Katsuya Maruyama

Increased Serum Levels of Pigment Epithelium-Derived Factor by Excessive Alcohol Consumption—Detection and Identification by a Three-Step Serum Proteome Analysis

BACKGROUNDnThe search for biological markers of alcohol abuse is of continual interest in experimental and clinical alcohol research. We previously used gel-free proteome analysis methods such as the ProteinChip(®) system and the ClinProt™ system to search for new serum markers for alcoholism and found several novel marker candidates. As serum contains thousands of proteins and peptides that are present in a large dynamic concentration, depletion of the abundant proteins and further fractionation of the remainder is necessary to get into the deep proteome. We recently described a simple and highly reproducible three-step method for identifying potential disease-marker candidates among the low-abundance serum proteins.nnnMETHODSnTwo serum samples-one on admission and one after 8 weeks of abstinence-were obtained from 8 patients with alcohol dependency. The samples were subjected to a three-step serum proteome analysis. The steps were the following: first, immunodepletion of the 6 most abundant proteins; second, fractionation using reverse-phase high-performance liquid chromatography; and third, separation using one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Differences revealed by protein staining were further confirmed by Western blotting and by enzyme-linked immunosorbent assays (ELISA).nnnRESULTSnThree-step serum proteome analysis revealed that the serum levels of 5 proteins, alpha2-HS glycoprotein, apolipoprotein A-I, glutathione peroxidase 3, heparin cofactor II, and pigment epithelial-derived factor (PEDF), were significantly greater on admission than after 8 weeks of abstinence. We focused on PEDF because alterations in its levels in alcoholic subjects are not well known. Western blotting and ELISA confirmed the upregulation of PEDF. Serum PEDF levels were significantly greater in moderate to heavy habitual drinkers (14.2 ± 7.7 μg/ml) than in healthy subjects without a drinking history (5.5 ± 3.0 μg/ml) (p < 0.001). The serum PEDF levels in subjects with nonalcoholic chronic liver diseases were comparable to the PEDF levels in healthy subjects.nnnCONCLUSIONnu2002 Three-step serum proteome analysis reveals that excessive alcohol drinking increases the PEDF level.

Blood Ethanol Levels of Nonabstinent Japanese Alcoholic Men in the Morning After Drinking and Their ADH1B and ALDH2 Genotypes

AIMSnGenetic polymorphisms of alcohol dehydrogenase-1B (ADH1B, rs1229984) and aldehyde dehydrogenase-2 (ALDH2, rs671) affect ethanol (EtOH) metabolism and susceptibility to alcoholism.nnnMETHODSnWe evaluated associations between ADH1B/ALDH2 genotypes and the blood EtOH levels of 805 Japanese alcoholic men in the morning after they had drunk within the previous 34 h.nnnRESULTSnAge-adjusted usual alcohol consumption did not differ according to ADH1B/ALDH2 genotypes. Higher blood EtOH levels persisted for longer periods in the ADH1B*1/*1 carriers (n = 246) than in the ADH1B*2 carriers (n = 559). Blood EtOH levels did not differ by ALDH2 genotype. The blood EtOH levels ≥ 0.3 mg/ml (criterion for drunk driving in Japanese law) were observed (40% vs. 14-17%, P < 0.0001) in a higher proportion of the ADH1B*1/*1 carriers than of the ADH1B*2 carriers after a 12.1-to-18-h interval since the last drink. Multivariate analyses showed that the EtOH levels heightened by 0.500 mg/ml in the presence of ADH1B*1*1 and by 0.248 mg/ml in the presence of cirrhosis, and lowered by 0.120 mg/ml per 10-year age increase, by 0.087 mg/ml per 10-kg body-weight increase and by 0.673 mg/ml per 10-h interval since the last drink. The odds ratio (95% confidence interval) for an EtOH level ≥ 0.3 mg/ml was 3.44 (2.34-5.04) in the presence of ADH1B*1/*1, 2.01 (1.28-3.14) in the presence of cirrhosis, 0.59 (0.49-0.71) per 10-year age increase, 0.80 (0.68-0.95) per 10-kg body-weight increase and 0.10 (0.07-0.15) per 10-h interval since the last drink.nnnCONCLUSIONnThe longer-than-expected EtOH lingering in the blood of the ADH1B*1/*1 alcoholics may exacerbate alcohol-related problems, including drunk driving.

