The Journal of Nutrition
Nutrition and Disease
Low Plasma Zinc Is Associated with Higher
Mitochondrial Oxidative Stress and Faster Liver
Fibrosis Development in the Miami Adult
Studies in HIV Cohort
1–4
Sabrina S Martinez,
5
Adriana Campa,
5
Yinghui Li,
5
Christina Fleetwood,
6
Tiffanie Stewart,
7
Venkataraghavan Ramamoorthy,
8
and Marianna K Baum
5
*
5
Robert Stempel College of Public Health and Social Work, Florida International University, Miami, FL;
6
Louis A Johnson Veterans
Affairs Medical Center, Clarksburg, WV;
7
Center for Nanoscience and Technology, University of Notre Dame, Notre Dame, IN; and
8
Department of Nutrition and Kinesiology, University of Central Missouri, Warrensburg, MO
Abstract
Background: Oxidative stress and reduced antioxidants may be a trigger for liver fibrogenesis. Reducing oxidative stress
through higher antioxidant concentration may be a potential antifibrotic target.
Objective: We aimed to investigate longitudinally whether plasma zinc, an antioxidant, is related to mitochondrial
oxidative stress and the progression of liver fibrosis in the Miami Adult Studies in HIV (MASH) cohort.
Methods: A prospective observational cohort study was conducted in 487 predominantly African American HIV-
monoinfected and HIV/hepatitis C virus (HCV)–coinfected adults with a mean 6 SD age of 47.08 6 7.67 y from the MASH
cohort and followed for a median of 34 mo. Blood was collected for plasma zinc and measures were used to calculate the
fibrosis-4 (FIB-4) score (aspartate amino transferase, alanine aminotransferase, and platelets). Plasma zinc deficiency was
defined as <0.75 mg/L. Total DNA was extracted from peripheral blood mononuclear cells and mitochondrial DNA
(mtDNA) 8-hydroxyguanosine (8-oxo-dG) was determined. Adjusted mixed models were used to assess the relations
between zinc, stage of liver disease, and oxidative stress over time and compared between HIV and HIV/HCV groups.
Results: Zinc concentrations (b: 20.368, SE = 0.172; P = 0.033) and deficiency were associated with lower FIB-4 scores over time
(b: 0.381, SE = 0.118; P = 0.001). Compared with those who were not zinc deficient, zinc-deficient participants had an increased
risk of having more-progressed liver disease (OR: 1.91; 95% CI: 1.15, 3.16; P = 0.012). Higher mtDNA 8-oxo-dG was associated
with zinc deficiency (b: 0.049, SE = 0.024; P = 0.044) and higher FIB-4 scores over time (b: 0.597, SE = 0.168, P < 0.001).
Conclusions: Lower plasma zinc concentrations were associated with liver fibrosis progression and mitochondrial
oxidative stress in the HIV and HIV/HCV groups. Zinc may play a role in the impact of liver disease outcomes. J Nutr
2017;147:556–62.
Keywords: zinc, HIV, HIV/HCV coinfection, mtDNA 8-oxo-dG, oxidative stress
Introduction
Liver disease in people living with HIV is a major public health
issue because it is one of the most common causes of non-AIDS–
related death (1). It is estimated that approximately one-quarter
of HIV-infected adults in the United States are coinfected with
hepatitis C virus (HCV)
9
(2). HIV and HCV coinfection has been
associated with faster progression of liver disease to cirrhosis and
hepatocellular carcinoma (3, 4). Liver disease progression
includes fibrosis, which involves a process of accumulation of
collagen and other extracellular matrix proteins that eventually
weaken the structure of the liver and reduce the amount of
functional tissue (5). Liver fibrosis is a continuous process
marked by molecular-, tissue-, and cellular-level activities that
4
The views expressed in this article are of those of the authors. The sponsor had
no role in the design and conduct of the study; collection, management, analysis,
and interpretation of the data; preparation, review, or approval of the manuscript;
or decision to submit the manuscript for publication.
*To whom correspondence should be addressed. E-mail: baumm@fiu.edu.
9
Abbreviations used: ALT, alanine aminotransferase; AST, aspartate amino-
transferase; Ct, threshold cycle; FIB-4, fibrosis-4; HCV, hepatitis C virus; mtDNA,
mitochondrial DNA; PBMC, peripheral blood mononuclear cell; ROS, reactive
oxygen species; SOD, superoxide dismutase; 8-oxo-dG, 8-hydroxyguanosine.
1
A preliminary version of this analysis was presented at the HEP DART
Conference, Maui, Hawaii, 6–10 December 2015.
