and was isolated by ion-exchange chromatography (Dowex 50 and Dowex I) or by paper chromatography with pyridine/n-butanol/ water (1/4/l, v/v/v) as a solvent (17). [4A-3H]NADH was prepared as described (18). Metabolite levels. Metobolites were determined from the neutralized HCIO, supernatant. Lactate, pyruvate, fl-hydroxybutyrate, acetoacetate, glucose, glycerol 3-phosphate, and ethanol were measured by standard enzymatic procedures (19). The concentration of xylitol was determined with sorbitol dehydrogenase from sheep liver after the method used by Touster and Montesi (20). RESULTS

Metabolic Changes The metabolic changes observed in the present study (Table I) are similar to those reported by others (1, 7, 21, 22). The rate of xylitol oxidation was the same in starved and Ts-treated rats (1.6 and 1.5 pmol/min g wet wt., respectively) and somewhat lower in the ethanol-induced rats (1.1 clmol/min g wet wt). Ethanol did not change the oxidation rate of xylitol in Ts-

DURING

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699

treated rats, but caused a 35-40% inhibition of the xylitol oxidation in starved and ethanol-induced rats. These results indicate that the capacity of the mitochondria to oxidize cytosolic reducing equivalents is only rate limiting for xylitol metabolism in starved and ethanol-induced rats in the presence of ethanol. The larger capacity of mitochondria to oxidize cytosolic NADH in Ts-treated rats is also reflected in the cytosolic NAD-redox state, as judged from the lactate/pyruvate concentration ratio. This ratio was increased three to four times by xylitol metabolism in cells from starved and ethanol-induced rats but unchanged in cells from Ts-treated rats (results not shown). Glucose was the major end product of xylitol in cells from starved and Ts-treated as well as ethanol-induced rats. The rate of gluconeogenesis was correlated to the xylitol oxidation rate and thus decreased in starved and ethanol-induced rats, when xylitol was metabolized in the presence of ethanol. Lactate was taken up by cells from all groups except in those from starved rats, when xylitol was metabolized in the presence of ethanol. The production of glycerol 3-phosphate was increased when xylitol was metabolized together with ethanol in both starved and ethanol-induced rats but was, as anticipated (14), small in T3 rats both in the presence and absence of ethanol. The rate of ethanol oxidation (Tabie II) in fasted rats was 2.3 pmol/min g wet wt (23) and in Ta-treated and ethanol-induced rats around 1.8 pmol/min g wet wt both in the presence and absence of xylitol. Hensgens et al. also found a reduction of 20% in the ethanol oxidation by T3 treatment (24).

Fate of Labeling from [2-SHjX&tol The yield of tritium in water, lactate, and glucose (Table III) shows that the relative amount of tritium found in water in T3-treated and ethanol-induced rats was larger when [2-SH]xylitol and ethanol were metabolized together, than when

induced

-1.2 -2.3 -3.3

-1.7 -3.6 -5.1

-2.3 -4.3 -6.7

-2.4 -4.9 -7.8

-1.6 -2.9 -4.6

represent

+ 0.3 (4) + 0.7 (5) f 0.3 (4)

+ 0.6 (4) f 1.1 (5) f 0.6 (4)

rt 0.5 (7) + 0.7 (8) + 1.2 (4)

k 0.2 (5) + 0.7 (7) + 0.8 (4)

+ 0.5 (8) + 0.9 (9) k 0.9 (4)

Figures

I

CHANGES

f 0.7 (7) 2 1.8 (9)

AXylitol -2.5 -5.0

as described under Methods.

15 30 45

Xylitol

+ ethanol

15 30 45

Xylitol

15 30 45

Xylitol

+ ethanol

15 30 45

Xylitol

15 30 45

Xylitol

+ ethanol

15 30

(min)

Xylitol

Substrate

Note. The experiments were performed Glycerol-P is L-glycerol 3-phosphate.

