Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

Vatsalya Vatsalya; Ruchita Agrawal; Jane Frimodig; Shweta Srivastava; Melanie L. Schwandt

doi:10.1155/2020/7835875

Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

Vatsalya, Vatsalya;Agrawal, Ruchita;Frimodig, Jane;Srivastava, Shweta;Schwandt, Melanie L. 2020-06-09 00:00:00 Hindawi Advances in Virology Volume 2020, Article ID 7835875, 9 pages https://doi.org/10.1155/2020/7835875 Research Article Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients 1,2,3,4 5 1,2 6 Vatsalya Vatsalya , Ruchita Agrawal, Jane Frimodig, Shweta Srivastava, and Melanie L. Schwandt Department of Medicine, University of Louisville, Louisville, KY, USA Robley Rex VA Medical Center, Louisville, KY, USA University of Louisville, Alcohol Research Center, Louisville, KY, USA National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA Department of Psychiatry, University of Louisville, Louisville, KY, USA Environmental Health Institute, University of Louisville, Louisville, KY, USA Correspondence should be addressed to Vatsalya Vatsalya; v0vats01@exchange.louisville.edu Received 19 February 2020; Accepted 30 April 2020; Published 9 June 2020 Academic Editor: Finn S. Pedersen Copyright © 2020 Vatsalya Vatsalya et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Alcohol use disorder (AUD) patients comorbid with hepatitis C virus (HCV) infection (HCV + AUD) could have progressively severe clinical sequels of liver injury and inﬂammation. Serum zinc and several polyunsaturated fatty acids (PUFAs) get dys- regulated in AUD as well as HCV. However, the extent of dysregulation of PUFAs and zinc deﬁciency and their interaction in HCV + AUD as a comorbid pathology has not been studied. We examined the role of dysregulation of FAs and low zinc in HCV + AUD patients. 138 male and female participants aged 21–67 years were grouped as HCV-only (Gr. 1; n � 13), HCV + AUD (Gr. 2; n � 25), AUD without liver injury (Gr. 3; n � 37), AUD with liver injury (Gr. 4; n � 51), and healthy volunteers (Gr. 5 or HV; n � 12). Drinking history, individual demographic measures, fasting fatty acids, liver function, and zinc were measured and analyzed. HCV + AUD patients showed the highest ALT level compared to the rest of the groups. Serum zinc concentrations were the lowest, and the proinﬂammatory shift was the highest (characterized by ω6 : ω3 ratio) in the HCV + AUD patients. Total ω3, eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA5,3) were the lowest in HCV + AUD patients. Total ω3, α-linoleic acid (α-LA) along with covariable number of drinking days past 90 days (NDD90), eicosapentaenoic acid (EPA), and doco- sapentaenoic acid (DPA5,3) independently showed signiﬁcant association with low zinc in the HCV + AUD patients. Heavy drinking pattern showed that NDD90 has a signiﬁcant mediating role in the representation of the relationship between candidate ω3 PUFAs and zinc uniquely in the HCV + AUD patients. Low serum zinc showed a distinctively stronger association with total and candidate ω3s in the HCV + AUD patients compared to the patients with HCV or AUD alone, supporting dual mechanism involved in the exacerbation of the proinﬂammatory response in this comorbid cohort. 'is trial is registered with NCT#00001673. a major form of liver disease that is responsible for the 1. Introduction mortality and morbidity in the USA and worldwide [4]. Hepatitis C virus (HCV) is the most common chronic blood- With no FDA approved drug for the treatment of ALD, borne infection, aﬀects 5 million Americans, and causes very treatment development for the alcohol-related liver disease high morbidity and mortality [1]. Hepatitis C virus (HCV) remains an unmet need [5]. AUD diagnosed individuals has lasting morbidity and lasting eﬀect on the liver and with HCV infection (HCV + AUD) have reported a pro- overall health [2]. Alcohol use disorder (AUD) is known to pensity for liver injury compared with AUD patients, with a be consequential in alcoholic liver disease (ALD) [3]. ALD is prevalence of HCV and AUD comorbidity being 16.3% [6]. 2 Advances in Virology Fatty acid synthesis has been shown to regulate Hepatitis these participants was as follows: Gr. 1: HCV-only (n � 13); C Virus (HCV) entry and production (Yang, 2008), though Gr. 2: HCV + AUD (n � 25); Gr. 3: AUD without liver injury HCV has also been involved in altering fatty acid meta- (n � 37); Gr. 3: AUD with liver injury (n � 51); Gr. 5: healthy bolism thereby causing lipid accumulation in the liver volunteers (HV, n � 12). All heavy drinkers met the diag- (Yamaguchi, 2005). Fatty acids are also variable with alcohol nosis criteria of AUD according to the DSM-IV, based on use; they can be either anti- or proinﬂammatory in nature the SCID I interview. Patients were not enrolled in the study [7]. 'e role of zinc in antiviral immunity is given in [8], and if they had a diagnosis for severe psychiatric illness, suicidal, changes in zinc levels in HCV-infected patients at pre-and or violent tendencies or showed agitation requiring im- posttreatment evaluations were given in [9]. Recent studies mediate clinical treatment. Patients with other forms of have shown that zinc deﬁciency is commonly found in AUD, signiﬁcant psychiatric illnesses (unless stable, and not re- AUD with early stage of ALD, and AUD with viral infections quiring medication such as antidepressants, lithium, neu- comorbidity [9–11]. Zinc plays an important role as a co- roleptics, naltrexone, acamprosate, disulﬁram, factor in the downstream regulation of proinﬂammatory benzodiazepines, or antiepileptic compounds within the last four weeks) were not enrolled. Advanced lung disease, (ω6) and anti-inﬂammatory (ω3) PUFAs [12]. Studies on low zinc and ω3 PUFAs have been well studied separately in unstable cardiovascular disease, renal failure (creatinine AUD patients with or without liver injury as well as HCV- clearance 30 ml/min), advanced liver disease (hepatocellular infected patients. However, the representation and rela- carcinoma, clinically evident alcoholic hepatitis, and cir- tionship of lower zinc and ω3 PUFAs is not clear in AUD rhosis), and HIV were other exclusionary criteria. Other patients who are also infected with HCV. exclusions on the day of assessment were (1) pregnancy Hepatitis C infection and excessive alcohol exposure are (negative test required) or ongoing breastfeeding and/or (2) synergistic in their destructive eﬀects on the liver [13]. a positive urine screen for any illicit drug. Importantly, these Identifying the early changes in the rearrangement of fatty patients had no clinical signs of alcoholic liver disease, and acids, inﬂammation, and injury-associated nutritional since the HCV diagnosed patients had the ﬁrst-time diag- measures during patient evaluation could elucidate the nosis, there was no standard of care indication to perform a biopsy (as this study is not a treatment evaluation study). comorbid condition of HCV and AUD. Markers of heavy drinking [14] and proﬁle have shown high predictability Patients did not show any clinical evidence of liver disease, with the exacerbation of liver injury in early stage of ALD. and overt clinical liver disease was an exclusion criterion in Nonetheless, the role of such heavy drinking markers in the the study; detailed information on eligibility criteria has proinﬂammatory response as characterized by the dysre- been published previously [15]. gulation of ω3 PUFAs and low zinc has not been investigated in HCV-infected AUD patients. 'ese gaps remain an open 2.2. Clinical Data and Laboratory Measures. 'is study was a area to investigate at both the clinical level and preclinical level. one-time study at the admission clinical observational in- vestigation. Demographic information included age, sex, We aimed to characterize the variability in the fatty acid height, weight, and BMI. Blood samples were collected for a proﬁle (both speciﬁc and group-based) participating in inﬂammation and changes in serum zinc levels in newly comprehensive metabolic panel (CMP) that included a liver panel, fatty acid panel (FAP), and serum trace metals (zinc acquired-HCV patients with comorbid AUD diagnosis. We also evaluated the association of HCV-viral load and serum for this study). Serum Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), AST/ALT ratio, Total zinc and PUFAs in HCV patients with or without AUD to show the extent of linked response in this comorbid patient bilirubin (Tbili), and albumin (ALB) measures were eval- uated for the extent of liver injury. Alanine Aminotrans- cohort. ferase (ALT) level was used as a reference to assess liver injury (Medline Plus-National Institutes of Health, 2014); 2. Methodology 40 IU/L for ALT was used as the upper limit of normal, and values ≥41 IU/L indicated liver injury as per the guideline 2.1. Patient Recruitment. 