2
Supported by the National Institute on Drug Abuse, grant no. 1R01DA023405,
and the National Institute on Alcoholism and Alcohol Abuse, grant no.
1R01AA018011.
3
Author disclosures: SS Martinez, A Campa, Y Li, C Fleetwood, T Stewart, V
Ramamoorthy, and MK Baum, no conflicts of interest.
ã 2017 American Society for Nutrition.
556 Manuscript received November 4, 2016. Initial review completed December 2, 2016. Revision accepted January 31, 2017.
First published online February 22, 2017; doi:10.3945/jn.116.243832.
are influenced by oxidative stress and lower antioxidant defenses
(6). HIV and HCV, in their destructive interaction with immune
mechanisms systemically and locally, greatly increase oxidative
stress, which contributes to liver fibrogenesis (7, 8).
Zinc is an essential nutrient and an antioxidant that is part of
>300 enzymes in the body. It is needed for proper immune
function, and its deficiency has been associated with oxidative
stress (9). A considerable amount of zinc is found in the nucleus,
where it plays a role in the stability of genes and their expression
(10). Studies have shown that >50% of people living with HIV
have low plasma zinc concentrations (11–13). We and others
have shown that zinc has been low not only in HIV-infected
cohorts, but also in those with HCV infections, as well as in
those with HIV/HCV coinfection (14–16). Zinc may directly
and indirectly affect liver fibrosis. Zinc affects the activity and
availability of proteins, including the enzymes needed for the
production and destruction of collagen, thus directly affecting
the process of fibrosis (17–20). Zinc administration during
in vitro and in vivo studies diminished the action of one of the
essential enzymes for collagen formation, prolyl hydroxylase
(17, 21). Zinc possesses anti-inflammatory and antioxidant
characteristics that may indirectly affect hepatic stellate cells
(22). In vitro zinc deficiency activated hepatic stellate cells to
form collagen (20) and augmented the oxidative stress stimu-
lated by hepatotoxins (23). Zinc therapy in both HIV and HCV
separately improved disease outcome measures (24, 25).
Oxidative stress may be generated through mitochondrial
alterations. Mitochondrial DNA (mtDNA) is more susceptible
to oxidative damage than is nuclear DNA because it lacks
introns, the structural protection and repairing capabilities of
nuclear DNA. The mtDNA position is contiguous to the electron
transport system, which is a major source of reactive oxygen
species (ROSs); in addition, it lacks protective histones and
nucleotide excision repair capacity (26, 27). 8-Hydroxyguanosine
(8-oxo-dG), a common DNA lesion, is considered to be a marker
of DNA damage (28, 29), and higher concentrations of mtDNA
8-oxo-dG have been associated with higher mutation, deletion,
fragmentation of DNA, and diminished mtDNA (30, 31). A
greater amount of 8-oxo-dG lesions has been shown to be produced
from mtDNA than from nuclear DNA (32, 33). Mitochondrial
dysfunction in HIV is commonly present as a result of both HIV
itself and antiretroviral therapy (34).
Although oxidative stress appears to be one of the triggers of
fibrogenesis, studies on the effect of antioxidants are lacking. To
our knowledge, most of the studies conducted on the relation
between zinc status and liver disease have been observational
and cross-sectional, and the majority of the zinc supplementa-
tion trials in HIV and HCV have been of too short a duration to
observe an effect on liver fibrosis. Therefore, long-term longi-
tudinal studies are needed to elucidate the role of zinc status in
generating excess oxidative stress and its effect on liver fibrosis.
Because the reduction of oxidative stress has the potential to be
an antifibrotic target for interventions, we longitudinally inves-
tigated the association between plasma zinc concentrations and
mitochondrial oxidative stress and liver fibrosis in those who
were HIV monoinfected and HIV/HCV coinfected in the Miami
Adult Studies in HIV cohort.
Methods
Study design
After we obtained informed consent, a total of 487 participants were
enrolled into observational studies from October 2009 through October
2012 and followed for a median of 34 mo. Participants were eligible if
they were HIV monoinfected or HIV/HCV coinfected by clinical
documentation; were >18–60 y of age; had a BMI (in kg/m
2
) >18 but
<40; had controlled comorbid diseases (diabetes, symptomatic cardio-
vascular disease, hyperlipidemia, and metabolic syndrome); were free of
hepatitis B virus and hepatic encephalopathy, carcinoma, or cirrhosis;
were not heavy tobacco smokers; and were not pregnant. The study
protocol was approved by the Florida International University Institu-
tional Review Board.
