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Cobalamin (Vitamin B₁₂) and Holotranscobalamin Changes in Plasma and Liver Tissue in Alcoholics with Liver Disease

Departments of Preventive Medicine and Community Health, Medicine, and Liver Center, University of Medicine and Dentistry, New Jersey Medical School, Newark, New Jersey

Address reprint requests to: Herman Baker, Ph.D., UMDNJ, New Jersey Medical School, 65 Bergen Street, Martland GB-159, Newark, NJ 07107-3001

	ABSTRACT

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Objective: We wanted to know if alterations in plasma cobalamin (B₁₂) concentration and B₁₂ carriers, e.g., holotranscobalamins (holo TC), occur in blood and liver tissue from patients with severe alcoholic liver disease. Our purpose was to test the hypothesis that liver disease may disrupt B₁₂ distribution.

Method: Total B₁₂, as well as B₁₂ bound to transcobalamin I, II, III (holo TC), were measured to determine their concentration in plasma and in liver tissue; Poteriochromonas malhamensis—a protozoan reagent served to measure only metabolically active (true) B₁₂. Total B₁₂ as distributed in holo TC in plasma and liver tissue of healthy subjects (controls) were compared to patients with severe alcoholic liver disease.

Results: Severe liver disease initiates highly elevated B₁₂ levels in plasma and a lowered liver tissue total B₁₂ concentration. The percent of B₁₂ distributed to holo TC II is significantly depleted during liver disease. In contrast, holo TC I and III are elevated in plasma during liver disease and contain more B₁₂ than controls. Total B₁₂ and B₁₂ distributed to TC are lower in diseased liver tissue.

Conclusion: Severe alcoholic liver disease involves leakage of total B₁₂ from liver tissue into the plasma. Holo TC I and III concentration increases in plasma; this preserves the high plasma B₁₂ from being excreted. However, plasma holo TC II B₁₂ distribution is decreased, indicating that there is a depression of exogenous B₁₂ entering the plasma and tissues. In severe liver disease, liver tissue B₁₂ binding and storage by TC is disrupted and causes B₁₂ to leak out of the liver into the circulation. Eventually liver disease could produce enough severe tissue B₁₂ deficits to cause metabolic dysfunction despite elevated plasma total B₁₂. Elevation of plasma B₁₂, accompanied by a lowering of holo TC II distribution, seemed to be a useful index of liver disease severity suggesting preventative treatment.

Key words: vitamin B₁₂, holotranscobalamins, liver disease

	INTRODUCTION

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Cobalamin (B₁₂) transport proteins exist in human fluids, e.g., gastric juice, blood, saliva, and bile; they are also found in B₁₂ auxotrophic microorganisms [1,2]. Human plasma contains at least three B₁₂-binding proteins: transcobalamin (TC) I, II, III. TC I contains the highest portion of endogenous B₁₂ and slowly delivers B₁₂ from the peripheral body tissues to the liver [1,3,4]. TC III, not yet functionally understood, perhaps acts as a scavenger for metabolically useless and potentially harmful cobalamin analogs in blood [3]. However, some studies indicate that TC III delivers B₁₂ rapidly and exclusively to the liver [1]. Plasma TC II transports B₁₂ from within the enterocyte to the circulation. Once B₁₂ is released from intrinsic factor in the distal ileum, the newly absorbed B₁₂ binds to TC II in plasma (holo TC II) and enters the portal circulation to deliver B₁₂ to all tissues for metabolic use; most cells depend on TC II for their B₁₂ delivery [1]. TC II exclusively transports only metabolically active cobalamins i.e., those containing 5,6 dimethylbenzimidazole as the nucleotide moiety—the only known function of TC II [1,3,4].

Measurements of B₁₂ concentration reported in blood and liver from alcoholics with liver disease may be falsely high because they also include endogenous metabolically inactive cobalamin analogs that contaminate active B₁₂ [5]. We wanted to know whether alteration in content of only true metabolically active cobalamin carried by TC I, II, III occurred in blood and liver tissue in patients with severe alcoholic liver disease.

	SUBJECTS AND METHODS

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After informed consent was obtained, blood was drawn from an antecubital vein into Vacutainers (Becton Dickenson, Sunnyvale, CA) containing EDTA as anticoagulant. After centrifugation, clear plasma was processed for measuring total B₁₂ and holo TC; specimens were stored at -20°C. Total B₁₂, the sum contained in all holo TC, and B₁₂ specifically contained in each TC (holo TC I, II, III) were measured in plasma of 52 healthy volunteers (controls), 11 patients having histologic evidence of cirrhosis and 18 with end stage liver disease; all were males aged 22 to 60. The controls were without history of hepatic, hematologic, gastrointestinal disease, previous surgery, history of chronic drug or alcohol intake. The 11 alcoholic patients with cirrhosis had chronic liver disease but not severe enough to warrant liver transplant. The 18 patients with end stage liver disease did exhibit life-threatening loss of liver function, e.g., irreversible liver failure with steatosis, ascites and a catabolic state with elevated aminotransferases and decreased albumin production; all were awaiting liver transplant. Because of severe life-threatening liver malfunction, 41 male patients aged 26 to 43 having end stage liver disease later had their liver transplanted with an undamaged liver by described procedures [6]. Samples of their excised damaged liver tissue were processed for measuring total B₁₂ contained in tissue holo TC I, II, III. These B₁₂ measurements were compared to histologically normal liver tissue obtained during autopsy from 12 male subjects without history of hematologic, liver, or renal disease (control tissue). Processed liver tissue was stored at -20°DC.