'e primary patient cohort of this that existed till 2014. 'e standard lower limit of normal for study was AUD patients with HCV infection; other cohorts serum zinc was 71 mcg/dL in this study. 'e comprehensive were included for comparison purposes only. 'is investi- fatty acid panel measured speciﬁc and total ω3 and ω6 FA gation was performed as one of the several projects under a levels using gas chromatography/mass spectrometry. 'e larger clinical study approved by the Institutional Review screening was performed to identify patients with HCV, Board of the National Institute on Alcohol Abuse and Al- which was conducted with COBAS Ampliprep/COBAS coholism (NIAAA), National Institutes of Health (NIH), Taqman HCV test methodology; HCV diagnosis was a ﬁrst- Bethesda, MD, USA (ClinicalTrials.gov identiﬁer # NCT00001673). 'is trial was approved under a screening time report in Gr. 1 and Gr. 2. Genotyping and RNA quantiﬁcation for HCV were also performed. No additional protocol 05-AA-0121 of NIAAA. All the study participants signed the informed consent prior to the involvement in the proteomic or rapid turnover proteins analyses were in the scope of this study. All ranges and guidelines for testing were study. Data were collected and analyzed at the NIH, assigned and all the study tests were performed by the Bethesda, MD, USA, and University of Louisville, Louisville, Department of Laboratory Medicine, NIH, Bethesda, MD, KY, USA. 138 male and female participants aged 21–65 years USA. were enrolled for this study (Table 1). 'e distribution of Advances in Virology 3 Table 1: Demographic and drinking proﬁle of HCV patients without or with AUD, AUD patients (without HCV) without or with liver injury, and healthy volunteers. HCV patients AUD Healthy Groups/ AUD without liver AUD with liver HCV-only (Gr. 1) HCV + AUD (Gr. 2) volunteers measures injury injury (n � 13) (n � 25) (n � 12) (Gr. 5) (Gr. 3) (n � 42) (Gr. 4) (n � 46) Demographics Male � 11 Male � 18 Male � 28 Male � 34 Male � 5 Sex Female � 2 Female � 7 Female � 14 Female � 12 Female � 7 Age (years) 47.83± 7.7 47.25± 5.8 39.33± 10.83 43.76± 10.0 40.98± 11.7 Height (cm) 173.25± 9.9 176.60± 9.11 170.81± 9.97 173.65± 7.6 169.00± 10.9 Weight (kg) 73.97± 13.11 78.65± 12.39 79.23± 19.46 77.46± 12.4 68.06± 13.2 BMI 24.65± 3.57 25.22± 3.87 26.49± 4.7 25.66± 3.5 23.58± 5.3 Drinking proﬁle LTDH (years) 17.60± 11.06 16.86± 11.5 13.10± 8.4 15.11± 10.2 NA TD90 159.25± 46.7 1118.36± 443.08 841.81± 458.1 1052.57± 442.7 NA AvgDPDD90 5.50± 1.9 14.52± 5.08 12.38± 6.0 13.85± 5.1 NA NDD90 33.5± 19.3 78.24± 18.1 67.57± 21.9 76.46± 16.9 NA HDD90 20.00± 16.5 76.04± 18.45 62.69± 21.6 73.43± 19.5 NA BMI: body mass index, LTDH: lifetime drinking history, TD90: total drinks in the past 90 days, AvgDPDD90: average drinks per drinking day in the past 90 days, NDD90: number of drinking days in the past 90 days, HDD90: heavy drinking days in the past 90 days. Data were represented as mean± SD and statistical signiﬁcance was set at p< 0.05. NA: not applicable. described the goodness of model ﬁt (adjusted R Drinking history was collected. Heavy drinking is de- ). SPSS 26.0 ﬁned as an intake of 15 alcoholic drinks or more per week for (IBM, Chicago, IL) and Microsoft Excel 2016 (MS Corp., males and eight drinks or more per week for females (https:// Redmond, WA) were used for data analyses. Data are www.cdc.gov/alcohol/faqs.htm). All AUD patients enrolled presented as mean± standard deviation (M± SD) for con- in this study met the criteria for heavy drinking per the tinuous variables. Center for Disease Control recommendation. Timeline followback past 90 days (TLFB90) questionnaire has been a 3. Results validated and well-established instrument to collect self- 3.1. Demographics, Drinking Proﬁle, and HCV Markers. reported data on the total number of drinks for each day in In our study cohort, 25 out of 38 (around 66%) HCV di- the past 90 days [16]. TLFB90 was used in our study to collect agnosed patients were comorbid with AUD. 'ere was not drinking history in all the groups. Markers of drinking that much diﬀerence in the demographic measures either nu- were derived from the TLFB90 questionnaire included total merically or statistically in the ﬁve study cohorts (Table 1). drinks in the past 90 days (TD90), the number of drinking AUD patients without liver injury (Gr. 3) were borderline days in the past 90 days (NDD90), drinks per drinking day in overweight and were drinking for the least number of years. the past 90 days (DPD90), average drinks per drinking day in HCV + AUD patients showed slightly higher levels of the past 90 days (AvgDD90), and heavy drinking days in the drinking markers compared to the AUD patients (Gr. 3 and past 90 days (HDD90). We also used the lifetime drinking Gr. 4). 'ere were only two females in the HCV group; thus, history (LTDH) questionnaire [17] and the number of years this ﬁnding limited the analyses of the sex diﬀerences within as other drinking measures of evaluation in this study this cohort (or with other groups). Only two HV subjects (https://pubs.niaaa.nih.gov/publications/AssessingAlcohol/ reported social drinking in the past 90 days; thus, data were measures.htm). We used the “Controlling Nutritional Status not added/evaluated in Table 1. All AUD patients drank Test” (CONUT) data to establish nutritional status. None of more than 10 drinks per day on average. the participants in this study showed signs of overt mal- 'ere was no signiﬁcant diﬀerence in the HCV RNA nutrition [18]. quantiﬁcation (or their converted international unit) values between HCV-only and HCV + AUD groups; however, these levels were higher in the HCV + AUD group (Supplemental 2.3. Statistical Analysis. Diﬀerences among demographics, Table S1A). Genotype 1A was the most prevalent genetic drinking history markers, zinc levels, fatty acid levels, and constitution (52.6%) among the patients diagnosed with ALT levels were evaluated with the use of one-way ANOVA HCV in this study (Supplemental Table S1B). for the ﬁve groups. Fisher’s exact test was used to compare group diﬀerences for categorical variables (e.g., sex). Uni- variate and multivariate linear regression analyses were 3.2. Liver Injury. ALT was signiﬁcantly higher in the conducted to analyze the associations between one variable HCV + AUD patients (Gr. 2), followed by the HCV-only versus one or multiple variables, respectively. Drinking patients (Gr. 1) and AUD patients with liver injury subse- history was used as a covariate where applicable. 'e sig- quently (Gr. 4) (Figure 1(a)). HCV + AUD patients showed niﬁcance level was set at p< 0.05. 'e association analysis numerically higher ALT levels than the HCV-only patients; 4 Advances in Virology Liver injury Liver injury 500 500 ∗∗ ∗∗ ∗∗ 400 400 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 300 300 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗ 200 200 100 100 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 5 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 5 Study groups Study groups (a) (b) Figure 1: Diﬀerences in the liver injury markers in all the study groups. (a) Serum ALT levels in all the study cohorts. (b) Serum AST levels in all the study groups. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. however, this increase was not statistically signiﬁcant. AUD (Supplemental Table S2). Lowering of the concentrations of patients with liver injury showed comparable levels of ALT the candidate ω3 PUFAs, speciﬁcally, EPA and DPA5,3ω, with HCV-only patients, and the levels between the two shows greater weightage in the proinﬂammatory shift more groups were not signiﬁcantly diﬀerent. AST levels did not than the increases in the proinﬂammatory ω6 PUFAs. With show any numerical or statistical diﬀerence between Gr.1 these ﬁndings, we further focused our analyses of the and Gr. 2 and Gr. 2 and Gr. 4 (Figure 1(b)). Mean AST was proinﬂammatory response originating from the lowering of the highest in Gr. 4; however, this could be due to one single ω3 PUFAs on Gr. 2. patient with AST approaching 400 u/L. Going forward with the analyses, we did not include the healthy volunteer (HV, 3.5. Association of PUFAs and Zinc in HCV and AUD Gr. 5) data. Comorbid Patients. Zinc levels did not show any association either with the ω6 : ω3 ratio (Figure 4(a)) or with the total ω6 FAs concentrations (Figure 4(b)). However, there was a 3.3. Zinc Deﬁciency and Proinﬂammatory Shift. Zinc deﬁ- trend level of association between the serum zinc level and ciency and FA dysregulation are already well known in liver ω3 levels (Figure 4(c)), which augmented to statistical sig- disease. 