Ethanol

T8 treated

Starved

Treatment

Time

METABOLIC

TABLE

-0.6 + 0.5 (3) -1.6 k 0.5 (4) -2.4 + 0.5 (4) -0.7 k 0.9 (3) -0.6 rt_ 0.2 (3) -1.2 + 0.4 (4)

(2)

+1.4 (2) +2.7 +_ 0.6 (4) +5.1 f 0.1 (3)

means * SD with the number of cell preparations

+6.1 f 1.2 (4) +8.8 + 0.6 (4)

+3.9

-1.5 + 0.7 (5) -2.7 f 0.5 (7) -3.4 + 0.5 (4)

-0.5 * 0.2 (3) -1.2 + 0.7 (7) -1.4 + 0.5 (4)

+3.1 + 1.0 (6) +6.0 f 1.1 (6) +7.0 f 1.3 (3) +3.7 k 0.8 (5) +6.2 + 1.4 (7) +8.1 + 1.5 (4)

0 0 0

-0.2 + 0.2 (7) -1.3 f 0.6 (6)

ALactate

+2.2 + 0.5 (5) +3.9 + 0.9 (6) +6.4 f 1.4 (4)

+2.2 f 0.7 (7) +6.8 f 2.5 (8)

AGlucose

rtmol10.1 ml packed cells

in parenthesis.

+0.44 f 0.07 (5) +0.49 f 0.06 (4)

-

+0.14 f 0.05 (4) +0.21 ic 0.10 (3)

-

+0.05 f 0.03 (5) +0.06 2 0.05 (4)

-

+0.02 f 0.02 (3)

+0.01 + 0.01 (4)

+0.42 + 0.27 (8) +0.62 + 0.34 (4)

-

+0.16 + 0.04 (5)

-

AGlycerol-P

z rj

5

4 2 a

PATHWAYS

OF REDUCING

EQUIVALENTS TABLE

DURING

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701

II

ETHANOL OXIDATION

Substrate Xylitol

+ ethanol

Ethanol

pmol EtOH/O.l

ml packed cells

Time (min)

Starred

Te treated

EtOH induced

15 30 45

- 3.6 + 0.2 (3) - 6.2 k 0.7 (3) - 8.4 k 0.5 (3)

- 2.8 k 1.1 (7) - 5.5 f 1.2 (8) - 9.7 f 1.6 (4)

-2.8 k 1.6 (4) -6.0 f 2.5 (5) -7.9 z!z 1.6 (4)

15 30 45

- 4.3 + 0.1 (3) - 7.2 k 0.2 (4) -10.0 + 1.4 (3)

- 2.8 f 0.5 (3) - 7.5 + 1.8 (4) -10.7 f 2.1 (3)

-2.7 (2) -4.6 + 1.7 (4) -7.8 k 1.9 (4)

Note. The experiments were performed as described the number of cell preparations in parenthesis.

[2-3H]xylitol was metabolized without ethanol. The difference was largest in cells from T3-treated rats (P < 0.001, Student’s t test). The relative amount of tritium in TABLE

under Methods. Figures

represent

means ? SD with

water increased with time (most in T3 rats, P c 0.001 from 15 to 45), while the relative amount of tritium in lactate decreased and the relative yield of tritium in glucose reIII

THE RELATIVE YIELDS OF TRITIUM IN WATER, LACTATE, AND GLUCOSE FROM [2-‘H]XYLIT~L

Treatment Starved

Substrate

Lactate

Glucose

[2-‘H]Xylitol

15 30 45

60 f 64 + 68 +

7 (6) 5 (7) 8 (3)

33 zt 10 (6) 29 f 8 (7) 22 + 9 (3)

5 f 2 (6) 6 k 2 (8) 10 * 2 (3)

[2+I]Xylitol + ethanol

15 30 45

62 k 11 (7) 63 k 10 (8) 71 f 6 (4)

32 k 9 (7) 29 +- 8 (6) 23 k 8 (4)

5 k 4 (8) 4 * 3 (9) 6 + 4 (4)

15 30 45

49 + 10 (6) 60 + 7 (8) 67 + 3 (4)

40 + 31 k 25 k

8 (6) 5 (8) 4 (4)

8 f 5 (6) 9 ?I 3 (8) 8 + 5 (4)

15 30 45

64 f 78 f 84 *

5 (7) 5 (8) 4 (4)

26 f 14 + 8 2

5 (7) 2 (8) 4 (4)

10 * 5 (7) 8 k 4 (8) 8 k 1 (4)

15 30 45

56 * 65 + 68 +

4 (4) 6 (5) 5 (4)

38 + 26 + 21 +

2 (4) 5 (5) 5 (4)

5 f 3 (4) 9 * 3 (5) 12 * 1 (4)

15 30 45

70 + 72 + 74 +

0 (3) 7 (5) 4 (4)

25 5~ 3 (3) 22 f 7 (5) 18 + 4 (5)

5 + 3 (4) 6 k 2 (5) 8 f 2 (4)