Assessments
Study visits were complet ed every 3 mo and data were collected on
demographics, medications, me dical history, and alcohol and sub-
stance abuse. Alcohol use information was obtained with the use of
the validated and standardized Alcohol Use Dis orders Identification
Test questionnaire that detects frequenc y of use, hazardous and
dependent drinking, and binge drinking (35). Fasting blood was
collected at baseline and ever y 6 mo for metabolic panel, oxidative
stress (mt DNA 8-oxo- dG), and plasma zinc. Plasma samples for zinc
concentrations and peripheral mononuclear cells (PBMCs) for
mtDNA 8-oxo- dG were s tored in a 280°C f reezer for batch analysi s.
Fibrosis-4 ( FIB-4), a noninvasi ve measure of liver fibrosis, was
calculated with the use of a formula that included participant age,
aspartate aminotransferase (AST) and alanine aminotransferase
(ALT) concentrations, and platelet counts, as follows: [age (years) 3
AST (U/L)]/[platelet counts (10
9
cells/L) 3 ALT
1/2
(U/L)]. FIB-4, at a
cutoff of >1.45, has a negat ive predictive value to exclude advanced
fibrosis (stages 4–6 of the Ishak scale) of 90% with a sensitivity of
70%. A cutoff of >3.25 has a positive predictive value of 65% and a
specificity of 97% to predict advanced disease (36). CD4 cell counts
and HIV viral load were obtained through medical records wit h the
participantÕspermission.
Laboratory assays
Plasma zinc concentrations. Determination of plasma zinc con cen-
trations was obtained through ve nipunctu re wit h the use of a BD trace-
element evacuated tube. The plasma sample was first digested with the
use of 0.2 mL + 0.2 mL HNO
3
+0.1mLH
2
O
2
in digestion tub es. The
solution was transferred to an inductively co upled plasma MS tube, to
which 20 mL yttrium and 9480 mLH
2
O was added, and it was then
analyzed via inductively coupled plasma MS. The cutof f for zinc
deficiency that was used for the analysis was <0.75 mg/L. Each
analytical batch included duplicate samples, $1 me thod blank, a
continuing calibration check samp le, and a quality cont rol sample. All
method blanks during analysis were below the method detection limits.
The readings of the co ntinuing calibration check samples were always
within acceptable range (85–115% of initial calibration). A certified
reference material was used as the quality cont rol sample throu ghout
the analysis, and the recoveries for these samples were always within
acceptable range (7 0–130%).
mtDNA 8-oxo-dG. Blood drawn into BD Vacutainer cell preparation
tubes was used to obtain PBMCs. This blood collection tube contains
sodium citrate anticoagulant and Ficoll-Paque density medium
separated by a gel barrier (37). Total DNA from the PBMCs was
obtained with the use of a QIAamp DNA blood mini kit (Qiagen).
The degree of oxidative damage was determined with the use of the
previously described quantitative real-time PCR method (38, 39).
Briefly, an iQ5 qPCR system (BioRad) was applied to detect the
amount of mtDNA damage caused by mutagenic 8-oxo-dG with the
use of 80 ng total genomic DNA extracted from PBMCs, and 1 of 2
samples was treated with human 8-oxoguanine glycosylase enzyme,
whichactsasbothanN-glycosylase and an apurinic lyase (40, 41).
Threshold cycle (Ct) values were obtained for both enzyme-treated
and enzyme-nontreated samples (Ct
2
and Ct
1
, respectively). T he
mean difference of Ct (Ct
2
2Ct
1
= DCt) was representative of the
amount of 8-oxo-dG and presented as DCt.Eachanalyticalbatch
included duplicate samples and a no-template control to detect
contaminating DNA. No significant amplifications were detected in
the no-template controls.
Zinc is associated with liver disease 557
Statistical analysis
Baseline demographics and characteristic s of the cohort were reported
with the use of frequenc ies and means 6 SDs. StudentÕs t te sts wer e
used to compare the HIV and HIV/HCV groups at baseline . A chi-
square test was used to compare the prevalence of zinc deficiency
between the HIV and HIV/HCV groups. Multivariable linear and
logistic regression analyses were used at baseline to examine the
relations and dose-respon se effect s between plasma z inc status, FIB-4,
and mtDNA 8-oxo-dG in the entire cohort and separately by
HIV/HCV and HIV groups, while controlling for age, sex, markers
of HIV disease progression (HIV viral load and CD4 cell count),
substance abuse, and energy and zinc intake. In addition, separate
models were analyzed by sex. Mixed models were use d to account for
repeated measures in order to assess z inc status, FIB-4, and mtDNA
8-oxo-dG over time in the entire cohort and separately by HIV/HCV
coinfection status, while adjusting for the variables of interest. The
mixed-models method is a flexible approach that allows a wide variety
of correlation patterns to be modeled. In addi tion, the mode l uses a
random int ercept to account f or correlation between study visits per
person and random slope based on ti me since baseline. The term
‘‘mixed’’ r efe rs to both fixed and random effects in the same model.