'e current study focused primarily on the niﬁcance when NDD90 was added as a covariable in this HCV + AUD patients, and data from other cohorts were regression analysis. 'ese within-group associations were used for comparison purposes. Zinc deﬁciency was the most either not signiﬁcant or showed much lower correlations in in Gr. 2 (HCV + AUD cohort) followed by HCV-only pa- Gr. 1 and Gr. 4. With this information, we further evaluated tients (Gr. 1) (Table 2); however, this lowering in Gr. 2 was candidate ω3 PUFAs that are involved in the anti-inﬂam- not statistically signiﬁcant compared to Gr. 1 (Figure 2(a)). A matory response in the downstream fashion. similar tendency in the proinﬂammatory shift was found α-Linoleic acid showed a trend level of signiﬁcant as- between Gr. 2 and Gr. 1 response (as characterized by ω6 : ω3 sociation with lower zinc values in Gr. 2, which augmented ratio; Figure 2(b)). ω6 : ω3 ratio level in Gr. 2 was signiﬁ- when NDD90 was added as a covariable in the same analysis cantly higher (p � 0.001) than that in Gr. 4. Noteworthy, Gr. (Figure 5(a)). EPA (Figure 5(b)) and DPA5,3ω (Figure 5(c)) 4 ω6 : ω3 ratio was lower than Gr. 3, which was consistent showed a signiﬁcant association with the serum zinc con- with our previous report [19]. centrations independently. 'e association of zinc deﬁciency showed a close association with the candidate ω3 PUFAs in 3.4. Changes in Fatty Acid Spectrum. 'e signiﬁcantly higher Gr. 2. HCV RNA quantiﬁcation was signiﬁcantly associated ω6 : ω3 ratio in the HCV + AUD (Gr. 2) patients discussed with DPA5,3ω and added covariable NDD90 (p � 0.035) in above was due to a lowering of the total ω3 (Figure 3(a)), HCV + AUD patients, although in HCV patients it was only which was signiﬁcantly evident between Gr. 2 and Gr. 4. We trending (data not plotted). did not ﬁnd these diﬀerences in the total ω6 values (data not presented). 'us, we evaluated the lowering of FAs in Gr. 2 4. Discussion further. We assessed the total and individual ω3 FAs that contributed to the anti-inﬂammatory response. Both EPA Approximately two-thirds of the HCV diagnosed patients and DPA5,3ω were signiﬁcantly lower in Gr. 2 exhibited heavy alcohol drinking. Heavy drinking HCV (HCV + AUD) (Figures 3(b) and 3(c)). DHA values showed patients showed higher liver injury [20] as characterized by only a trend of lowering in the HCV + AUD patients (Gr. 2) the serum ALT levels. Notably, these patients did not show compared to the AUD with liver injury patients (Gr. 4) any outwardly diﬀerent drinking patterns than the AUD ALT U/L AST U/L Advances in Virology 5 Table 2: Liver injury, nutritional status, and serum nutrients in the study cohorts. HCV patients AUD Healthy volunteers Groups/measures HCV-only HCV + AUD AUD without liver injury AUD with liver injury (Gr. 5) (Gr. 1) (Gr. 2) (Gr. 3) (Gr. 4) Liver panel ALT (U/L) 102.62± 54.85 121.68± 74.26 26.40± 8.44 84.48± 33.55 23.92± 8.65 AST (U/L) 88.85± 43.82 112.84± 64.41 25.02± 12.99 122.65± 80.30 33.67± 21.1 AST : ALT ratio 0.89± 0.24 1.03± 0.42 1.01± 0.48 1.44± 0.83 1.39± 0.53 Tbili (μmol/L) 0.68± 0.23 0.71± 0.39 0.69± 0.61 0.72± 0.61 0.73± 0.70 ALB (g/dL) 4.02± 0.36 3.83± 0.32 4.10± 0.32 4.10± 0.46 4.05± 0.32 Serum zinc and CONUT Zn (mcg/dL) 70.23± 11.53 63.50± 17.40 79.20± 11.25 72.89± 26.80 72.10± 15.73 CONUT score 1.17± 0.94 1.78± 1.28 0.85± 1.13 1.23± 1.55 1.10± 1.31 ω3 and ω6 polyunsaturated fatty acids participating in inﬂammation in AUD and HCV + AUD patients. ALT: alanine transaminase; AST: aspartate transaminase; Tbili: Total bilirubin; ALB: albumin; Zn: serum zinc; CONUT: Controlling Nutritional Status. Zinc Proinﬂammatory shi 200 30 ∗∗ 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Study groups Study groups (a) (b) Figure 2: Serum zinc concentrations and plasma assessed proinﬂammatory shift in all the study groups. (a) Serum zinc level in all the study cohorts. (b) Proinﬂammatory shift as characterized by ω6 : ω3 ratio in all the study groups. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. ω3 FAs EPA DPA5, 3ω 750 500 ∗∗ ∗∗ ∗∗∗ ∗∗ 600 400 ∗∗ 300 200 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Study groups Study groups Study groups (a) (b) (c) Figure 3: Plasma ω3 PUFA measures in HCV-only (Gr. 1), HCV + AUD (Gr. 2), AUD without any liver injury (Gr. 3), and AUD with liver injury (Gr. 4). (a) Total ω3 concentration in Gr. 1 through Gr. 4. (b) Plasma eicosapentaenoic acid (EPA) concentration in Gr. 1 through Gr. 4. (c) ω3 docosapentaenoic acid (DPA5,3ω) concentrations in Gr. 1 through Gr. 4. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. patients who had a liver injury. HCV patients who did not HCV-infected AUD patients. Serum zinc levels in the HCV- drink also had clinically relevant levels of ALT; however, infected heavy drinkers were the lowest, consistent with a they were comparatively much lower than the ALT levels in higher proinﬂammatory shift. On the other hand, we found ω3 mmol/L Zinc mcg/dL EPA nmol/ml ω6:ω3 DPA5, 3ω nmol/mL 6 Advances in Virology HCV + AUD patients HCV + AUD patients HCV + AUD patients 30 10 r = 0.487, p = 0.058 24 8 0.8 2 p = NS #Adjusted R = 0.192, p = 0.041 p = NS 18 0.6 12 4 0.4 6 0.2 0 0 0 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 Zinc mcg/dL Zinc mcg/dL Zinc mcg/dL (a) (b) (c) Figure 4: Association of plasma-derived PUFAs involved in inﬂammation and serum zinc levels in HCV + AUD patients (Gr. 2). (a) Association of proinﬂammatory shift as characterized by the ω6 : ω3 ratio and serum zinc levels. (b) Association of total ω6 con- centrations and serum zinc levels. (c) Association of total ω3 concentrations and serum zinc levels. Data analysis showed correlation coeﬃcient: “r”; #adjusted analysis with NDD90 as a covariable: R . Statistical signiﬁcance was set at p≤ 0.05. NS: not signiﬁcant. HCV + AUD patients HCV + AUD patients HCV + AUD patients 200 200 200 r = 0.374, p = 0.072 160 2 160 160 #adjusted R = 0.218, p = 0.029 r = 0.404, p = 0.050 r = 0.425, p = 0.038 120 120 120 80 80 80 40 40 40 0 0 0 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 Zinc mcg/dL Zinc mcg/dL Zinc mcg/dL (a) (b) (c) Figure 5: Association of plasma-derived PUFAs involved in inﬂammation and serum zinc levels in HCV + AUD patients (Gr. 2). (a) Association of α-linoleic acid (αLA) concentrations and serum zinc levels. (b) Association of eicosapentaenoic acid (EPA) concentrations and serum zinc levels. (c) Association of ω3 docosapentaenoic acid (DPA5,3ω) concentrations and serum zinc levels. Data analysis showed a correlation coeﬃcient: r; #adjusted analysis with NDD90 as a covariable: R . Statistical signiﬁcance was set at p≤ 0.05. NS: not signiﬁcant. a lowered proinﬂammatory shift in AUD patients with liver [26]. In our study, ﬁndings on zinc deﬁciency were con- injury (Gr. 4), which is consistent with our previous study sistent in HCV-infected patients, both those who had only [19]. HCV infection alone and with AUD diagnosis, and those Several polyunsaturated fatty acids (PUFAs) including who also drank heavily with or without liver injury. How- EPA and DHA inhibit HCV replication in vitro using the ever, this deﬁciency in zinc was most severe in HCV + AUD HCV RNA replicon system considerably [21]. In our study, patients. Zinc metabolism in ALD gets altered [27], and zinc we found a generalized drop in the total ω3, EPA, and deﬁciency [28] causes major dysregulation in several DPA5,3ω and DHA PUFAs in both the HCV-only and pathological pathways involved in the initiation and pro- gression of ALD [29]. A therapeutic role for zinc supple- HCV + AUD groups. Previously, ω3 supplementation has shown eﬃcacy in the medical management of HCV patients mentation has shown great promise in ALD, especially in who priorly were nonresponders to the combination anti- alcoholic cirrhosis and alcoholic hepatitis [30, 31]. viral therapy [22]. We found that the HCV + AUD patients Zinc has a pivotal role in the hepatic regulation of fatty also had higher HCV RNA quantiﬁcation, which was sig- acids; it aids in the downstream process of anti-inﬂamma- niﬁcantly predictable by the DPA5,3ω level with respect to tory PUFAs [32, 33]. 