T3 treated

[2-‘HjXylitol + ethanol

Ethanol

Percentage

Time (min)

induced

[2-3H]Xylitol + ethanol

Note. The experiments were performed as described the number of cell preparations in parenthesis.

under Methods. Figures

represent

means + SD with

702

VIND [Z-‘H]

Xylitol

[z-3~]

AND GRUNNET

Xylitol*Ethanol

FIG. 1. The specific radioactivity of lactate (L) (in rmol 3H/pmol) as a function of time (t). The values of K and T were calculated by means of the specific radioactivity of lactate at 15 and 30 min from the equation L = K(l - e-‘/r). The K values in rmol 3H/ rmol were K, = 0.160 f 0.045 (7), K,I = 0.185 f 0.067 (7), K,,I = 0.417 + 0.089 (6), K,v = 0.198 + 0.081 (6), Kv = 0.235 f 0.052 (4), and Kvr = 0.114 + 0.058 (4), and the ‘T values in min were rr = 15 f 5 (7), rn = 31 + 25 (7), 7111= 29 f 13 (6), rrv = 14 f 7 (6), 7v = 25 + 5 (4), and rV1 = 16 f 7 (4) (mean + SD with the number of cell preparations in parenthesis). Lactate specific radioactivity at 45 min is the mean value of four experiments. Bars indicate SD.

mained the same for 45 min, except in cells from starved and ethanol-induced rats when [2-3H]xylitol was metabolized in the absence of ethanol. We did not find any tritium in ethanol from [2-3H]xylitol in cells from either starved, T3-treated, or ethanol-induced rats (results not shown) which shows that when xylitol was metabolized together with ethanol, the use of reducing equivalents in the reduction of acetaldehyde to ethanol (the flux denoted by y in Fig. 2) can be neglected. The labeling pattern of lactate during the incubation can be seen from Fig. 1. The incorporation of tritium in lactate during

[2-3H]xylitol metabolism follows the equation L = K(1 - e-“‘) (pm01 xylitol hydrogen/pmol lactate). From the experimental values of the lactate specific radioactivity at 15 and 30 min, we have calculated the steady-state values K and the initial labeling rates K/T and from those calculated the graphs, given in Fig. 1. The experimental values of lactate specific radioactivity at 45 min are in agreement with the calculated values. The relative specific radioactivity of lactate in cells from starved rats reached a steady state (K) at 0.160 + 0.045 pmol xylitol hydrogen/pmol lactate (n = 7), when [2-3H]xylitol was metabolized alone and a steady-state value about the same, when [2-3H]xylitol was metabolized in the presence of ethanol in spite of the increased amount of unlabeled reducing equivalents available. This might either reflect a nonalcohol dehydrogenase pathway for oxidation of ethanol or be due to a flux through lactate dehydrogenase, which is insufficient to equilibrate lactate rapidly with NAD3H (see Discussion). The Spec&

‘Radioactivity

of NAPH

Flux through lactate dehydrogenase and the specific radioactivity of NADH has been calculated on the basis of the model shown in Fig. 2. The model assumes one NAD pool and a’constant specific radioactivity of xylitol, which is set equal to 1. Assuming isotopic steady state, inflow

J HZ0

I

GLtJCOSE

FIG. 2. Model for flux of reducing equivalents from [2-3H]xylitol. a, b + y, z + d, and e represent formation of NADH from xylitol, ethanol, lactate, and endogenous substrates, respectively. a + b represents consumption of NADH in the microsomal and mitochondrial electron transport chains and f in the triosephosphate dehydrogenase reaction, respectively.

induced

Note. The calculations

Ethanol

Ts treated

Starved

Treatment

IV

0.419 f 0.085 (7) 0.414 + 0.075 (9) 0.419 k 0.031 (3) 0.146 k 0.060 (8) 0.160 + 0.049 (9) 0.191 k 0.027 (4)

15 30 45 15 30 45

[2-SH]Xylitol

[2-3H]Xylitol + ethanol

in the Results. Figures

represent

0.184 + 0.072 (4) 0.193 f 0.087 (5) 0.192 + 0.027 (4)

15 30 45

[2-sHylylito1 + ethanol

were performed

0.267 + 0.077 (4) 0.350 k 0.058 (5) 0.409 k 0.038 (4)

15 30 45

0.223 _+ 0.077 (6) 0.252 k 0.076 (7) 0.278 k 0.052 (4)

15 30 45

[2-SHJXylitol + ethanol

[2-SI]Xylitol

0.249 + 0.047 (5) 0.339 + 0.049 (7) 0.418 +- 0.071 (4)