Separate mixed models were also analyzed by sex. Zinc status was
analyzed as a continuous variable and a cate gorical variable for zin c
deficiency (1 = yes and 0 = no). The P values reported are 2 sided, and
P values of <0.05 were used to indicate statistical s ignificance. SAS
version 9.3 was used for all s tatistical analysis.
Results
Characteristics of the study population. A total of 487
participants from the Miami Adult Studies in HIV cohort were
included in the analyses, and their characteristics are displayed
in Table 1. The majority of the participants were male (65.1%),
African American (68.1%), and taking antiretroviral therapy
(80.1%). Approximately 29% of the participants were coinfected
with HIV and HCV. The prevalence of zinc deficiency was high
in the entire cohort (70.16%) and significantly higher in the
HIV/HCV group (82.3%) than it was in the HIV group (65.3%)
(P < 0.001) (Figure 1).Themeanplasmazincconcentrationwas
also significantly lower in the HIV/HCV group (0.62 6 0.17 mg/L)
than in the HIV group (0.73 6 0.28 mg/L; P = 0.001).
Relation between zinc status and liver fibrosis. Plasma
zinc concentrations we re significantly related to FIB-4
(P = 0.011) in the entire cohort at baseline (Table 2). For every
unit decrease in plasma zinc, there was a 0.63-unit increase in
the FIB-4 score. Those with zinc d eficiency had a 0.44-unit
increase in their FIB-4 scores compared with those without
zinc deficiency in the entire cohort (P = 0.002). When we
looked at the HIV group separately, there were n o signific ant
associations between plasma zinc or zinc deficiency and FIB-4
in the monoinfected group at bas eline. However, significant
associations were observed in the HIV/HCV participants
between plasma zinc and FIB-4 at baseline. Higher FIB-4
scores were associated with lower pla sma zinc as a continuous
variable (P = 0.001) and with zinc defici ency (P =0.031)inthis
group. We observed similar findi ngs in the coinfected group in
models analyzed by sex.
Participants with zinc deficiency compared with those without
zinc deficiency in the entire cohort were at a greater risk of having
an FIB-4 score of >1.45, indicative of mild liver disease (OR: 1.91;
95% CI: 1.15, 3.16; P = 0.012). The HIV/HCV group had a 3 times
greater risk of having an FIB-4 score >1.45 (OR: 3.05; 95% CI: 1.05,
8.87; P = 0.041) after adjusting for race, BMI, CD4 cell count,
HIV viral load, and alcohol and tobacco use (results not shown
in the tables).
Longitudinal analyses that used mixed models examining the
relation between plasma zinc status and FIB-4 over time while
adjusting for the variables of interest are also displayed in Table
2. In the entire cohort, plasma zinc concentrations (P = 0.033)
and zinc deficiency (P = 0.001) were associated with lower FIB-4
scores over time. Thus, over a median of 24 mo, for every unit
decrease in plasma zinc, there was an increase of 0.37 units in
FIB-4, which shows increasing liver fibrosis occurring in
conjunction with zinc deficiency over time. Similarly, those
with zinc deficiency over time had an increase of 0.38 units of
FIB-4 compared with those who were not zinc deficient.
Longitudinal analyses indicated that in those who were HIV
positive, there was a significant association over time between
TABLE 1 Baseline characteristics of the cohort
1
Variable Value
Sex
F 34.9 (170)
M 65.1 (317)
Age, y 47.1 6 7.67
Race/ethnicity
White non-Hispanic 6.79 (33)
White Hispanic 17.9 (87)
Black non-Hispanic 68.1 (331)
Black Hispanic 4.32 (21)
Other 2.80 (14)
HIV/HCV coinfected 28.8 (140)
CD4 cell count, cells/μL 502 6 347
HIV viral load, log
10
copies/mL 2.75 6 1.34
BMI, kg/m
2
27.4 6 5.18
ART 81.1 (391)
Plasma zinc, mg/L 0.70 6 0.26
Zinc deficiency
2
70.2 (301)
FIB-4 score
3
1.59 6 1.38
mtDNA 8-oxo-dG, DCt 0.36 6 0.31
1
Values are means 6 SDs or % (n), n = 487. ALT, alanine aminotransferase; ART,
antiretroviral therapy; AST, aspartate aminotransferase; Ct, threshold cycle; FIB-4,
fibrosis-4; HCV, hepatitis C virus; mtDNA, mitochondrial DNA; PLT, platelet count;
8-oxo-dG, 8-hydroxyguanosine.
