We have recently shown that the intake of 750 mL of wine (~70 g of ethanol) during meal absorption in normal subjects inhibits the fractional secretory rates of the two important hepatic proteins albumin and fibrinogen (De Feo et al. 1995). Ethanol appears to have acute and relatively specific effects on hepatic protein synthesis because fractional secretory rates of immunoglobulin G (IgG)⁴ and leucine estimates of whole-body protein synthesis were unaffected by alcohol ingestion (De Feo et al. 1995). It is likely that the repeated intake of large amounts of alcohol contributes to the pathogenesis of the complications of chronic alcoholism such as liver cirrhosis and muscle myopathy by impairing the metabolism of secretory liver proteins. In fact, experiments in animals demonstrated that the impairment in hepatic protein metabolism plays a key role in the development of alcoholic liver injury (French 1989, Lieber 1980). Furthermore, studies in humans suggested that the postprandial inhibition of albumin secretion might induce muscle wasting also in the absence of poor protein intake (Baumgartner et al. 1996, De Feo et al. 1992).

Although the role of albumin in whole-body amino acid and protein homeostasis is not well defined, it is known that albumin synthesis is stimulated by insulin (De Feo et al. 1993, Volpi et al. 1996) and/or meal ingestion of amino acids (Volpi et al. 1996) and that it is catabolized by nearly every tissue in the body (Yedgar et al. 1983). Approximately 30% of the increase in whole-body protein synthesis observed during meal absorption can be accounted for by the increase in albumin synthesis (De Feo et al. 1992). One potential nutritional role of albumin is as a temporary storage pool of amino acids, protecting some fraction of ingested amino acids from irreversible oxidation and thus ensuring their delivery to peripheral tissues for utilization in protein synthesis days after their intake. This hypothesis is consistent with studies in dogs, demonstrating that hepatic secreted plasma proteins may serve as an important source of amino acids for protein synthesis in muscle and other peripheral tissues (Elwyn 1970, Elwyn et al. 1968), and in elderly humans, in whom serum albumin concentration significantly associates with muscle mass (Baumgartner et al. 1996). Thus any factor(s) that might interfere with the synthesis of albumin during meal absorption (i.e., ethanol) could have significant effect on whole-body and particularly muscle protein homeostasis if such a process was extended over time.

The mechanism by which alcohol acutely impairs liver protein metabolism has been investigated in vitro. In cultured rat hepatocytes, ethanol oxidation leads to the consumption of NAD⁺, which is reduced to NADH. The subsequent shift in the redox equilibrium toward the reduced state (increased NADH/NAD⁺ ratio) is the most likely cause of the impairment in hepatic intermediate metabolism (French 1989, Lieber 1980). In fact, the administration of hydrogen ion acceptors such as methylene blue or high doses of hydrogen ion shuttles such as aspartate, that are able to reoxidize NADH, prevents ethanol inhibition of protein synthesis (Baraona et al. 1980). However these compounds are not safe for use in humans. Recent studies have shown that nicotinamide administration increases the intracellular NAD⁺ pool (Pociot et al. 1993) because nicotinamide is the direct precursor for NAD⁺ synthesis (Mayes 1990).

Table 1. Leucine infusion rates, 14CO2 excretion rates, plasma leucine and -ketoisocaproicacid (KIC) specific activities and whole-body leucine kinetics in normal humans in the postabsorptive period (basal) and during the intragastric (ig) administration of a mixed meal combined with the ingestion of water (control) or wine with (NA) or without (W) the administration of nicotinamide1,2 [View Table]

The present study was undertaken to test the hypothesis that administration of the NAD⁺ precursor, nicotinamide, by increasing the intracellular availability of NAD⁺, can reduce or reverse the inhibitory effect of acute ethanol ingestion on liver protein metabolism, specifically on the meal-induced increase in albumin synthesis.

SUBJECTS AND METHODS

All subjects were studied 3 d after consuming a weight-maintenance diet of 146 kJ/(kg·d) containing 55, 30 and 15% carbohydrate, fat and protein, respectively; no alcoholic beverages were permitted. After overnight fasting, the volunteers were admitted to the Clinical Research Unit of the Dipartimento di Medicina Interna e Scienze Endocrine e Metaboliche of the University of Perugia, Italy, at ~0730 h. At ~0800 h, a 23-gauge catheter was placed in an antecubital vein for the infusion (Harvard syringe pump, Harvard Apparatus, Ealing, South Natick, MA) of [1-¹⁴C]leucine (specific activity 2 MBq/mmol, Amersham International, Buckinghamshire, UK) and saline (0.5 mL/min, Vial Médical pump, Grenoble, France). A contralateral hand vein was cannulated in a retrograde fashion with a 20-gauge butterfly needle; the hand was maintained at 65°C in a thermoregulated Plexiglas box to permit intermittent sampling of arterialized-venous blood (McGuire et al. 1976). An 8F size nasogastric feeding tube was inserted for intragastric (ig) meal infusion.

