9. Shih YJ, Baynes RD, Hudson BG, Flowers CH, Skikne BS, Cook JD. Serum ... Cie`ncies de la Salut, Hospital Universitari de Sant Joan,. C/. Sant Joan s/n, ...
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from plasma (12 ). When sera taken from IDA patients are stored for a long time before being measured, there is a possibility that, in some assay systems, values may be underestimated because TfR free of Tf, which is found predominantly in IDA sera, readily loses its immunologic reactivity. References 1. Kohgo Y, Nishisato T, Kondo H, Tsushima N, Niitsu Y, Urushizaki I. Circulating transferrin receptor in human serum. Br J Haematol 1986;64: 277– 81. 2. Kohgo Y, Niitsu Y, Kondo H, Kohgo Y, Niitsu Y, Kondo H, et al. Serum transferrin receptor as a new index of erythropoiesis. Blood 1987;70: 1955– 8. 3. Cook JD, Skikne BS. Iron deficiency: definition and diagnosis. J Intern Med 1989;226:349 –55. 4. Ahn J, Johnstone RM. Origin of a soluble truncated transferrin receptor. Blood 1993;81:2442–51. 5. Cook JD. Iron-deficiency anemia. Baillieres Clin Haematol 1994;7:787– 804. 6. Ferguson BJ, Skikne BS, Simpson KM, Baynes RD, Cook JD. Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia. J Lab Clin Med 1992;119:385–90. 7. Punnonen K, Irjala K, Rajamaki A. Iron-deficiency anemia is associated with high concentrations of transferrin receptor in serum. Clin Chem 1994;40: 774 – 6. 8. Punnonen K, Irjala K, Rajamaki A, Rajamaki A. Serum transferrin receptor and its ratio to serum ferritin in diagnosis of iron-deficiency. Blood 1997; 89:1052–7. 9. Shih YJ, Baynes RD, Hudson BG, Flowers CH, Skikne BS, Cook JD. Serum transferrin receptor is a truncated form of tissue receptor. J Biol Chem 1990;265:19077– 81. 10. Rutledge EA, Root BJ, Lucas JJ, Enns CA. Elimination of the O-linked glycosylation site at Thr 104 results in the generation of a soluble human-transferrin receptor. Blood 1994;83:580 – 6. 11. Shih YJ, Baynes RD, Hudson BG, Cook JD. Characterization and quantitation of the circulating forms of serum transferrin receptor using domain-specific antibodies. Blood 1993;81:234 – 8. 12. Hikawa A, Nomata Y, Suzuki T, Ozasa H, Yamada O. Soluble transferrin receptor-transferrin complex in serum: measurement by latex agglutination nephelometric immunoassay. Clin Chim Acta 1996;254:159 –72. 13. Lash A, Saleem A. Iron metabolism and its regulation. A review. Ann Clin Lab Sci 1995;25:20 –30. 14. Uchida T. Overview of iron metabolism. Int J Hematol 1995;62:193–202.