'erefore, a low zinc level likely heavy drinking. Anti-HCV activities of the selective PUFAs lowered the downstream synthesis of omega-3 PUFAs have been reported previously [23]. Our study showed that, systematically in HCV + AUD patients (Supplemental in HCV + AUD patients, elevated RNA quantiﬁcation was Table S2). Previously, we have reported that AUD heavy shown compared to the HCV-only patients. drinkers with HIV comorbidity showed a close association Zinc depletion has been abundantly reported with liver of proinﬂammatory ω6 PUFA and low zinc [19]. In this study, we found that low zinc was closely associated with disease [24]; importantly, it is lower in the HCV diagnosed patients [25]. Zinc levels were also deﬁcient in AUD patients total ω3 PUFAs and EPA and DPA5,3ω [34] in HCV-in- who exhibited early-stage ALD [15]. In one report, altered fected heavy drinkers. One recent review has described the PUFAs were observed in HIV-HCV-infected heavy drinkers role of zinc as a cofactor in the modulation of desaturase α-Linoleic acid nmol/mL ω6:ω3 ratio EPA nmol/mL Total ω6 FAs mmol/L Total ω3 FAs mmol/L DPA5, 3 nmol/mL Advances in Virology 7 activity involved in FA absorption, oxidation, metabolism, 5. Conclusions and incorporation [12]. 'e importance of our study was Depletion in the concentrations of ω3 PUFAs, DPA5,3ω that low zinc and candidate ω3 PUFAs were signiﬁcantly and EPA, is involved in the proinﬂammatory shift in HCV associated with HCV + AUD patients only. 'e interaction diagnosed individuals, especially comorbid with AUD of hypozincemia and the lowering of anti-inﬂammatory FAs diagnosis. Clinically deﬁcient zinc concentrations are ob- in HCV-infected heavy drinkers likely played a pathological served in HCV patients, with further lowering in HCV- role in the proinﬂammatory shift. infected AUD patients. 'is deﬁciency in both ω3 PUFAs NDD90 as a heavy drinking marker showed a close and zinc concentrations is highly correlated. 'e assess- association with candidate ω3 fatty acids, especially ment of the number of drinking days (NDD90) could be a DPA5,3ω, in the expression of HCV RNA quantiﬁcation. Its good marker in HCV-infected heavy drinkers to identify role was also signiﬁcant in the association analyses for de- the changes that are involved in the proinﬂammatory ﬁcient ω3 PUFAs and low zinc. 'e connection of speciﬁc response. markers of heavy drinking with the exacerbation of meta- bolic dysregulation in liver injury has been recently high- lighted [35]. 'e number of drinking days past 90 days Abbreviations (NDD90) was a signiﬁcant marker that was observed in our AUD: Alcohol use disorder study. It seems that how many days an individual drinks ALD: Alcoholic liver disease heavily (NDD90) in an assessment period is consequential in ALB: Albumin the changes in the anti-inﬂammatory ω3 PUFAs in HCV- ALP: Alkaline phosphatase infected heavy drinkers, which was a new ﬁnding in our ALT: alanine aminotransferase study. AST: Aspartate aminotransferase Our study has several limitations. We had smaller AST: ALT: AST by ALT ratio number of females in each group; however, in HCV-in- CS: Clinically signiﬁcant fected groups, this diﬀerence was numerically signiﬁcant. DHA: Docosahexaenoic acid 'is study was a single time-point clinical observational DPA 5,3ω: Docosapentaenoic acid investigation on the characterization of low zinc and FA: Fatty acids omega-3 alterations in a unique HCV and AUD comorbid Gr. 1: HCV-only cohort. 'us, neither longitudinal interpretations or Gr. 2: HCV + AUD treatment outcomes of zinc or omega-3 supplementation Gr. 3: AUD without liver injury were not evaluated. 'e plasma or tissue levels of PGs, LTs, Gr. 3: AUD with liver injury TXs, and lipoxins were not measured, which could have Gr. 5: HV further identiﬁed speciﬁc role of the product-associated HCV: Hepatitis C virus proinﬂammatory response, synthesized during the HCV + AUD: Patients with HCV and AUD comorbidity downstream process of PUFA. 'ere was no biopsy HV: Healthy volunteers performed on the study patients since this was an ob- PUFAs: Polyunsaturated fatty acids servational study only. 'is study did not use a mecha- Tbili: Total bilirubin nistic approach, which was beyond the scope of this study TLFB90: Timeline followback past 90 days design. Dietary FAs were not studied in this investigation; TD90: Total drinks in the past 90 days however, none of the patients showed any malnutrition at AvgD90: Average drinks in the past 90 days intake (Table 2). We did not study the type of alcohol [36]; HDD90: Heavy drinking days in the past 90 days our study focused on the patterns and amount of alcohol NDD90: Number of drinking days in the past 90 days intake as per the guideline of NIAAA as described in the ω3-EPA: Eicosapentaenoic acid methods section. ω6 : ω3: Omega 6 by omega 3 ratio. Lowering of the concentrations in ω3 PUFAs, DPA5,3ω and EPA, likely plays a signiﬁcant role in the proinﬂammatory shift in HCV-infected AUD patients. Data Availability Serum zinc lowered to a clinically deﬁcient level in the same cohort. 'is deﬁciency was a strong predictor of the Data will be provided on request based on reasonable queries lowering of total and candidate ω3 PUFAs, exclusively, in only. the HCV + AUD patients. 'e number of drinking days (NDD90) could be a good marker in HCV-infected heavy Disclosure drinkers and a key to identify the changes that can incur in the ω3 PUFAs in such comorbid cohort. 'is report ex- 'is article is a work of the University of Louisville Alcohol tends the need to elucidate the anti-HCV mechanism in Research Center and National Institutes of Health. 'e comorbid AUD patients by a combination of speciﬁc anti- content is solely the responsibility of the authors and does inﬂammatory PUFAs [37] and zinc supplementation [38] not necessarily represent the oﬃcial views of the National that may lead to the development of novel molecular- Institutes of Health. 'is manuscript is in the public domain based therapeutic agents and direct therapeutic mecha- in the USA. E-mail address for the readers to contact the nistic studies in this special cohort. author to obtain the data is v0vats01@louisville.edu. 8 Advances in Virology [6] I. Novo-Veleiro, “Alcoholic liver disease and hepatitis C virus Conflicts of Interest infection,” World Journal of Gastroenterology, vol. 22, no. 4, p. 1411, 2016. All authors declare no conﬂicts of interest. [7] K. H. Zirnheld, D. R. Warner, J. B. Warner, J. E. Hardesty, C. J. McClain, and I. A. Kirpich, “Dietary fatty acids and Authors’ Contributions bioactive fatty acid metabolites in alcoholic liver disease,” Liver Research, vol. 3, no. 3-4, pp. 206–217, 2019. VV is the project PI and designed the study. VV and MLS [8] S. A. Read, S. Obeid, C. Ahlenstiel, and G. Ahlenstiel, “'e acquired and managed the data. VV and JF analyzed the role of zinc in antiviral immunity,” Advances in Nutrition, data. VV, RA, and SS interpreted the data analysis. VV, JF, vol. 10, no. 4, pp. 696–710, 2019. SS, and RA wrote the manuscript. VV, RA, SS, and MLS [9] T. Suda, O. Okawa, R. Shirahashi, N. Tokutomi, and M. Tamano, “Changes in serum zinc levels in hepatitis C critically reviewed the manuscript and contributed scien- patients before and after treatment with direct-acting antiviral tiﬁcally. All authors have approved the submission version agents,” Hepatology Research, vol. 10, no. 4, pp. 696–710, 2019. of this manuscript. [10] V. Vatsalya, M. C. Cave, R. Kumar et al., “Alterations in serum zinc and polyunsaturated fatty acid concentrations in treat- Acknowledgments ment-naive HIV-diagnosed alcohol-dependent subjects with liver injury,” AIDS Research and Human Retroviruses, vol. 35, 'e authors thank the clinical staﬀ of the Clinical Center of no. 1, pp. 92–99, 2019. NIH for providing the patientcare. 'e authors also ac- [11] J. A. Barocas, K. S. Armah, D. M. Cheng et al., “Zinc deﬁciency knowledge the research staﬀ of the Alcohol Research Center and advanced liver ﬁbrosis among HIV and hepatitis C co- infected anti-retroviral na¨ıve persons with alcohol use in of the University of Louisville. 'e manuscript has been Russia,” PloS One, vol. 14, no. 6, 2019. edited by Jane Frimodig. 'e study was supported by the [12] D. Tsoukalas, A. K Alegakis, P Fragkiadaki et al., “Application NIH: Z99-AA999999 (VV), U01-AA026936-02, of metabolomics part II: focus on fatty acids and their me- U01AA026980-2, and 1R01AA023681-05 (CJM). 'e re- tabolites in healthy adults,” International Journal of Molecular search reported in this publication was supported by an Medicine, vol. 43, pp. 233–242, 2019. Institutional Development Award (IDEA) from the National [13] M. Shoreibah, B. S. Anand, and A. K. Singal, “Alcoholic Institute of General Medical Sciences of the National In- hepatitis and concomitant hepatitis C virus infection,” World stitutes of Health under grant number: P20GM113226 Journal of Gastroenterology, vol. 20, no. 34, p. 11929, 2014. (CJM) and the National Institute on Alcohol Abuse and [14] L. C. Sobell and M. B. Sobell, “Timeline follow-back,” in Alcoholism of the National Institutes of Health under award Measuring Alcohol Consumption, pp. 41–72, Springer, Berlin, number: P50AA024337 (CJM). Germany, 1992. [15] V. 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Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2020 Vatsalya Vatsalya et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 1687-8639
