Other fat-soluble vitamin deficiencies were observed in 77% of the study participants. The most common was vitamin D deficiency as assessed by serum 25-hydroxyvitamin D, observed in 22 (71%) of patients (Table 2). Nine (29%) patients had decreased serum vitamin A levels, whereas 2 patients (6%) had decreased serum vitamin E levels (Table 2). Two patients had both vitamin D and vitamin E deficiency, whereas 7 patients had vitamin D and vitamin A deficiency. In total, 56% of patients with vitamin A deficiency, 77% of patients with 25-hydroxyvitamin D deficiency, and 100% of patients with vitamin E deficiency had elevated PIVKA-II.
When we evaluated the relation between diagnosis and increased INR and plasma PIVKA-II levels (Table 3), we found a consistently higher frequency of increased PIVKA-II level compared with INR increases across all diagnoses. This may be a direct reflection of severity of cholestasis with a strong correlation among serum bile acids, serum conjugated bilirubin, and PIVKA-II (P = 0.002 and P < 0.001, respectively) (Table 4).
To assess whether markers of the severity of an individual's cholestatic liver disease such as serum conjugated bilirubin and elevated INR were associated with elevated plasma PIVKA-II, Spearman correlations were performed between plasma PIVKA-II, serum ucOC, and other measured parameters. Plasma PIVKA-II directly correlated with PT (P = 0.02), INR (P = 0.02), and the severity of cholestasis as measured by serum conjugated bilirubin (P < 0.001) and serum bile acids (P = 0.002) (Table 4, Fig. 1). Additional positive correlations were observed between plasma PIVKA-II and serum ucOC, serum AST, ALT, and alkaline phosphatase. There was an inverse correlation between plasma PIVKA-II and serum 25-hydroxyvitamin D levels (P = 0.01) (Fig. 1). No significant correlations were found between plasma PIVKA-II and other measured laboratories (Table 4).
Twenty-nine patients had serum analyzed for ucOC, with a mean ucOC of 25% (median 21.3%, range 0%–57.5%). In total, 14 (48%) patients had abnormal values. One (14%) of the unsupplemented individuals had abnormal ucOC values (defined as >35%, value 40.8%), whereas 13 (59%) patients taking supplemental vitamin K had abnormal ucOC values (defined as >20%, range 21%–57.5%). Of those patients with abnormal ucOC values, 100% of unsupplemented patients and 77% of supplemented patients had elevated PIVKA-II. Spearman correlations were performed between serum ucOC and other measured parameters. Serum ucOC directly correlated with plasma PIVKA-II (P = 0.003) and serum albumin (P = 0.004), and inversely correlated with serum vitamin A (P < 0.01). No significant correlation was observed between PT, INR, or markers of cholestasis such as conjugated bilirubin or bile acids.
Because most patients (77%) were taking oral vitamin K supplements, Spearman correlations were also determined between supplemental vitamin K intake and other measured parameters. Vitamin K intake was positively correlated with serum bile acids and alkaline phosphatase, and the correlation between vitamin K intake and serum conjugated bilirubin approach significance (Table 5), indicating patients with more significant cholestatic liver disease were more likely to be taking higher dosages of supplemental vitamin K. However, no correlation was observed between vitamin K intake and PT, INR, plasma PIVKA-II, or serum ucOC (Table 5). Subsequently, Spearman correlations between plasma PIVKA-II, serum ucOC and other laboratories were reanalyzed, controlling for vitamin K intake. Apart from the correlation between PIVKA-II and alkaline phosphatase, all previously correlated parameters persisted, with the correlations between plasma PIVKA-II, serum ucOC, conjugated bilirubin, and bile acids remaining most significant (data not shown). Similarly, significant correlations between serum ucOC with previously noted parameters remained when controlling for vitamin K intake (data not shown).
Fat and fat-soluble vitamin malabsorption in cholestatic liver disease results from intraluminal bile salt concentrations falling below the critical micellar level. Deficiencies of fat-soluble vitamin deficiencies are a well-known complication of cholestasis. The results of the current study indicate that vitamin K deficiency, as assessed by plasma PIVKA-II, affects two thirds of cholestatic children and adults, with 77% having deficiencies of other fat-soluble vitamins. Of note, since vitamin E/lipid ratios were not measured, we may have underestimated vitamin E deficiency in our population.
Increased undercarboxylated prothrombin or decreased vitamin K levels have previously been identified in adults with cholestatic liver disease (13–15). Although there were discrepancies between measured vitamin K levels and PT, a limitation of these studies is that plasma vitamin K levels may not be representative of total body vitamin K stores (5). Most studies on adults have included only primary biliary cirrhosis, and therefore, the findings may not be generalizable to other cholestatic liver diseases, particularly those causing childhood cholestasis. There is a single study evaluating vitamin K deficiency in childhood cholestasis in which elevated plasma PIVKA-II was found in 48% of children of mild to moderate cholestatic liver disease, with only 4.7% having prolonged PT. Plasma PIVKA-II was positively correlated with markers of cholestasis and severity of liver disease as measured by the Child-Turcotte-Pugh classification (16). The present study similarly indicates that vitamin K deficiency, as determined by elevated PIVKA-II levels, is common in cholestatic liver disease despite oral vitamin K supplementation. Prothrombin time, a surrogate marker of vitamin K status, identified only 43% of those with increased plasma PIVKA-II, suggesting that PT underestimates the prevalence of vitamin K deficiency.