15 30 45

[2-3Hy(ylitol

as described

N pmol aH/pmol

Substrate

Time (min)

16 k 3 (3) 19 + 2 (4) 19 f 2 (4) 4 + 2 (4) 4 f 3 (4) 7 + 1 (3)

8 (6) 5 (7) 7 (4) 4 (4)

62 T 71 -t 80 + 45 +

54 k 7 (5) 60 -c 5 (4) 69 -t 72 k 73 +

3.2 (6) 4.7 (7) 4.5 (4) 1.0 (4) 1.3 (5) 1.1 (4)

5.1 + 7.8 + 6.8 + 3.4 + 4.6 + 4.5 +

0.9 (4) 1.7 (5) 0.5 (4)

means + SD with the number of cell preparations

2.8 + 4.7 + 4.9 +

2 (4) 8 (5) 2 (4)

18 + 4 (8) 16 + 3 (4)

3 (7) 5 (4)

52 -c 58 k 3.7 (7) 3.4 (4)

9.7 + 11.7 +

in parenthesis.

13 f 2 (4)

14 f 8 (7) 16 + 5 (8)

18 k 5 (6)

42 xk 6 (5)

6.7 (5)

8.7 +

0.9 (6) 2.5 (8) 2.4 (3)

7 k 4 (8) 8 _+ 3 (9) 10 + 4 (4)

2.2 + 5.1 + 7.8 +

60 -+ 10 (7) 60 -t 10 (8) 67 -t 8 (4)

Glucose (%I

4.9 + 2.7 (7) 7.4 + 3.3 (6) 15.8 + 14.5 (4)

(o/o)

11 f 7 (7) 13 k 4 (9) 17 f 5 (3)

Hz0

ANDTHECALCULATEDRELATIVE

56 + 4 (6) 58 -+ 5 (7) 61 k 7 (3)

2 o,kmol/O.l ml packed cells)

LACTATEDEHYDROGENASE(Z), YIELDS OF TRITIUM IN WATER AND GLUCOSE FROM [2-~H]XYLITOL

THECALCULATED SPECIFIC RADIOACTIVITYOFNADH(N),THEFLUXTHROUGH

TABLE

d w

3 g $ t; g

; g

g 8

z o2

3

3 % Q 8 c z$ c kl

ifi

g 22

F

704

VIND

AND GRUNNET

of tritium to NAD+ equals outflow of tritium from NADH. The model shown in Fig. 2 gives a’ + L(x + d) = N(a + b +f+ or a’=N(a+b+f)+(Nx-L(x+d)).

x),

N= [l]

N is the relative “average” specific radioactivity of cytosolic NADH in a given time period and L is the relative “average” specific radioactivity of lactate in the same time period. a’ is the flux of tritium from [2-3H]xylitol, and a the total flux of reducing equivalents from [2-3H]xylitol, a = a’, when the specific radioactivity of xylitol is set equal to 1. The total amount of reducing equivalents in micromoles transferred to NAD+ is a + b +f, a from xylitol, b from ethanol, and f from other reduced substrates. The value off must be at least large enough to account for the hydrogen from the carbon necessary for the formation of glucose from substrates other than xylitol and was calculated as follows. (a) If gluconeogenesis was larger than corresponding to the metabolism of xylitol, the assumption was made that all xylitol metabolized was converted to glucose. The formation of glucose from other substrates (e) thus equals Aglucose - 5/6a. This glucose formation gives rise to the formation off = 2 (AGlucose - 5/6u) cytosolic reducing equivalents. (b) In the few cases where gluconeogenesis was less than corresponding to the oxidation of xylitol, some of the xylitol has been converted through triosephosphate dehydrogenase and thus donated another reducing equivalent to NAD+. In this casef = d + 2(5/6u - Aglucose), where d is the experimentally determined formation of reducing equivalents from the oxidation of lactate. For the time intervals O-30 and O-45 min, f was calculated as the sum of the f values in each of the time intervals O-15, 15-30, and 30-45 min. Flux through lactate dehydrogenase is denoted x, and X = Nx - L(x + d), where X is the experimentally

accumulation of xylitol hydrogen in lactate (equaling inflow minus outflow of tritium). Substituting into Eq. [l] with a specific radioactivity of xylitol = 1, gives

PI determined

U-h

a+b+f’