eISSN: 1687-8647
DOI: 10.1155/2020/7835875
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Abstract

Hindawi Advances in Virology Volume 2020, Article ID 7835875, 9 pages https://doi.org/10.1155/2020/7835875 Research Article Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients 1,2,3,4 5 1,2 6 Vatsalya Vatsalya , Ruchita Agrawal, Jane Frimodig, Shweta Srivastava, and Melanie L. Schwandt Department of Medicine, University of Louisville, Louisville, KY, USA Robley Rex VA Medical Center, Louisville, KY, USA University of Louisville, Alcohol Research Center, Louisville, KY, USA National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD, USA Department of Psychiatry, University of Louisville, Louisville, KY, USA Environmental Health Institute, University of Louisville, Louisville, KY, USA Correspondence should be addressed to Vatsalya Vatsalya; v0vats01@exchange.louisville.edu Received 19 February 2020; Accepted 30 April 2020; Published 9 June 2020 Academic Editor: Finn S. Pedersen Copyright © 2020 Vatsalya Vatsalya et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Alcohol use disorder (AUD) patients comorbid with hepatitis C virus (HCV) infection (HCV + AUD) could have progressively severe clinical sequels of liver injury and inﬂammation. Serum zinc and several polyunsaturated fatty acids (PUFAs) get dys- regulated in AUD as well as HCV. However, the extent of dysregulation of PUFAs and zinc deﬁciency and their interaction in HCV + AUD as a comorbid pathology has not been studied. We examined the role of dysregulation of FAs and low zinc in HCV + AUD patients. 138 male and female participants aged 21–67 years were grouped as HCV-only (Gr. 1; n � 13), HCV + AUD (Gr. 2; n � 25), AUD without liver injury (Gr. 3; n � 37), AUD with liver injury (Gr. 4; n � 51), and healthy volunteers (Gr. 5 or HV; n � 12). Drinking history, individual demographic measures, fasting fatty acids, liver function, and zinc were measured and analyzed. HCV + AUD patients showed the highest ALT level compared to the rest of the groups. Serum zinc concentrations were the lowest, and the proinﬂammatory shift was the highest (characterized by ω6 : ω3 ratio) in the HCV + AUD patients. Total ω3, eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA5,3) were the lowest in HCV + AUD patients. Total ω3, α-linoleic acid (α-LA) along with covariable number of drinking days past 90 days (NDD90), eicosapentaenoic acid (EPA), and doco- sapentaenoic acid (DPA5,3) independently showed signiﬁcant association with low zinc in the HCV + AUD patients. Heavy drinking pattern showed that NDD90 has a signiﬁcant mediating role in the representation of the relationship between candidate ω3 PUFAs and zinc uniquely in the HCV + AUD patients. Low serum zinc showed a distinctively stronger association with total and candidate ω3s in the HCV + AUD patients compared to the patients with HCV or AUD alone, supporting dual mechanism involved in the exacerbation of the proinﬂammatory response in this comorbid cohort. 'is trial is registered with NCT#00001673. a major form of liver disease that is responsible for the 1. Introduction mortality and morbidity in the USA and worldwide [4]. Hepatitis C virus (HCV) is the most common chronic blood- With no FDA approved drug for the treatment of ALD, borne infection, aﬀects 5 million Americans, and causes very treatment development for the alcohol-related liver disease high morbidity and mortality [1]. Hepatitis C virus (HCV) remains an unmet need [5]. AUD diagnosed individuals has lasting morbidity and lasting eﬀect on the liver and with HCV infection (HCV + AUD) have reported a pro- overall health [2]. Alcohol use disorder (AUD) is known to pensity for liver injury compared with AUD patients, with a be consequential in alcoholic liver disease (ALD) [3]. ALD is prevalence of HCV and AUD comorbidity being 16.3% [6]. 2 Advances in Virology Fatty acid synthesis has been shown to regulate Hepatitis these participants was as follows: Gr. 1: HCV-only (n � 13); C Virus (HCV) entry and production (Yang, 2008), though Gr. 2: HCV + AUD (n � 25); Gr. 3: AUD without liver injury HCV has also been involved in altering fatty acid meta- (n � 37); Gr. 3: AUD with liver injury (n � 51); Gr. 5: healthy bolism thereby causing lipid accumulation in the liver volunteers (HV, n � 12). All heavy drinkers met the diag- (Yamaguchi, 2005). Fatty acids are also variable with alcohol nosis criteria of AUD according to the DSM-IV, based on use; they can be either anti- or proinﬂammatory in nature the SCID I interview. Patients were not enrolled in the study [7]. 'e role of zinc in antiviral immunity is given in [8], and if they had a diagnosis for severe psychiatric illness, suicidal, changes in zinc levels in HCV-infected patients at pre-and or violent tendencies or showed agitation requiring im- posttreatment evaluations were given in [9]. Recent studies mediate clinical treatment. Patients with other forms of have shown that zinc deﬁciency is commonly found in AUD, signiﬁcant psychiatric illnesses (unless stable, and not re- AUD with early stage of ALD, and AUD with viral infections quiring medication such as antidepressants, lithium, neu- comorbidity [9–11]. Zinc plays an important role as a co- roleptics, naltrexone, acamprosate, disulﬁram, factor in the downstream regulation of proinﬂammatory benzodiazepines, or antiepileptic compounds within the last four weeks) were not enrolled. Advanced lung disease, (ω6) and anti-inﬂammatory (ω3) PUFAs [12]. Studies on low zinc and ω3 PUFAs have been well studied separately in unstable cardiovascular disease, renal failure (creatinine AUD patients with or without liver injury as well as HCV- clearance 30 ml/min), advanced liver disease (hepatocellular infected patients. However, the representation and rela- carcinoma, clinically evident alcoholic hepatitis, and cir- tionship of lower zinc and ω3 PUFAs is not clear in AUD rhosis), and HIV were other exclusionary criteria. Other patients who are also infected with HCV. exclusions on the day of assessment were (1) pregnancy Hepatitis C infection and excessive alcohol exposure are (negative test required) or ongoing breastfeeding and/or (2) synergistic in their destructive eﬀects on the liver [13]. a positive urine screen for any illicit drug. Importantly, these Identifying the early changes in the rearrangement of fatty patients had no clinical signs of alcoholic liver disease, and acids, inﬂammation, and injury-associated nutritional since the HCV diagnosed patients had the ﬁrst-time diag- measures during patient evaluation could elucidate the nosis, there was no standard of care indication to perform a biopsy (as this study is not a treatment evaluation study). comorbid condition of HCV and AUD. Markers of heavy drinking [14] and proﬁle have shown high predictability Patients did not show any clinical evidence of liver disease, with the exacerbation of liver injury in early stage of ALD. and overt clinical liver disease was an exclusion criterion in Nonetheless, the role of such heavy drinking markers in the the study; detailed information on eligibility criteria has proinﬂammatory response as characterized by the dysre- been published previously [15]. gulation of ω3 PUFAs and low zinc has not been investigated in HCV-infected AUD patients. 'ese gaps remain an open 2.2. Clinical Data and Laboratory Measures. 'is study was a area to investigate at both the clinical level and preclinical level. one-time study at the admission clinical observational in- vestigation. Demographic information included age, sex, We aimed to characterize the variability in the fatty acid height, weight, and BMI. Blood samples were collected for a proﬁle (both speciﬁc and group-based) participating in inﬂammation and changes in serum zinc levels in newly comprehensive metabolic panel (CMP) that included a liver panel, fatty acid panel (FAP), and serum trace metals (zinc acquired-HCV patients with comorbid AUD diagnosis. We also evaluated the association of HCV-viral load and serum for this study). Serum Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), AST/ALT ratio, Total zinc and PUFAs in HCV patients with or without AUD to show the extent of linked response in this comorbid patient bilirubin (Tbili), and albumin (ALB) measures were eval- uated for the extent of liver injury. Alanine Aminotrans- cohort. ferase (ALT) level was used as a reference to assess liver injury (Medline Plus-National Institutes of Health, 2014); 2. Methodology 40 IU/L for ALT was used as the upper limit of normal, and values ≥41 IU/L indicated liver injury as per the guideline 2.1. Patient Recruitment. 