At ~0900 h (time, 0 min), a primed-constant intravenous infusion of L-[1-¹⁴C]leucine [prime, 333 kBq/min (9 µCi); infusion rate, 11.1 kBq/min (0.3 µCi/min)], was started and continued for 8 h. At time 240 min, after blood and breath sampling, the ig infusion of a liquid mixed meal was started (1.75 mL/min) and continued throughout the study (Vial Médical pump). The meal provided 2642 kJ (17% from amino acids, 33% from lipids and 50% from carbohydrates). It was prepared by mixing a complete formula of nonessential and essential amino acids (Isopuramin Plus 10%, Bieffe Medical, Modena, Italy; ig unlabeled leucine infusion rate reported in Table 1) with 84 g of glucose and a mixed oil solution (Lipofundin S, B. Braun, Melsungen, Germany). Either 150 mL of natural spring water (Santa Chiara, Motette srl, Scheggia, Italy) (control group), or 150 mL of white wine (12% v/v ethanol content, Pergoleto Lungarotti, Cantine Lungarotti srl, Torgiano, Italy) (W and NA groups) were given to the volunteers at the beginning of the meal infusion. Then, 50 mL water (control group) or 50 mL wine (W and NA groups) was given every 15 min (from 255 to 420 min) for a total of 750 mL water to the control group, and 750 mL wine to the W and NA groups. At 240 min, the subjects of the NA group were given two tablets of nicotinamide (250 mg per tablet, IDI, Pomezia, Italy), and three other tablets were dissolved into their meal (~1.78 g/L of meal) and infused, for a total amount of 1.25 g nicotinamide.

Blood (16 mL) and breath samples were collected at 15, 0, 180, 200, 220, 240, 420, 440, 460 and 480 min to measure the plasma concentrations of glucose, lactate, pyruvate, insulin, isoleucine, leucine, -ketoisocaproic acid (KIC), albumin, fibrinogen and IgG, the plasma specific activity of leucine and KIC, the rates of expired total CO₂, total ¹⁴CO₂ and CO₂ specific activity, and the specific activity of leucine derived from albumin, fibrinogen and IgG hydrolysis. Plasma ethanol concentrations (1 mL blood) were measured every 60 min from 240 to 480 min.

The estimates of whole-body leucine kinetics were determined on the data obtained during the last hour of each study period (postabsorptive state: 180-240 min; absorptive state: 420-480 min) at the isotopic and metabolic steady state, using the four-compartment model (Schwenk et al. 1985). The rate of total leucine appearance [Total Ra, µmol/(kg·min)] was calculated using the following formula: where i is the labeled leucine infusion rate [dpm/(kg·min)] and SA_KIC is the plasma KIC specific activity (dpm/µmol). During meal administration, the rate of endogenous leucine appearance [Endogenous Ra, µmol/(kg·min)], an index of whole-body proteolysis, was calculated as follows: where D_Leu is the ig leucine infusion rate. The rate of leucine oxidation [Ox, µmol/(kg·min)] was calculated using the precursor-product model: where CO₂ is the ¹⁴CO₂ excretion rate [dpm/(kg·min)] and  is the correction factor for the CO₂ recovery, assuming values of 0.70 and 0.82 in the postabsorptive and absorptive states (Hoerr et al. 1989), respectively. The rate of nonoxidative leucine disposal [NOLD, µmol/(kg·min)], index of whole-body protein synthesis and net leucine balance [Balance, µmol/(kg·min)] were estimated as follows:

The fractional secretory rates (FSR, %/h) of plasma proteins were calculated using the precursor-product model: where SA_{Leu protein}/t is the incorporation rate of labeled leucine into proteins from 180 to 240 min (postabsorptive state) and from 420 to 480 min (absorptive state), and SA_{KIC plasma} is the mean plasma KIC specific activity during the same time periods. The use of plasma KIC specific activity as precursor pool specific activity for hepatic protein synthesis in the postabsorptive and the absorptive states has been recently validated (De Feo et al. 1995, Volpi et al. 1996).

Table 2. Albumin, fibrinogen and immunoglobulin G (IgG) tracer incorporation rates (SA/t) and fractional secretory rates (FSR) in normal humans in the postabsorptive period (basal) and during the intragastric (ig) administration of a mixed meal combined with the ingestion of either water (control) or wine with (NA) or without (W) the administration of nicotinamide1 [View Table]

RESULTS

Plasma ethanol concentrations were undetectable at 240 min in the three groups. In the second study period (absorptive state), they remained undetectable in the control group until the end of the study, whereas they increased significantly in the W and NA groups without differences between the two groups (Fig. 1).

The plasma lactate/pyruvate ratio (mol/mol) did not differ among the three groups in the postabsorptive state and did not change in the control group during meal absorption, whereas it increased by~200% in the W group and only ~100% in the NA group, with a significant difference between these two groups (Fig. 1).