Plasma Homocysteine Concentrations in Patients with Liver Cirrhosis, Nata`lia Ferre´,1 Frederic Go´ mez,1 Jordi Camps,1* Josep M. Simo´ ,1 Michelle M. Murphy,2 Joan Ferna´ ndez-Ballart,2 and Jorge Joven1 (1 Centre de Recerca Biome`dica and 2 Unitat de Medicina Preventiva, Facultat de Medicina i Cie`ncies de la Salut, Institut de Recerca en Cie`ncies de la Salut, Hospital Universitari de Sant Joan, C/. Sant Joan s/n, 43201-Reus, Catalunya, Spain; * author for correspondence: fax 34-977-312569, e-mail jcamps@ grupsgs.com) Plasma homocysteine (tHcy) is a marker of folate or cobalamin deficiency states (1 ) and a risk factor for cardiovascular diseases (2 ), and is altered by renal insufficiency (3 ). Increased tHcy in liver diseases may also play a role in hepatic disorders (4, 5 ). Chronic treatment of experimental animals with ethanol or CCl4 is associated with hyperhomocysteinemia, and the hepatoprotective effect of S-adenosylmethionine on experimental cirrhosis is accompanied by a normalization of methionine metabolism and a decrease in tHcy concentration (4, 5 ). Studies
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in cultured hepatocytes suggest a role of the liver in metabolism of Hcy (6 ). However, the presence and degree of hyperhomocysteinemia in patients with liver disease and its modulation by chronic alcohol intake are, as yet, not well defined. We studied 76 patients with liver cirrhosis (55 men and 21 women; age range, 57 ⫾ 11 years) who were being treated in the outpatient clinic of Hospital Universitari de Sant Joan. The diagnosis of cirrhosis was based on liver biopsy or on clinical evidence, including echography to evaluate splenomegaly and portal vein dilation and fiberoptic gastroscopy to detect the presence of gastroesophageal varices. The etiology of cirrhosis was alcoholic in 48 patients (63%) and nonalcoholic in 28 (37%). Twenty-nine of the 48 alcoholic patients studied had stopped alcohol consumption at least 3 months before the study; the other 19 had continued drinking. The severity of the liver disease was measured in all patients by the Child–Pugh score (7 ). This classification estimates the severity of cirrhosis based on biochemical and clinical indices. The concept is to give a numeric score to certain aspects of liver function (plasma albumin and bilirubin concentrations, prothrombin time, and the degree of ascites and hepatic encephalopathy). Adding these numbers together provides a final score of liver cell function (7 ). The control group consisted of 83 healthy volunteers (36 men and 47 women; age range, 42 ⫾ 14 years) participating in an epidemiologic study being conducted in our area. In all participants, venous blood was collected, after an overnight fast, into sodium EDTA-containing tubes kept at 4 °C for tHcy, folate, and cobalamin determinations, or into tubes with no anticoagulants added for the other biochemical tests. The tubes were centrifuged at 2500g at 4 °C, and plasma or serum was stored at ⫺80 °C. This handling minimizes preanalytical errors in tHcy measurements (8 ). tHcy was measured by fluorescence polarization immunoassay (9 ). Folate and cobalamin concentrations were measured by ion-capture and microparticle immunoassay, respectively, in an AxSYM® analyzer (Abbott Laboratories). Serum alanine aminotransferase, alkaline phosphatase, and ␥-glutamyltransferase activities and albumin and bilirubin concentrations were measured by standard techniques (ITC Diagnostics). Hemoglobin and erythrocyte mean corpuscular volume were measured in a Coulter® STKS counter (Beckman Coulter). Means were compared by a linear multivariate model taking into account age and gender. Pearson correlation coefficients were used to evaluate the degree of association between pairs of variables. When the distribution of variables was skewed, statistical analysis was performed using log-transformed data. All calculations were performed with the SPSS 10.0 statistical package. As expected, mean serum bilirubin concentrations and mean alanine aminotransferase activities were higher in cirrhotic patients than in the controls (Table 1). The mean corpuscular volume of red blood cells and mean serum ␥-glutamyltransferase activity were significantly increased, especially in nonabstaining alcoholic cirrhotics.