'e primary patient cohort of this that existed till 2014. 'e standard lower limit of normal for study was AUD patients with HCV infection; other cohorts serum zinc was 71 mcg/dL in this study. 'e comprehensive were included for comparison purposes only. 'is investi- fatty acid panel measured speciﬁc and total ω3 and ω6 FA gation was performed as one of the several projects under a levels using gas chromatography/mass spectrometry. 'e larger clinical study approved by the Institutional Review screening was performed to identify patients with HCV, Board of the National Institute on Alcohol Abuse and Al- which was conducted with COBAS Ampliprep/COBAS coholism (NIAAA), National Institutes of Health (NIH), Taqman HCV test methodology; HCV diagnosis was a ﬁrst- Bethesda, MD, USA (ClinicalTrials.gov identiﬁer # NCT00001673). 'is trial was approved under a screening time report in Gr. 1 and Gr. 2. Genotyping and RNA quantiﬁcation for HCV were also performed. No additional protocol 05-AA-0121 of NIAAA. All the study participants signed the informed consent prior to the involvement in the proteomic or rapid turnover proteins analyses were in the scope of this study. All ranges and guidelines for testing were study. Data were collected and analyzed at the NIH, assigned and all the study tests were performed by the Bethesda, MD, USA, and University of Louisville, Louisville, Department of Laboratory Medicine, NIH, Bethesda, MD, KY, USA. 138 male and female participants aged 21–65 years USA. were enrolled for this study (Table 1). 'e distribution of Advances in Virology 3 Table 1: Demographic and drinking proﬁle of HCV patients without or with AUD, AUD patients (without HCV) without or with liver injury, and healthy volunteers. HCV patients AUD Healthy Groups/ AUD without liver AUD with liver HCV-only (Gr. 1) HCV + AUD (Gr. 2) volunteers measures injury injury (n � 13) (n � 25) (n � 12) (Gr. 5) (Gr. 3) (n � 42) (Gr. 4) (n � 46) Demographics Male � 11 Male � 18 Male � 28 Male � 34 Male � 5 Sex Female � 2 Female � 7 Female � 14 Female � 12 Female � 7 Age (years) 47.83± 7.7 47.25± 5.8 39.33± 10.83 43.76± 10.0 40.98± 11.7 Height (cm) 173.25± 9.9 176.60± 9.11 170.81± 9.97 173.65± 7.6 169.00± 10.9 Weight (kg) 73.97± 13.11 78.65± 12.39 79.23± 19.46 77.46± 12.4 68.06± 13.2 BMI 24.65± 3.57 25.22± 3.87 26.49± 4.7 25.66± 3.5 23.58± 5.3 Drinking proﬁle LTDH (years) 17.60± 11.06 16.86± 11.5 13.10± 8.4 15.11± 10.2 NA TD90 159.25± 46.7 1118.36± 443.08 841.81± 458.1 1052.57± 442.7 NA AvgDPDD90 5.50± 1.9 14.52± 5.08 12.38± 6.0 13.85± 5.1 NA NDD90 33.5± 19.3 78.24± 18.1 67.57± 21.9 76.46± 16.9 NA HDD90 20.00± 16.5 76.04± 18.45 62.69± 21.6 73.43± 19.5 NA BMI: body mass index, LTDH: lifetime drinking history, TD90: total drinks in the past 90 days, AvgDPDD90: average drinks per drinking day in the past 90 days, NDD90: number of drinking days in the past 90 days, HDD90: heavy drinking days in the past 90 days. Data were represented as mean± SD and statistical signiﬁcance was set at p< 0.05. NA: not applicable. described the goodness of model ﬁt (adjusted R Drinking history was collected. Heavy drinking is de- ). SPSS 26.0 ﬁned as an intake of 15 alcoholic drinks or more per week for (IBM, Chicago, IL) and Microsoft Excel 2016 (MS Corp., males and eight drinks or more per week for females (https:// Redmond, WA) were used for data analyses. Data are www.cdc.gov/alcohol/faqs.htm). All AUD patients enrolled presented as mean± standard deviation (M± SD) for con- in this study met the criteria for heavy drinking per the tinuous variables. Center for Disease Control recommendation. Timeline followback past 90 days (TLFB90) questionnaire has been a 3. Results validated and well-established instrument to collect self- 3.1. Demographics, Drinking Proﬁle, and HCV Markers. reported data on the total number of drinks for each day in In our study cohort, 25 out of 38 (around 66%) HCV di- the past 90 days [16]. TLFB90 was used in our study to collect agnosed patients were comorbid with AUD. 'ere was not drinking history in all the groups. Markers of drinking that much diﬀerence in the demographic measures either nu- were derived from the TLFB90 questionnaire included total merically or statistically in the ﬁve study cohorts (Table 1). drinks in the past 90 days (TD90), the number of drinking AUD patients without liver injury (Gr. 3) were borderline days in the past 90 days (NDD90), drinks per drinking day in overweight and were drinking for the least number of years. the past 90 days (DPD90), average drinks per drinking day in HCV + AUD patients showed slightly higher levels of the past 90 days (AvgDD90), and heavy drinking days in the drinking markers compared to the AUD patients (Gr. 3 and past 90 days (HDD90). We also used the lifetime drinking Gr. 4). 'ere were only two females in the HCV group; thus, history (LTDH) questionnaire [17] and the number of years this ﬁnding limited the analyses of the sex diﬀerences within as other drinking measures of evaluation in this study this cohort (or with other groups). Only two HV subjects (https://pubs.niaaa.nih.gov/publications/AssessingAlcohol/ reported social drinking in the past 90 days; thus, data were measures.htm). We used the “Controlling Nutritional Status not added/evaluated in Table 1. All AUD patients drank Test” (CONUT) data to establish nutritional status. None of more than 10 drinks per day on average. the participants in this study showed signs of overt mal- 'ere was no signiﬁcant diﬀerence in the HCV RNA nutrition [18]. quantiﬁcation (or their converted international unit) values between HCV-only and HCV + AUD groups; however, these levels were higher in the HCV + AUD group (Supplemental 2.3. Statistical Analysis. Diﬀerences among demographics, Table S1A). Genotype 1A was the most prevalent genetic drinking history markers, zinc levels, fatty acid levels, and constitution (52.6%) among the patients diagnosed with ALT levels were evaluated with the use of one-way ANOVA HCV in this study (Supplemental Table S1B). for the ﬁve groups. Fisher’s exact test was used to compare group diﬀerences for categorical variables (e.g., sex). Uni- variate and multivariate linear regression analyses were 3.2. Liver Injury. ALT was signiﬁcantly higher in the conducted to analyze the associations between one variable HCV + AUD patients (Gr. 2), followed by the HCV-only versus one or multiple variables, respectively. Drinking patients (Gr. 1) and AUD patients with liver injury subse- history was used as a covariate where applicable. 'e sig- quently (Gr. 4) (Figure 1(a)). HCV + AUD patients showed niﬁcance level was set at p< 0.05. 'e association analysis numerically higher ALT levels than the HCV-only patients; 4 Advances in Virology Liver injury Liver injury 500 500 ∗∗ ∗∗ ∗∗ 400 400 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ 300 300 ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗ 200 200 100 100 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 5 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 5 Study groups Study groups (a) (b) Figure 1: Diﬀerences in the liver injury markers in all the study groups. (a) Serum ALT levels in all the study cohorts. (b) Serum AST levels in all the study groups. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. however, this increase was not statistically signiﬁcant. AUD (Supplemental Table S2). Lowering of the concentrations of patients with liver injury showed comparable levels of ALT the candidate ω3 PUFAs, speciﬁcally, EPA and DPA5,3ω, with HCV-only patients, and the levels between the two shows greater weightage in the proinﬂammatory shift more groups were not signiﬁcantly diﬀerent. AST levels did not than the increases in the proinﬂammatory ω6 PUFAs. With show any numerical or statistical diﬀerence between Gr.1 these ﬁndings, we further focused our analyses of the and Gr. 2 and Gr. 2 and Gr. 4 (Figure 1(b)). Mean AST was proinﬂammatory response originating from the lowering of the highest in Gr. 4; however, this could be due to one single ω3 PUFAs on Gr. 2. patient with AST approaching 400 u/L. Going forward with the analyses, we did not include the healthy volunteer (HV, 3.5. Association of PUFAs and Zinc in HCV and AUD Gr. 5) data. Comorbid Patients. Zinc levels did not show any association either with the ω6 : ω3 ratio (Figure 4(a)) or with the total ω6 FAs concentrations (Figure 4(b)). However, there was a 3.3. Zinc Deﬁciency and Proinﬂammatory Shift. Zinc deﬁ- trend level of association between the serum zinc level and ciency and FA dysregulation are already well known in liver ω3 levels (Figure 4(c)), which augmented to statistical sig- disease. 