Effect of a Comprehensive Lifestyle Modification Program on the Bone Density of Male Heavy Drinkers

BACKGROUNDnHeavy alcohol drinking is implicated in osteoporosis. Although abstinence is rapidly followed by a restoration of osteoblastic activity, little is known about the contributions of alcohol-related factors or the effectiveness of a lifestyle modification program (LMP) on bone density.nnnMETHODSnWe conducted a study of 138 male alcoholic patients to investigate whether drinking history and concurrent factors were associated with the bone density of the calcaneus. A 2.5-months LMP in an institutionalized setting was completed by 20 of them, and its effect on bone density, serum parathyroid hormone (PTH), and 1.25-(OH)(2) vitamin D levels were assessed.nnnRESULTSnThe patients had a high prevalence of daytime drinking (93.5%), continuous drinking (84.1%), and current smoking (82.0%) with mean duration of alcohol abuse of 30.0 +/- 12.8 years. The patients had lower bone density than a reference control group (Z-scores: -0.45 +/- 1.02). Multiple stepwise regression analysis identified age, poor activities of daily living (ADL), continuous drinking, absence of liver cirrhosis, depression, and dementia as determinants of low bone density. The bone density of the 20 participants in the LMP improved 2.3% (p = 0.0003) with a more ameliorating effect on bone density than a conventional abstinence therapy (p = 0.014 for interventional effect). The upper normal range of PTH levels at baseline were significantly decreased, and 1.25-(OH)(2) vitamin D levels also had a trend toward decrease during the abstinence.nnnCONCLUSIONSnAlcoholic patients may have many complications such as poor ADL and dementia, which are independently associated with decreased bone density. The results of this study support the idea that comprehensive approach to lifestyle factors to minimize risk of osteoporosis is the best way to improve bone density.

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes Are Strong Determinants of the Serum Triglyceride and Cholesterol Levels of Japanese Alcoholic Men.

Background Elevated serum triglyceride (TG) and high-density-lipoprotein cholesterol (HDL-C) levels are common in drinkers. The fast-metabolizing alcohol dehydrogenase-1B encoded by the ADH1B*2 allele (vs. ADH1B*1/*1 genotype) and inactive aldehyde dehydrogenase-2 encoded by the ALDH2*2 allele (vs. ALDH2*1/*1 genotype) modify ethanol metabolism and are prevalent (≈90% and ≈40%, respectively) in East Asians. We attempted to evaluate the associations between the ADH1B and ALDH2 genotypes and lipid levels in alcoholics. Methods The population consisted of 1806 Japanese alcoholic men (≥40 years) who had undergone ADH1B and ALDH2 genotyping and whose serum TG, total cholesterol, and HDL-C levels in the fasting state had been measured within 3 days after admission. Results High serum levels of TG (≥150 mg/dl), HDL-C (>80 mg/dl), and low-density-lipoprotein cholesterol (LDL-C calculated by the Friedewald formula ≥140 mg/dl) were observed in 24.3%, 16.8%, and 15.6%, respectively, of the subjects. Diabetes, cirrhosis, smoking, and body mass index (BMI) affected the serum lipid levels. Multivariate analysis revealed that the presence of the ADH1B*2 allele and the active ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) for a high TG level (2.22 [1.67–2.94] and 1.39 [0.99–1.96], respectively), and decreased the OR for a high HDL-C level (0.37 [0.28–0.49] and 0.51 [0.37–0.69], respectively). The presence of the ADH1B*2 allele decreased the OR for a high LDL-C level (0.60 [0.45–0.80]). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs for high TG levels and lowest OR for a high HDL-C level. The genotype effects were more prominent in relation to the higher levels of TG (≥220 mg/dl) and HDL-C (≥100 mg/dl). Conclusions The fast-metabolizing ADH1B and active ALDH2, and especially a combination of the two were strongly associated with higher serum TG levels and lower serum HDL-C levels of alcoholics. The fast-metabolizing ADH1B was associated with lower serum LDL-C levels.