The calculated values of N are shown in Table IV. Calculation of the Relative Yield of Tritium in Water and Glucose As the oxidation of NADH ultimately results in transfer of the hydrogen to 02, the transfer of radioactivity to water should equal the specific radioactivity of NADH multiplied by the net formation of reducing equivalents minus the amount used to reductive biosynthesis (glucose and lactate). Incorporation of tritium into glyceraldehyde 3-phosphate via the triosephosphate dehydrogenase reaction is N X ft of which half is lost to water upon glucose formation in the aldolase reaction. The relative yield of tritium in water and glucose is thus: 9%of tritium

in water

= Na + b + f 12) 1oo , U 9%tritium

in glucose = Nxf/2100 u

,

assuming equal specific radioactivity of the 4A- and 4B-hydrogen of NADH. As can be seen (Table IV), the calculated values of the tritium incorporation into water are grossly in accordance with the measured values (Table III), whereas the calculated incorporation into glucose is higher than the experimental values by a factor of approximately 2. The discrepancy will be discussed later. Lactate Dehydrogenuse Flux From Eq. [2] above, x is obtained as X + Ld x=N-L L is the “average” specific radioactivity

of

PATHWAYS

OF REDUCING

EQUIVALENTS

lactate in the time period considered (0 - T), and is calculated as T

L=L s

K(1 - e-“‘)dt T = K( T + TemT”- T) T ’

The K and T values are obtained from Fig. 1 and N is calculated as described above. The calculated values of x are shown in Table IV. DISCUSSION

Flux through Lactate Dehydrogenase and the Spec& Radioactivity of NADH Ethanol metabolism gives rise to the formation of cytosolic NADH and is therefore expected to cause a dilution of the NAD3H, produced by the oxidation of [23H]xylitol. This is in fact observed in cells from starved rats, where the relative radioactivity of NADH decreased as much as might be expected due to ethanol oxidation (Table IV). In contrast, the steadystate value of the specific radioactivity of lactate was about the same in the presence and absence of ethanol (Fig. 1). This apparent discrepancy is, however, explained by the different flux through lactate dehydrogenase in the absence and presence of ethanol (1.5 and 3.2 rmol/min g wet wt), respectively (Table IV). It thus appears that the flux through lactate dehydrogenase is insufficient to equilibrate the C4hydrogen of NADH and the CB-hydrogen of lactate. In cells from T3-treated and ethanol-fed rats, the specific radioactivity of NADH is decreased less than expected from the formation of unlabeled NADH by ethanol oxidation, and the flux through lactate dehydrogenase is decreased when ethanol is metabolized together with xylitol, whereas an increase was found in cells from starved rats (Table IV). An explanation for these findings might be that ethanol is metabolized partly via a

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705

nonalcohol dehydrogenase pathway (e.g., MEOS) (25) in cells from T3-treated or ethanol-fed rats, thereby providing less NADH to the cytosol. Evidence supporting this suggestion is that T3 treatment and prolonged ethanol feeding causes an increase in the MEOS activity and a decrease in the alcohol dehydrogenase activity (12, 24, 26) and that xylitol causes a loss of adenine nucleotides (21), thereby deinhibiting NADPH cytochrome c reductase and one or more NADPH-generating enzymes (27). Also, the simultaneous oxidation of xylitol and ethanol will create a massive supply of NADH to cytochrome b,, catalyzed by cytochrome bs reductase (4), which preferentially use NADH instead of NADPH (6) and whose reduction is dependent on the ratio NADPH/NADH (5). In agreement with this suggestion, xylitol decreased the inhibition of ethanol oxidation by 4-methylpyrazole, an alcohol dehydrogenase inhibitor, in cells from ethanol-induced rats incubated with 50 mM ethanol (S. E. Damgaard, this laboratory, unpublished results). The calculated flux through lactate dehydrogenase in the present experiments range from 1 to 6 pmol/min g wet wt. Flux through lactate dehydrogenase is thus only a small fraction of the maximal enzyme activity, which is ca. 200 pmol/min g wet wt (28). No physiological effecters of lactate dehydrogenase are known, and it may be anticipated that flux through the enzyme is rate-limited by the concentrations of the substrates, pyruvate, and NADH. In the present experiments, the pyruvate concentrations were at or below the K, value for pyruvate (0.14-1.0 mM) (29,30) and higher in the absence than in the presence of ethanol (results not shown). The cytosolic NADH concentration was very much higher in the presence of ethanol than in its absence (as judged from the lactate/pyruvate ratio). Thus no correlation was found between either of the two substrates and the flux through lactate dehydrogenase (cf. Table IV) and the latter might well have been determined by both the concentration of pyruvate and NADH.