Methods for extracting holo TC from plasma using microfine glass powder were used as described [7,8]. The glass powder binds plasma holo TC II taking it out of solution while holo I and III remain in the supernatant. The supernatant, containing holo I and III, was assayed for B₁₂; the B₁₂ content is then subtracted from total B₁₂ (see below) concentration in plasma. After subtracting holo TC I and III B₁₂ concentration from total B₁₂, the remaining B₁₂ represents B₁₂ concentration of holo TC II. A separate plasma specimen was used to measure total B₁₂, i.e., B₁₂ combined with all TC. We modified the plasma method [7,8] for determining holo TC in liver tissue; a sample of homogenized liver in saline (0.5 mg/ml) suffices for this purpose. Total B₁₂ and B₁₂ concentration in holo TC in plasma and liver tissue were analyzed using Poteriochromonas malhamensis (ATCC #11532 formally Ochromonas malhamensis) as the reagent [9]. This protozoan responds only to metabolically active forms of B₁₂; it does not measure functionally inactive B₁₂ analogs as do other techniques [5,9–14].

Protein concentration of each liver specimen was determined [15] to serve as a standard of reference; B₁₂ content per mg of tissue protein was calculated. Results for all analyses were expressed as standard of the mean (±SD) by standard methods. Student t-test was also applied to estimate significance of differences (p); the means in each group were compared to control values. Percentage of total B₁₂ distributed to a specific TC was calculated by differences between values for total B₁₂ and the B₁₂ sequestered by the specific TC.

	RESULTS

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Plasma from patients with alcoholic liver disease revealed significantly higher levels of total plasma B₁₂ than did healthy subjects .Depression of the percent of total B₁₂ carried by plasma TC II was also significant during liver disease. Conversely, the B₁₂ concentration and percent of B₁₂ sequestered on TC I and III was significantly elevated (Table 1). Diseased liver tissue showed a different B₁₂ concentration pattern than plasma. Total liver B₁₂ concentration, as well as B₁₂ contained in all holo TC, were significantly depressed. A greater percent of B₁₂ sequestration by TC I and III and a lesser percent of B₁₂ concentration sequestered by TC II was seen in liver tissue (Table 2).

	DISCUSSION

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As mentioned, overestimates of blood B₁₂ concentration seen in liver necrosis have been noted. This could be due to the use of microbial or radio-dilution procedures which measure the sum of true B₁₂ and non-metabolic B₁₂ analogs concentrated in blood and tissue [5]. Such a problem is irrelevant in the present study. As noted, we used P. malhamensis, which measures only true metabolically active B₁₂, including active B₁₂ bound to transcobalamins (Tables 1, 2). Metabolically inactive B₁₂ analogs, e.g., cobalamins not having a 5,6 dimethylbenzimidazole nucleotide moiety, can not be measured by this protozoan [9,12]. When we mention B₁₂ here, we refer to only metabolically active B₁₂ and not inactive B₁₂ analogs which are concentrated in liver and blood [5,10,12–14]. Assuming the B₁₂ in the holo TC I and III combination is not exclusive for only TC I, then our results (Tables 1, 2) show that holo TC III can carry true metabolically active B₁₂ and does not only scavenge B₁₂ analogs as supposed by others [3].

We agree (Table 1) with others [21] who suggest that lower percentage of B₁₂ carried by TC II points to negative B₁₂ balance in alcoholics. In that context, one should also note that elevated plasma B₁₂ (Table 1) leaked from the liver (Table 2) is also a useful biomarker. Total B₁₂ leakage seems responsible for a decrease of B₁₂ bound by TC in the diseased liver (Table 2). These events may add more insult to the diseased liver and cause it to severely malfunction and thus perhaps warrant a liver transplant. Could a direct chronic flooding of the depleted liver with high dose intramuscular B₁₂ reverse oncoming liver failure by mass action of B₁₂? Such intervention might temporarily bypass the need for B₁₂ carriers. When the B₁₂ tissue level, and perhaps folate too, are fully saturated, a sharp rise in hepatic DNA synthesis with ensuing liver regeneration may occur [22] and so lessen liver malfunction. Increased synthesis of TC might occur and permit exogenous B₁₂ to be carried to cells again.

Received February 1, 1997. Accepted October 1, 1997.

	REFERENCES

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