Macrocytosis, Macrocytic Anemia, and Genetic Polymorphisms of Alcohol Dehydrogenase-1B and Aldehyde Dehydrogenase-2 in Japanese Alcoholic Men

BACKGROUNDnOxidation of ethanol by alcohol dehydrogenase (ADH) generates acetaldehyde (AcH), which is converted to acetate by aldehyde dehydrogenase-2 (ALDH2). Roughly 40% of East Asians are ALDH2-deficient due to an inactive enzyme encoded by the ALDH2*2 allele. ALDH2-deficient individuals have a dramatically elevated risk of esophageal cancer from alcohol consumption.nnnMETHODSnWe investigated the relationship between ALDH2*2, ADH1B*2 (encoding a highly active ADH) and erythrocyte abnormalities, in a population of Japanese alcoholic men (N = 1,238).nnnRESULTSnMacrocytosis (mean corpuscular volume [MCV] ≥100 fl) and macrocytic anemia (MCV ≥100 fl and hemoglobin <13.5 g/dl) were found in 62.4 and 24.1% of the subjects, respectively. Age-adjusted daily alcohol consumption did not differ according to ADH1B and ALDH2 genotypes. However, macrocytosis and macrocytic anemia were strongly associated with the ALDH2*1/*2 genotype multivariate odds ratios (ORs; 95% confidence interval [CI] = 2.85 [1.95 to 4.18] and 3.68 [2.64 to 5.15], respectively, versus ALDH2*1/*1). In comparison with the ADH1B*1/*1 and ALDH2*1/*1 genotype combination, the ADH1B*1/*1 and ALDH2*1/*2 genotype combination and the ADH1B*2 allele and ALDH2*1/*2 genotype combination increased stepwise the ORs (95% CI) for macrocytosis (1.65 [0.92 to 2.94] and 4.07 [2.33 to 7.11], respectively, p for difference in OR = 0.015) and macrocytic anemia (2.80 [1.52 to 5.15] and 5.32 [3.29 to 8.62], respectively, p for difference in OR = 0.045). Genotype effects were more prominent on the risks of the more advanced erythrocyte abnormalities. Older age, cigarette smoking, and low body mass index independently increased the risks of the erythrocyte abnormalities. Consumption of beer, which contains folate, decreased the risks, whereas consumption of alcoholic beverages lacking folate did not.nnnCONCLUSIONSnThese results suggest that the erythrocyte abnormalities in alcoholics are attributable to high AcH exposure as well as to nutritional deficiencies and may be prevented by folate.

The measurement of a fibrinogen α C-chain 5.9 kDa fragment (FIC 5.9) using MALDI-TOF MS and a stable isotope-labeled peptide standard dilution

BACKGROUNDnWe previously identified a 5.9 kDa peptide fragment of fibrinogen α C-chain (FIC 5.9) as a novel biomarker candidate for heavy drinking. In an effort to improve FIC 5.9 measurement for potential use in clinical diagnostics, we combined the ClinProt System and a stable isotope-labeled peptide standard dilution as a simple and reproducible system for measuring FIC 5.9.nnnMETHODSnWe analyzed 104 serum samples that were obtained from patients with alcohol dependency, from patients with chronic hepatitis C, and from healthy volunteers. Serum FIC 5.9 levels were measured using the ClinProt system with and without a stable isotope-labeled synthetic FIC 5.9 as an internal standard.nnnRESULTSnThe within-day and between-day CVs were significantly smaller with stable isotope dilution mass spectrometry (SID-MS) than with conventional MALDI-TOF MS. Of the two different MALDI-TOF MS platforms, we obtained concordant results with SID-MS. Furthermore, only SID-MS detected a small but significant difference between the serum FIC 5.9 levels in the chronic hepatitis C group and the controls.nnnCONCLUSIONSnMALDI-TOF MS with a stable isotope-labeled peptide spike can determine serum FIC 5.9 levels more precisely than conventional MS. This will make inter-laboratory FIC 5.9 comparisons possible.