706

VIND

AND GRUNNET TABLE

V

THE CORRECTED, CALCULATED RELATIVE YIELDS OF TRITIUM IN WATER AND GLUCOSE FROM [~~H]XYLIT~L AND THE RELATIVE YIELDS OF TRITIUM DETERMINED EXPERIMENTALLY

Treatment Starved

T3 treated

Ethanol

induced

Percentage *H in glucose

Percentage

‘H in Hz0

Time (min)

Found

Calculated

[2-8H]Xylitol

15 30 45

60 64 68

62 65 70

5 6 10

6 7 9

[2JH]Xylitol + ethanol

15 30 45

62 63 71

64 64 73

5 4 6

4 4 6

[2-3H]Xylitol

15 30 45

49 60 6’7

51 61 66

8 9 8

9 9 8

[2-3H]Xylitol + ethanol

15 30 45

64 78 84

69 79 87

10 8 8

7 8 7

[2-‘H]Xylitol

15 30 45

56 65 68

53 66 70

5 9 12

8 10 10

[2-3H]Xylitol + Ethanol

15 30 45

70 72 74

71 74 77

5 6 8

2 2 4

Substrate

Found

Calculated

Note. The calculations were performed as described in the Results with the modification that N X f/4/a x 166% of the tritium was transferred from glucose to water thus assuming the specific radioactivity of the 4B-hydrogen of NADH to be half that of the 4A-hydrogen. The figures represent means.

The Labeling of Water The relative amount of tritium found in water was larger, when [2-3H]xylitol and ethanol were metabolized together, than when [2-3H]xylitol was metabolized alone (Table III). The difference was largest in cells from T3-treated rat (P c 0.001) (Student’s t test) and the ethanol-induced rats (P < 0.05). This is in agreement with the assumption made before that part of the ethanol in cells from T3-treated and ethanol-induced rats will be oxidized via a nonalcohol-dehydrogenase pathway and that NAD 3H might be one of the electron donors (4, 31) with the result that some of the tritium will be transported to Oz out-

side the mitochondria and without the limitation of the mitochondrial shuttle systems for reducing equivalents. The calculated yields of 3H in water (Table IV) is in accordance with the relative yield of ‘H found in our experiments (Table III).

The Labeling of Glucose The calculated yield of 3H in glucose (Table IV) differs from the relative yield in our experiments by approximately a factor of 2 (Table III). The most obvious explanation is that the specific radioactivity of the 4B-H of NADH is less than that of the 4A-H (32). Xylitol dehydrogenase is an A-specific dehydro-

PATHWAYS

OF REDUCING

EQUIVALENTS

genase (33) and triosephosphate dehydrogenase is B specific. If a specific radioactivity of the 4B-H of NADH of about half that of the 4A-H is assumed, agreement between the calculated and observed values are obtained (Table V). Conclusions and Assumptions The simple model proposed for the metabolism of cytosolic reducing equivalents (Fig. 2) accounts satisfactorily for the flux of the CB-hydrogen of xylitol to the major products, under the experimental conditions used. Thus, the results of the present experiments do not contradict the concept of a single cytosolic pool of NAD+, and indicate that flux through the various dehydrogenases is insufficient to equilibrate fully the 4A and 4B hydrogen of cytosolic NADH. Flux through the lactate dehydrogenase reaction was calculated to be only a few percent of the maximal activity of the enzyme. Furthermore, evidence was obtained for the participation, in Ts-treated and ethanol-induced animals, of nonalcohol dehydrogenase catalyzed oxidation of ethanol in the presence of xylitol. The present approach assumes metabolic and isotopic steady state. Metabolic steady state is probably approximated in the experiments and isotopic steady state attained within a few minutes (36), i.e., a sufficiently short time compared to the experimental periods so that the initial transient incorporation of isotope into intermediates can be neglected. Possible tritium isotope discrimination effects in the pathways studied have been neglected. However, the specific radioactivity of the xylitol sta,yed approximately constant throughout the experiments, the tritium isotope effect in the lactate dehydrogenase reaction is below 1.3 (34,35), and generally quite small overall isotope discrimination effects are found in metabolic studies of deuterium- or tritium-labeled substrates. ACKNOWLEDGMENTS We are indebted to Ruth Jdrgensen and Marte Nord for expert technical assistance. This work was

DURING supported skningsrad

ALCOHOL

METABOLISM

707

by Statens laegevidenskahelige ForJ. nr. 512-15523 and J. nr. 512-20106. REFERENCES

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