'e current study focused primarily on the niﬁcance when NDD90 was added as a covariable in this HCV + AUD patients, and data from other cohorts were regression analysis. 'ese within-group associations were used for comparison purposes. Zinc deﬁciency was the most either not signiﬁcant or showed much lower correlations in in Gr. 2 (HCV + AUD cohort) followed by HCV-only pa- Gr. 1 and Gr. 4. With this information, we further evaluated tients (Gr. 1) (Table 2); however, this lowering in Gr. 2 was candidate ω3 PUFAs that are involved in the anti-inﬂam- not statistically signiﬁcant compared to Gr. 1 (Figure 2(a)). A matory response in the downstream fashion. similar tendency in the proinﬂammatory shift was found α-Linoleic acid showed a trend level of signiﬁcant as- between Gr. 2 and Gr. 1 response (as characterized by ω6 : ω3 sociation with lower zinc values in Gr. 2, which augmented ratio; Figure 2(b)). ω6 : ω3 ratio level in Gr. 2 was signiﬁ- when NDD90 was added as a covariable in the same analysis cantly higher (p � 0.001) than that in Gr. 4. Noteworthy, Gr. (Figure 5(a)). EPA (Figure 5(b)) and DPA5,3ω (Figure 5(c)) 4 ω6 : ω3 ratio was lower than Gr. 3, which was consistent showed a signiﬁcant association with the serum zinc con- with our previous report [19]. centrations independently. 'e association of zinc deﬁciency showed a close association with the candidate ω3 PUFAs in 3.4. Changes in Fatty Acid Spectrum. 'e signiﬁcantly higher Gr. 2. HCV RNA quantiﬁcation was signiﬁcantly associated ω6 : ω3 ratio in the HCV + AUD (Gr. 2) patients discussed with DPA5,3ω and added covariable NDD90 (p � 0.035) in above was due to a lowering of the total ω3 (Figure 3(a)), HCV + AUD patients, although in HCV patients it was only which was signiﬁcantly evident between Gr. 2 and Gr. 4. We trending (data not plotted). did not ﬁnd these diﬀerences in the total ω6 values (data not presented). 'us, we evaluated the lowering of FAs in Gr. 2 4. Discussion further. We assessed the total and individual ω3 FAs that contributed to the anti-inﬂammatory response. Both EPA Approximately two-thirds of the HCV diagnosed patients and DPA5,3ω were signiﬁcantly lower in Gr. 2 exhibited heavy alcohol drinking. Heavy drinking HCV (HCV + AUD) (Figures 3(b) and 3(c)). DHA values showed patients showed higher liver injury [20] as characterized by only a trend of lowering in the HCV + AUD patients (Gr. 2) the serum ALT levels. Notably, these patients did not show compared to the AUD with liver injury patients (Gr. 4) any outwardly diﬀerent drinking patterns than the AUD ALT U/L AST U/L Advances in Virology 5 Table 2: Liver injury, nutritional status, and serum nutrients in the study cohorts. HCV patients AUD Healthy volunteers Groups/measures HCV-only HCV + AUD AUD without liver injury AUD with liver injury (Gr. 5) (Gr. 1) (Gr. 2) (Gr. 3) (Gr. 4) Liver panel ALT (U/L) 102.62± 54.85 121.68± 74.26 26.40± 8.44 84.48± 33.55 23.92± 8.65 AST (U/L) 88.85± 43.82 112.84± 64.41 25.02± 12.99 122.65± 80.30 33.67± 21.1 AST : ALT ratio 0.89± 0.24 1.03± 0.42 1.01± 0.48 1.44± 0.83 1.39± 0.53 Tbili (μmol/L) 0.68± 0.23 0.71± 0.39 0.69± 0.61 0.72± 0.61 0.73± 0.70 ALB (g/dL) 4.02± 0.36 3.83± 0.32 4.10± 0.32 4.10± 0.46 4.05± 0.32 Serum zinc and CONUT Zn (mcg/dL) 70.23± 11.53 63.50± 17.40 79.20± 11.25 72.89± 26.80 72.10± 15.73 CONUT score 1.17± 0.94 1.78± 1.28 0.85± 1.13 1.23± 1.55 1.10± 1.31 ω3 and ω6 polyunsaturated fatty acids participating in inﬂammation in AUD and HCV + AUD patients. ALT: alanine transaminase; AST: aspartate transaminase; Tbili: Total bilirubin; ALB: albumin; Zn: serum zinc; CONUT: Controlling Nutritional Status. Zinc Proinﬂammatory shi 200 30 ∗∗ 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Study groups Study groups (a) (b) Figure 2: Serum zinc concentrations and plasma assessed proinﬂammatory shift in all the study groups. (a) Serum zinc level in all the study cohorts. (b) Proinﬂammatory shift as characterized by ω6 : ω3 ratio in all the study groups. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. ω3 FAs EPA DPA5, 3ω 750 500 ∗∗ ∗∗ ∗∗∗ ∗∗ 600 400 ∗∗ 300 200 0 0 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Gr. 1 Gr. 2 Gr. 3 Gr. 4 Study groups Study groups Study groups (a) (b) (c) Figure 3: Plasma ω3 PUFA measures in HCV-only (Gr. 1), HCV + AUD (Gr. 2), AUD without any liver injury (Gr. 3), and AUD with liver injury (Gr. 4). (a) Total ω3 concentration in Gr. 1 through Gr. 4. (b) Plasma eicosapentaenoic acid (EPA) concentration in Gr. 1 through Gr. 4. (c) ω3 docosapentaenoic acid (DPA5,3ω) concentrations in Gr. 1 through Gr. 4. Data were presented as mean± standard deviation. Statistical signiﬁcance was set at p≤ 0.05. patients who had a liver injury. HCV patients who did not HCV-infected AUD patients. Serum zinc levels in the HCV- drink also had clinically relevant levels of ALT; however, infected heavy drinkers were the lowest, consistent with a they were comparatively much lower than the ALT levels in higher proinﬂammatory shift. On the other hand, we found ω3 mmol/L Zinc mcg/dL EPA nmol/ml ω6:ω3 DPA5, 3ω nmol/mL 6 Advances in Virology HCV + AUD patients HCV + AUD patients HCV + AUD patients 30 10 r = 0.487, p = 0.058 24 8 0.8 2 p = NS #Adjusted R = 0.192, p = 0.041 p = NS 18 0.6 12 4 0.4 6 0.2 0 0 0 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 Zinc mcg/dL Zinc mcg/dL Zinc mcg/dL (a) (b) (c) Figure 4: Association of plasma-derived PUFAs involved in inﬂammation and serum zinc levels in HCV + AUD patients (Gr. 2). (a) Association of proinﬂammatory shift as characterized by the ω6 : ω3 ratio and serum zinc levels. (b) Association of total ω6 con- centrations and serum zinc levels. (c) Association of total ω3 concentrations and serum zinc levels. Data analysis showed correlation coeﬃcient: “r”; #adjusted analysis with NDD90 as a covariable: R . Statistical signiﬁcance was set at p≤ 0.05. NS: not signiﬁcant. HCV + AUD patients HCV + AUD patients HCV + AUD patients 200 200 200 r = 0.374, p = 0.072 160 2 160 160 #adjusted R = 0.218, p = 0.029 r = 0.404, p = 0.050 r = 0.425, p = 0.038 120 120 120 80 80 80 40 40 40 0 0 0 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 Zinc mcg/dL Zinc mcg/dL Zinc mcg/dL (a) (b) (c) Figure 5: Association of plasma-derived PUFAs involved in inﬂammation and serum zinc levels in HCV + AUD patients (Gr. 2). (a) Association of α-linoleic acid (αLA) concentrations and serum zinc levels. (b) Association of eicosapentaenoic acid (EPA) concentrations and serum zinc levels. (c) Association of ω3 docosapentaenoic acid (DPA5,3ω) concentrations and serum zinc levels. Data analysis showed a correlation coeﬃcient: r; #adjusted analysis with NDD90 as a covariable: R . Statistical signiﬁcance was set at p≤ 0.05. NS: not signiﬁcant. a lowered proinﬂammatory shift in AUD patients with liver [26]. In our study, ﬁndings on zinc deﬁciency were con- injury (Gr. 4), which is consistent with our previous study sistent in HCV-infected patients, both those who had only [19]. HCV infection alone and with AUD diagnosis, and those Several polyunsaturated fatty acids (PUFAs) including who also drank heavily with or without liver injury. How- EPA and DHA inhibit HCV replication in vitro using the ever, this deﬁciency in zinc was most severe in HCV + AUD HCV RNA replicon system considerably [21]. In our study, patients. Zinc metabolism in ALD gets altered [27], and zinc we found a generalized drop in the total ω3, EPA, and deﬁciency [28] causes major dysregulation in several DPA5,3ω and DHA PUFAs in both the HCV-only and pathological pathways involved in the initiation and pro- gression of ALD [29]. A therapeutic role for zinc supple- HCV + AUD groups. Previously, ω3 supplementation has shown eﬃcacy in the medical management of HCV patients mentation has shown great promise in ALD, especially in who priorly were nonresponders to the combination anti- alcoholic cirrhosis and alcoholic hepatitis [30, 31]. viral therapy [22]. We found that the HCV + AUD patients Zinc has a pivotal role in the hepatic regulation of fatty also had higher HCV RNA quantiﬁcation, which was sig- acids; it aids in the downstream process of anti-inﬂamma- niﬁcantly predictable by the DPA5,3ω level with respect to tory PUFAs [32, 33]. 