The specific activity of leucine derived from the hydrolysis of the three plasma proteins increased linearly during the last hour of each study period (data not shown).

Meal administration (Table 2, Fig. 2) changed albumin fractional secretory rate (P < 0.01 vs. basal) with a significant time by group interaction (P < 0.001). Albumin fractional secretory rate increased similarly in the control and NA groups to rates that did not differ from one another, whereas it decreased in the W group (P < 0.001 vs. control and NA). Fibrogen fractional secretory rate was unaffected by meal absorption in the control and NA groups, whereas it decreased in the W group (P < 0.01 vs. control and NA).

IgG fractional secretory rate increased during meal absorption (P < 0.001 vs. basal), without differences among the three groups (Table 2, Fig. 2).

DISCUSSION

The lactate/pyruvate ratio is a good index of the intrahepatic redox state, because any variations in the NADH/NAD⁺ ratio are reflected by an according shift in the lactate:pyruvate equilibrium (i.e., an increase in NADH will increase the reduction of pyruvate to lactate) (French 1989, Lieber 1980). The increase in lactate/pyruvate ratio with ethanol ingestion and the significant reduction of these changes with the simultaneous administration of nicotinamide provide evidence that the ethanol-induced decrease in albumin and fibrinogen synthesis is directly or indirectly related to a change in the redox state of the liver. In vitro studies suggest that the synthesis of liver proteins is reduced as a result of an ethanol-induced depletion of ATP (Lieber 1980, Masson et al. 1993). The oxidation of both ethanol and its primary metabolite, acetaldehyde, increases the NADH/NAD⁺ ratio, which secondarily decreases the activity of key enzymes on ATP-producing pathways such as glycolysis (Berry et al. 1994), -oxidation (French 1989) and the tricarboxylic acid cycle (Lieber 1980). This relative depletion of ATP could be compounded by the shunting of glycerol-3-phosphate to triglyceride synthesis as a result of increased acetyl-CoA availability from ethanol oxidation (French 1989).

A second potential mechanism for the effect of nicotinamide is at the level of hepatic protein secretion (Sorrell et al. 1983). In cultured hepatocytes, acetaldehyde generated from ethanol oxidation by reacting with tubulin impairs tubulin polymerization and inhibits the secretory process (Tuma et al. 1987). We measured the FSR of secretory proteins in the plasma space; therefore any processes that inhibited secretion alone would prevent the entry of labeled protein into the sampling space and thus reduce its FSR. Because NAD⁺ is the coenzyme for aldehyde dehydrogenase, nicotinamide administration would accelerate acetaldehyde oxidation by increasing the intracellular NAD⁺ availability and thus maintain the secretory process of newly synthesized hepatic proteins. Although we cannot exclude this mechanism as a contributing factor, it is less attractive because of the very high concentrations of ethanol and acetaldehyde required in both in vivo and in vitro animal studies to demonstrate an effect on tubulin polymerization. Additional studies will be required to specifically address this issue.

Finally, on the basis of in vitro data, it is possible to exclude a direct effect of nicotinamide on liver protein synthesis and/or secretion, because the addition of the vitamin to liver cell cultures did not change either the secretion rate or the intracellular mRNA levels of albumin and ₁-acid glycoprotein (Barraud et al. 1995).

We would suggest that the nicotinamide administration during ethanol ingestion maintained the intracellular NAD⁺ concentration, reducing the increase in the NADH/NAD⁺ (and in the lactate/pyruvate) ratio, and thus maintained the production of ATP, albumin and fibrinogen.

In any case, the main result of this study is the nicotinamide-induced reversal of the alterations due to acute ethanol metabolism on postprandial liver protein secretion. Such an observation may have practical importance and application. Chronic alcohol abusers are reported to have an inverse correlation between alcohol intake and plasma fibrinogen (Krobot et al. 1992) and albumin concentrations (Lindholm et al. 1991) and are at increased risk for alcohol-induced myopathy (Preedy and Peters 1990). These complications could be explained if ethanol ingestion on a habitual basis chronically inhibits albumin and fibrinogen synthesis and if albumin is an important nutrient pool for many essential amino acids. Our results suggest that these alterations might be prevented with the administration of nicotinamide. In addition, it is possible that the impairment in hepatic protein metabolism may play a key role in the development of alcoholic liver injury (Lieber 1980). Were this the case, could it also be prevented with the administration of nicotinamide? The health care implications and costs in health care dollars and loss of productivity as a result of ethanol-induced hepatic damage are profound (Lieber 1995). The low cost of nicotinamide and the absence of side effects at doses twice as high as those used in the present study might make it a very cost-effective intervention strategy with a wide therapeutic safety margin in the prevention of liver disease in alcoholics who refuse to abstain from ethanol ingestion.

ACKNOWLEDGMENTS