Alcoholic Ketosis: Prevalence, Determinants, and Ketohepatitis in Japanese Alcoholic Men

AIMSnAlcoholic ketosis and ketoacidosis are metabolic abnormalities often diagnosed in alcoholics in emergency departments. We attempted to identify determinants or factors associated with alcoholic ketosis.nnnMETHODSnThe subjects of this cross-sectional survey were 1588 Japanese alcoholic men (≥40 years) who came to an addiction center within 14 days of their last drink.nnnRESULTSnThe results of the dipstick urinalyses revealed a prevalence of ketosis of 34.0% (±, 21.5%; +, 8.9%; and 2+/3+; 3.6%) in the alcoholics. Higher urine ketone levels were associated with higher serum total bilirubin, aspartate transaminase (AST), alanine transaminase and gamma-glutamyl transpeptidase levels. A multivariate analysis by the proportional odds model showed that the odds ratio (95% confidence interval) for an increase in ketosis by one category was 0.94 (0.84-1.06) per 10-year increase in age, 0.93 (0.89-0.97) per 1-day increase in interval since the last drink, 1.78 (1.41-2.26) in the presence of slow-metabolizing alcohol dehydrogenase-1B (ADH1B*1/*1), 1.61 (1.10-2.36) and 1.30 (1.03-1.65) when the beverage of choice was whiskey and shochu, respectively (distilled no-carbohydrate beverages vs. the other beverages), 2.05 (1.27-3.32) in the presence of hypoglycemia <80 mg/dl, 0.91 (0.88-0.94) per 1-kg/m(2) increase in body mass index (BMI), 1.09 (1.00-1.18) per +10 cigarettes smoked, and 2.78 (2.05-3.75) when the serum total bilirubin level was ≥2.0 mg/dl, and 1.97 (1.47-2.66) when the serum AST level was ≥200 IU/l.nnnCONCLUSIONnKetosis was a very common complication and frequently accompanied by alcoholic liver injury in our Japanese male alcoholic population, in which ADH1B*1/*1 genotype, consumption of whiskey or shochu, hypoglycemia, lower BMI and smoking were significant determinants of the development of ketosis.

Alcohol Dehydrogenase-1B (rs1229984) and Aldehyde Dehydrogenase-2 (rs671) Genotypes and Alcoholic Ketosis Are Associated with the Serum Uric Acid Level in Japanese Alcoholic Men.

AIMSnTo identify determinants of hyperuricemia in alcoholics.nnnMETHODSnThe serum uric acid (UA) levels of 1759 Japanese alcoholic men (≥40 years) were measured on their first visit or within 3 days after admission; ADH1B and ALDH2 genotyping on blood DNA samples were performed. Dipstick urinalyses for ketonuria and serum UA measurements were simultaneously performed for 621 men on their first visit.nnnRESULTSnSerum UA levels of >416 μmol/l (7.0 mg/dl) and ≥535 μmol/l (9.0 mg/dl) were observed in 30.4 and 7.8% of the subjects, respectively. Ketonuria was positive in 35.9% of the subjects, and a multivariate analysis revealed that the ketosis level was positively associated with the UA level. The presence of the ADH1B*2 allele and the ALDH2*1/*1 genotype increased the odds ratio (OR; 95% confidence interval) among subjects with a high UA level of >416 μmol/l (vs. ≤416 μmol/l; 2.04 [1.58-2.65] and 1.48 [1.09-2.01], respectively) and those with a high UA level of ≥535 μmol/l (vs. ≤416 μmol/l; 2.29 [1.42-3.71] and 3.03 [1.51-6.08], respectively). The ADH1B*2 plus ALDH2*1/*1 combination yielded the highest ORs (2.86 [1.61-5.10] and 6.21 [1.49-25.88] for a UA level of >416 μmol/l and ≥535 μmol/l, respectively), compared with the ADH1B*1/*1 plus ALDH2*1/*2 combination. The presence of diabetes and the consumption of Japanese sake rather than beer were negatively associated with the UA levels.nnnCONCLUSIONSnThe faster metabolism of ethanol and acetaldehyde by the ADH1B*2 allele and ALDH2*1/*1 genotype and higher ketosis levels were associated with higher UA levels in alcoholics, while diabetes and the consumption of sake were negative determinants.