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Technical Briefs
Table 1. Summary of results. Hcy (mol/L) Folate, nmol/L Cobalamin, ng/L Hemoglobin, g/L MCV, fL Albumin, g/L Bilirubin, mol/L ALT, kat/L ALP, kat/L GGT, kat/L a
Controls (n ⴝ 83)
Nonalcoholic LCa (n ⴝ 28)
Abstaining LC (n ⴝ 29)
Nonabstaining LC (n ⴝ 19)
9.3 ⫾ 2.5 25.5 ⫾ 9.7 530 ⫾ 180 149 ⫾ 14 90.7 ⫾ 5.2 42 ⫾ 2 9⫾6 0.40 ⫾ 0.35 2.62 ⫾ 0.76 0.45 ⫾ 0.28
10.3 ⫾ 3.2 17.9 ⫾ 9.0c 776 ⫾ 449 135 ⫾ 20 97.3 ⫾ 7.7b 39 ⫾ 8 24 ⫾ 18b 1.51 ⫾ 1.11b 2.27 ⫾ 1.91 1.14 ⫾ 1.47b
12.5 ⫾ 9.5 17.1 ⫾ 9.4b 623 ⫾ 304 135 ⫾ 32 93.5 ⫾ 11.4 42 ⫾ 7 29 ⫾ 45b 0.85 ⫾ 0.86b 1.85 ⫾ 0.90b 0.80 ⫾ 0.40b
14.9 ⫾ 9.4b 10.6 ⫾ 6.0b 855 ⫾ 472c 151 ⫾ 17 102.4 ⫾ 6.4b 39 ⫾ 7 35 ⫾ 33b 0.99 ⫾ 0.64b 2.57 ⫾ 2.53 4.09 ⫾ 3.37b
LC, liver cirrhosis; MCV, mean erythrocyte corpuscular volume; ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, ␥-glutamyl transferase. Significance with respect to controls: b P ⬍0.001; c P ⬍0.01.
b,c
Mean plasma folate concentrations were mildly decreased in nonalcoholic and abstaining cirrhotics and more severely decreased in nonabstaining alcoholic cirrhotics. The mean plasma cobalamin concentration was significantly increased in nonabstaining cirrhotics, presumably because of hepatic release from injured hepatocytes (10 ). Mean tHcy concentrations were similar in the controls and the nonalcoholic cirrhotics, but were significantly higher in nonabstaining alcoholic cirrhotics. An intermediate situation applied to abstaining alcoholic cirrhotics, in whom the differences from controls did not reach statistical significance (Table 1). There was a significant inverse correlation between tHcy and folate concentrations in controls (r ⫽ ⫺0.35; P ⬍0.001), but not in cirrhotic patients (r ⫽ ⫺0.17; P, not significant). tHcy concentration (log transformed) was inversely correlated (r ⫽ ⫺0.35; P ⬍0.05) with the severity of the liver disease measured by the Child–Pugh score in alcoholic cirrhotics (Fig. 1). The present study shows that the mean tHcy concen-
Fig. 1. Relationship between plasma tHcy concentration (log transformed) and Child–Pugh score in patients with cirrhosis.
tration in patients with liver cirrhosis is related to the etiology of their disease, alcohol intake habits at the time of the study, and the severity of their liver impairment. Chronic alcoholism may influence alterations in Hcy by promoting hepatic folate deficiency (1 ). However, the lack of correlation between tHcy and folate concentrations in cirrhotic patients suggests that other factors may also play a role. Studies in experimental models have shown that chronic alcohol intake may increase tHcy concentration through a direct inhibitory effect of ethanol on hepatic methionine synthase activity, which would cause a compensatory increase of betaine/homocysteine methyltransferase activity and Hcy synthesis (3, 11 ). The inverse correlation of tHcy with Child–Pugh score may be explained by a reduction in the functional mass of liver cells in the most severe cirrhotic patients. Hyperhomocysteinemia and the related disturbances of methionine metabolism may inhibit cell proliferation (12 ) and produce hypomethylation and, consequently, destabilization of DNA (5 ) in patients with liver disease. The intracellular concentration of glutathione may decrease, thus increasing lipid peroxide production and collagen synthesis (13 ). An interesting recent study showed that Hcy directly induces the expression of procollagen type I and tissue inhibitor of metalloproteinases-1 genes in hepatocytes and stellate cells in vitro (14 ). This suggests that Hcy may be an effective inducer of liver fibrogenesis. We conclude that the tHcy concentration is influenced by alcohol intake and the degree of liver impairment in cirrhosis. These results raise questions regarding the utility of tHcy as a marker of folate deficiency in patients with impaired liver function. However, tHcy measurement may be relevant in further research on the biochemistry of chronic liver impairment.
This study was supported, in part, by a grant from Fondo de Investigaciones Sanitarias (00/0954). N.F. was recipient of a fellowship from the Generalitat de Catalunya (FI/FIAP-99).
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