Deficiencies of other fat-soluble vitamins were present in 77% of the study patients, with vitamin D deficiency in 71% and vitamin A deficiency in 29%. Vitamin E deficiency occurred less frequently. Overall, the prevalence of vitamin D deficiency in the present study is higher than in previous reports (2,4,13,15,17,18). This discrepancy is likely due to our use of a more stringent definition of vitamin D deficiency defined as 25-hydroxyvitamin D levels <32 ng/mL. Values of 25-hydroxyvitamin D >32 ng/mL are now recognized by authorities in the field as the level required for optimal calcium absorption and bone health, and thus this value has become the standard for vitamin D sufficiency (19). Studies in children, adolescents, and adults have shown an inverse relation between serum PTH and 25-hydroxyvitamin D at levels below 32 ng/mL (20–23). The prevalence of vitamin A and E deficiency in our study is consistent with some previously reported frequencies in patients with cholestatic liver disease (2,4,13,15,18), whereas others have reported a higher prevalence (3,17,24–27). These differences may relate to the methods used to assess deficiency, particularly for vitamin E. Cholestatic liver disease can lead to hyperlipidemia, which may artificially raise vitamin E levels to normal values because vitamin E is carried in the high-density lipoprotein and low-density lipoprotein fraction of lipoproteins, thus masking true vitamin E deficiency (28,29). The measurement of the ratio of serum vitamin E to total lipids is a more accurate indicator of vitamin E status (29), and because serum total lipids, cholesterol, and triglycerides were not measured in our study, vitamin E deficiency may be underestimated in our study population. In the present study, vitamin A deficiency may be overestimated because vitamin A levels were not normalized for serum retinol binding protein, which may be reduced in decompensated liver disease (30). Because few patients had evidence of advanced hepatic decompensation as assessed by reduced serum albumin concentrations, this may not represent a significant problem in our study population.
Previous studies have found associations between vitamin K deficiency, either measured by low plasma vitamin K levels (15) or elevated undercarboxylated prothrombin (13,16), and the severity of cholestasis in adults as determined by elevated conjugated bilirubin, serum bile acids, and alkaline phosphatase. This study confirmed these findings, and we identified correlations between plasma PIVKA-II and serum aminotransferases. In the present study, the 100% concordance between prolonged PTs and elevated plasma PIVKA-II levels, significant correlation between plasma PIVKA-II and serum ucOC, another vitamin K–dependent protein, and the presence of other fat-soluble vitamin deficiencies in 81% of the patients with increased plasma PIVKA-II strongly suggests that abnormal PIVKA-II was the result of vitamin K deficiency. Although we cannot categorically conclude that malabsorption was the cause of the elevated PIVKA-II, given the above associations, this is the most plausible explanation.
Not surprisingly, vitamin K intake was positively correlated with the severity of cholestasis, suggesting that clinicians recommend supplementation in the presence of increasing cholestasis; however, plasma PIVKA-II elevation was common in these patients despite vitamin K therapy. It would be expected that a negative correlation between vitamin K intake and PT, INR, and PIVKA-II levels would be identified, but in fact there were no correlations found. This suggests that even with supplementation, intake is not sufficient to overcome the absorptive defect in these patients and current practices of oral vitamin K supplementation may be inadequate to maintain vitamin K nutriture in cholestatic liver disease. A new mixed micellar preparation of vitamin K1 (Kanakion MM) is available in Europe, but unfortunately, absorption of this formulation in cholestatic infants remains poor and unpredictable (31). In addition, few clinicians recommend parenteral vitamin K unless extremely prolonged PTs develop. Future studies examining the dosage and route of vitamin K administration are, therefore, indicated.
Clinical characteristics and medical management may differ between adult and pediatric patients. In the present study, there was a trend for the pediatric patients to be more cholestatic, with higher conjugated bilirubin and serum bile acids, factors that are highly correlated with PIVKA-II elevation. Additionally, pediatric patients were more likely to be receiving supplemental vitamin K replacement. Only 1 adult patient with Alagille syndrome studied at the pediatric center was receiving vitamin K therapy.
This study has some limitations. The sample size is small, particularly in regards to the adult population. Although the study had sufficient power to evaluate correlations between plasma PIVKA-II and markers of cholestasis within the study population, differences between various cholestatic conditions and adult and pediatric patients and predictors of plasma PIVKA-II elevation within these subgroups could not be assessed. The study patients are a convenience sample and may not be completely representative of children and adults with cholestasis; however, there is no reason to believe that our patients would not be representative of all pediatric and adult cholestatic patients. Measurement of serum lipids and retinal binding protein were not performed, which may have influenced the identified prevalence of deficiencies of vitamin E and A in this population. Finally, many patients (58%) were taking antibiotics at the time of study enrollment, which may alter intestinal bacteria and thus influence menoquinone production. However, most of these patients (89%) were receiving oral vitamin K supplementation and it is doubtful that the antibiotics had any measurable effect on their vitamin K metabolism.
In summary, despite vitamin K supplementation elevated plasma PIVKA-II is common in cholestatic liver disease and frequently accompanies other fat-soluble vitamin deficiencies, suggesting continued vitamin K deficiency in these patients. Patients with advanced cholestasis, with increased serum conjugated bilirubin and serum bile acids, are at highest risk for PIVKA-II elevation. Prothrombin time may underestimate the prevalence of vitamin K deficiency as determined by elevated plasma PIVKA-II. Better strategies for vitamin K assessment and guidelines for specific dosing in cholestatic liver disease should be developed.
The authors would like to acknowledge the following individuals for their thoughtful comments, advice, and their assistance in this project: the physicians and nurses of the Division of Gastroenterology, Hepatology, and Nutrition at CCHMC, Kenneth Sherman, MD, Stephen Zucker, MD, Caren Gundberg, PhD, the General Clinical Research Center nurses and staff, and all of the parents and patients.
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Keywords:© 2009 Lippincott Williams & Wilkins, Inc.
Cholestasis; PIVKA-II; Vitamin K