A transient increase in cerebrospinal fluid tau level after epileptic seizure in an elderly patient.

1. Phan A, Touzet S, Dalle S et al. Acral lentiginous melanoma: A clinicoprognostic study of 126 cases. Br J Dermatol 2006;155:561–569. 2. Kuchelmeister C, Schaumburg-Lever G, Garbe C. Acral cutaneous melanoma in Caucasians: Clinical features, histopathology and prognosis in 112 patients. Br J Dermatol 2000;143:275–280. 3. Banfield CC, Redburn JC, Dawber RPR. The incidence and prognosis of nail apparatus melanoma. A retrospective study of 105 patients in four English regions. Br J Dermatol 1998;139:276–279. 4. De Giorgi V, Stante M, Carelli G et al. Subungual melanoma: An insidious erythematous nodule on the nail bed. Arch Dermatol 2005;141:398–399. 5. Soon SL, Solomon AR, Papadopoulos D et al. Acral lentiginous melanoma mimicking benign disease: The Emory experience. J Am Acad Dermatol 2003;48:183–188. 6. Harrington P, O’Kelly A, Trail IA et al. Amelanotic subungual melanoma mimicking pyogenic granuloma in the hand. J R Coll Surg Edinb 2002;47:638– 640. 7. Kato T, Suetake T, Sugiyama Yet al. Epidemiology and prognosis of subungual melanoma in 34 Japanese patients. Br J Dermatol 1996;134:383–387. 8. Quinn MJ, Thompson JE, Crotty K et al. Subungual melanoma of the hand. J Hand Surg 1996;21:506–511. 9. Richard MA, Grob JJ, Avril MF et al. Delays in diagnosis and melanoma prognosis (I): The role of patients. Int J Cancer 2000;89:271–279. 10. Richard MA, Grob JJ, Avril MF et al. Delays in diagnosis and melanoma prognosis (II): The role of doctors. Int J Cancer 2000;89:280–285. 11. Thai K, Young R, Sinclair RD. Nail apparatus melanoma. Australas J Dermatol 2001;42:71–81. 12. Levit EK, Kagen MH, Scher RH et al. The ABC rule for clinical detection of subungual melanoma. J Am Acad Dermatol 2000;42:269–274.

Higher Serum Free Glycerol Levels in a Group of Alcoholics than in Controls

BACKGROUNDnThe biomarkers of excessive alcohol intake reported thus far have not always been reliable. We discovered the presence of free glycerol (FG) by high-performance liquid chromatography (HPLC) in the serum of alcoholic patients but not in healthy persons, and a higher percentage of the alcoholics were positive for serum FG than for gamma-glutamyl transpeptidase, mean corpuscular volume, or carbohydrate-deficient transferrin (%CDT). Therefore, in this study, we investigated whether the same results as with HPLC could be obtained by measuring FG with an easy-to-use autoanalyzer and whether the serum FG measured by this method would be useful as a biomarker of excessive alcohol intake.nnnMETHODSnFirst, the measurements of serum FG made by HPLC and by a biochemical method with an autoanalyzer were tested for a correlation, and then fasting serum FG was measured with the autoanalyzer in 3 groups: a group of Japanese male alcoholics who drank until just before admission, a group of Japanese male patients with chronic viral hepatitis, and a group of Japanese healthy male volunteers.nnnRESULTSnThere was a strong positive correlation between the serum FG values measured by HPLC and by the autoanalyzer in the alcoholic group. The values in the alcoholic group were significantly higher than in the viral hepatitis group and healthy control group. We set the cutoff serum FG value for discriminating between the alcoholic group and the other 2 groups in the receiver operating characteristic analysis at 0.9xa0mg/dl, and that cutoff value provided a sensitivity of 80% for identifying the alcoholic group and a specificity of 84 and 94% in relation to the viral hepatitis group and the healthy volunteer group, respectively. Among various clinical tests in the alcoholic group, serum FG showed the highest rate of abnormally high value.nnnCONCLUSIONSnMeasurement of serum FG with an autoanalyzer may be useful as a biomarker of excessive alcohol intake.