'erefore, a low zinc level likely heavy drinking. Anti-HCV activities of the selective PUFAs lowered the downstream synthesis of omega-3 PUFAs have been reported previously [23]. Our study showed that, systematically in HCV + AUD patients (Supplemental in HCV + AUD patients, elevated RNA quantiﬁcation was Table S2). Previously, we have reported that AUD heavy shown compared to the HCV-only patients. drinkers with HIV comorbidity showed a close association Zinc depletion has been abundantly reported with liver of proinﬂammatory ω6 PUFA and low zinc [19]. In this study, we found that low zinc was closely associated with disease [24]; importantly, it is lower in the HCV diagnosed patients [25]. Zinc levels were also deﬁcient in AUD patients total ω3 PUFAs and EPA and DPA5,3ω [34] in HCV-in- who exhibited early-stage ALD [15]. In one report, altered fected heavy drinkers. One recent review has described the PUFAs were observed in HIV-HCV-infected heavy drinkers role of zinc as a cofactor in the modulation of desaturase α-Linoleic acid nmol/mL ω6:ω3 ratio EPA nmol/mL Total ω6 FAs mmol/L Total ω3 FAs mmol/L DPA5, 3 nmol/mL Advances in Virology 7 activity involved in FA absorption, oxidation, metabolism, 5. Conclusions and incorporation [12]. 'e importance of our study was Depletion in the concentrations of ω3 PUFAs, DPA5,3ω that low zinc and candidate ω3 PUFAs were signiﬁcantly and EPA, is involved in the proinﬂammatory shift in HCV associated with HCV + AUD patients only. 'e interaction diagnosed individuals, especially comorbid with AUD of hypozincemia and the lowering of anti-inﬂammatory FAs diagnosis. Clinically deﬁcient zinc concentrations are ob- in HCV-infected heavy drinkers likely played a pathological served in HCV patients, with further lowering in HCV- role in the proinﬂammatory shift. infected AUD patients. 'is deﬁciency in both ω3 PUFAs NDD90 as a heavy drinking marker showed a close and zinc concentrations is highly correlated. 'e assess- association with candidate ω3 fatty acids, especially ment of the number of drinking days (NDD90) could be a DPA5,3ω, in the expression of HCV RNA quantiﬁcation. Its good marker in HCV-infected heavy drinkers to identify role was also signiﬁcant in the association analyses for de- the changes that are involved in the proinﬂammatory ﬁcient ω3 PUFAs and low zinc. 'e connection of speciﬁc response. markers of heavy drinking with the exacerbation of meta- bolic dysregulation in liver injury has been recently high- lighted [35]. 'e number of drinking days past 90 days Abbreviations (NDD90) was a signiﬁcant marker that was observed in our AUD: Alcohol use disorder study. It seems that how many days an individual drinks ALD: Alcoholic liver disease heavily (NDD90) in an assessment period is consequential in ALB: Albumin the changes in the anti-inﬂammatory ω3 PUFAs in HCV- ALP: Alkaline phosphatase infected heavy drinkers, which was a new ﬁnding in our ALT: alanine aminotransferase study. AST: Aspartate aminotransferase Our study has several limitations. We had smaller AST: ALT: AST by ALT ratio number of females in each group; however, in HCV-in- CS: Clinically signiﬁcant fected groups, this diﬀerence was numerically signiﬁcant. DHA: Docosahexaenoic acid 'is study was a single time-point clinical observational DPA 5,3ω: Docosapentaenoic acid investigation on the characterization of low zinc and FA: Fatty acids omega-3 alterations in a unique HCV and AUD comorbid Gr. 1: HCV-only cohort. 'us, neither longitudinal interpretations or Gr. 2: HCV + AUD treatment outcomes of zinc or omega-3 supplementation Gr. 3: AUD without liver injury were not evaluated. 'e plasma or tissue levels of PGs, LTs, Gr. 3: AUD with liver injury TXs, and lipoxins were not measured, which could have Gr. 5: HV further identiﬁed speciﬁc role of the product-associated HCV: Hepatitis C virus proinﬂammatory response, synthesized during the HCV + AUD: Patients with HCV and AUD comorbidity downstream process of PUFA. 'ere was no biopsy HV: Healthy volunteers performed on the study patients since this was an ob- PUFAs: Polyunsaturated fatty acids servational study only. 'is study did not use a mecha- Tbili: Total bilirubin nistic approach, which was beyond the scope of this study TLFB90: Timeline followback past 90 days design. Dietary FAs were not studied in this investigation; TD90: Total drinks in the past 90 days however, none of the patients showed any malnutrition at AvgD90: Average drinks in the past 90 days intake (Table 2). We did not study the type of alcohol [36]; HDD90: Heavy drinking days in the past 90 days our study focused on the patterns and amount of alcohol NDD90: Number of drinking days in the past 90 days intake as per the guideline of NIAAA as described in the ω3-EPA: Eicosapentaenoic acid methods section. ω6 : ω3: Omega 6 by omega 3 ratio. Lowering of the concentrations in ω3 PUFAs, DPA5,3ω and EPA, likely plays a signiﬁcant role in the proinﬂammatory shift in HCV-infected AUD patients. Data Availability Serum zinc lowered to a clinically deﬁcient level in the same cohort. 'is deﬁciency was a strong predictor of the Data will be provided on request based on reasonable queries lowering of total and candidate ω3 PUFAs, exclusively, in only. the HCV + AUD patients. 'e number of drinking days (NDD90) could be a good marker in HCV-infected heavy Disclosure drinkers and a key to identify the changes that can incur in the ω3 PUFAs in such comorbid cohort. 'is report ex- 'is article is a work of the University of Louisville Alcohol tends the need to elucidate the anti-HCV mechanism in Research Center and National Institutes of Health. 'e comorbid AUD patients by a combination of speciﬁc anti- content is solely the responsibility of the authors and does inﬂammatory PUFAs [37] and zinc supplementation [38] not necessarily represent the oﬃcial views of the National that may lead to the development of novel molecular- Institutes of Health. 'is manuscript is in the public domain based therapeutic agents and direct therapeutic mecha- in the USA. E-mail address for the readers to contact the nistic studies in this special cohort. author to obtain the data is v0vats01@louisville.edu. 8 Advances in Virology [6] I. Novo-Veleiro, “Alcoholic liver disease and hepatitis C virus Conflicts of Interest infection,” World Journal of Gastroenterology, vol. 22, no. 4, p. 1411, 2016. All authors declare no conﬂicts of interest. [7] K. H. Zirnheld, D. R. Warner, J. B. Warner, J. E. Hardesty, C. J. McClain, and I. A. Kirpich, “Dietary fatty acids and Authors’ Contributions bioactive fatty acid metabolites in alcoholic liver disease,” Liver Research, vol. 3, no. 3-4, pp. 206–217, 2019. VV is the project PI and designed the study. VV and MLS [8] S. A. Read, S. Obeid, C. Ahlenstiel, and G. Ahlenstiel, “'e acquired and managed the data. VV and JF analyzed the role of zinc in antiviral immunity,” Advances in Nutrition, data. VV, RA, and SS interpreted the data analysis. VV, JF, vol. 10, no. 4, pp. 696–710, 2019. SS, and RA wrote the manuscript. VV, RA, SS, and MLS [9] T. Suda, O. Okawa, R. Shirahashi, N. Tokutomi, and M. Tamano, “Changes in serum zinc levels in hepatitis C critically reviewed the manuscript and contributed scien- patients before and after treatment with direct-acting antiviral tiﬁcally. All authors have approved the submission version agents,” Hepatology Research, vol. 10, no. 4, pp. 696–710, 2019. of this manuscript. [10] V. Vatsalya, M. C. Cave, R. Kumar et al., “Alterations in serum zinc and polyunsaturated fatty acid concentrations in treat- Acknowledgments ment-naive HIV-diagnosed alcohol-dependent subjects with liver injury,” AIDS Research and Human Retroviruses, vol. 35, 'e authors thank the clinical staﬀ of the Clinical Center of no. 1, pp. 92–99, 2019. NIH for providing the patientcare. 'e authors also ac- [11] J. A. Barocas, K. S. Armah, D. M. Cheng et al., “Zinc deﬁciency knowledge the research staﬀ of the Alcohol Research Center and advanced liver ﬁbrosis among HIV and hepatitis C co- infected anti-retroviral na¨ıve persons with alcohol use in of the University of Louisville. 'e manuscript has been Russia,” PloS One, vol. 14, no. 6, 2019. edited by Jane Frimodig. 'e study was supported by the [12] D. Tsoukalas, A. 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Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

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Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

Dysregulation in Plasma ω3 Fatty Acids Concentration and Serum Zinc in Heavy Alcohol-Drinking HCV Patients

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