2
Defined as plasma zinc concentration ,0.75 mg/L.
3
Calculated with the following formula: [age (years) 3 AST (U/L)]/[PLT (10
9
cells/L) 3
ALT
1/2
(U/L)].
FIGURE 1 Prevalence of zinc deficiency among the entire cohort
(n = 487) and HIV-monoinfected (347) and HIV/HCV-coinfected groups
(n = 140). Values are percentages (SDs) of those with zinc deficiency
(plasma zinc concentrations ,0.75 mg/L). *Different from HIV, P , 0.05;
chi-square test used in the analysis. HCV, hepatitis C virus.
558 Martinez et al.
zinc deficiency and FIB-4 (P = 0.038); however, continuous zinc
concentrations were not significantly associated with FIB-4. Both
continuous lower plasma zinc concentrations (P = 0.003) and zinc
deficiency were related to higher FIB-4 concentrations in the
HIV/HCV group (P = 0.045). When models were analyzed separately
by sex, only men in the HIV/HCV group were found to have plasma
zinc concentrations and zinc deficiency significantly associated with
FIB-4 over time (P =0.0001andP = 0.036, respectively).
Relation between zinc status and 8-oxo-dG. A dose-
response effect was also observed between 8-oxo-dG and
plasma zinc status at baseline in the entire cohort (Table 3).
For every unit decrease in plasma zinc there was an increase of
0.196 units in the 8-oxo-dG for the overall cohort (P < 0.001).
Those with zinc deficiency had higher 8-oxo-dG by 0.088 units
than did those who were not zinc deficient. Plasma zinc was
associated with 8-oxo-dG (Table 3) in the HIV group (P = 0.009),
and there was a trend toward significance between zinc de-
ficiency and 8-oxo-dG (P = 0.08). Similar findings were observed
in the regression models among men living with HIV, with
plasma zinc being associated with 8-oxo-dG (P = 0.037);
however, this relation with 8-oxo-dG as a marker of oxidative
stress was not observed when analyzed with the use of zinc
deficiency as a categorical variable. There were no significant
findings between plasma zinc and 8-oxo-dG at baseline in those
in the HIV/HCV group, in the entire cohort or models of coinfected
women; however, in men in the HIV/HCV group, there was a
significant relation between plasma zinc concentrations and 8-oxo-
dG at baseline (P =0.037).
The relation between plasma zinc status and mtDNA 8-oxo-
dG when using mixed models longitudinally and controlling for
variables of interest is displayed in Table 3. Regardless of HCV
serostatus, zinc deficiency was associated with higher 8-oxo-dG
concentrations (P = 0.044), showing increasing oxidative stress
with increasing 8-oxo-dG concentrations over time. When the
analyses were conducted separately for the HIV and HIV/HCV
groups, there were no significant findings with plasma zinc and
8-oxo-dG over time; however, in men in the HIV/HCV group,
plasma zinc was significantly related to 8-oxo-dG over time
(P = 0.032). Models that included only women were not
significantly associated with 8-oxo-dG over time.
Relation between liver fibrosis and 8-oxo-dG. The relation
between FIB-4 scores and 8-oxo-dG at baseline and longitudinally is
shown in Table 4. At baseline, there was a significant relation
between FIB-4 and 8-oxo-dG in the entire cohort (P =0.017)that
was strengthened when only men were considered in the model
(P = 0.004). Over time, FIB-4 scores were positively associated with
8-oxo-dG in the entire cohort (P < 0.001), showing that for every
1-unit increase of FIB-4 score, a 0.60-unit increase in 8-oxo-dG
occurred. Similar findings were observed in the HIV/HCV group
(P < 0.023). When only men were analyzed, FIB-4 was associated
with 8-oxo-dG longitudinally in the combined cohort (P <0.001)
and in the HIV/HCV group (P = 0.023). When only the HIV group
or only women were considered, the relations between FIB-4 and
8-oxo-dG were not significant.
Discussion
The results of this study indicate that lower plasma concentrations
of zinc (an antioxidant) are associated with higher FIB-4 scores, a
marker of liver fibrosis, and that, over time, zinc concentrations
deteriorate as liver fibrosis increases in a cohort of adults with HIV
and HIV/HCV coinfection. Moreover, zinc deficiency compared
with no zinc deficiency increased the risk of having more progressed
liver disease. Plasma zinc concentrations were also longitudinally
associated with mtDNA 8-oxo-dG, a biomarker of oxidative stress
in the mitochondria. These relations were also more prevalent in the
models that included men only, confirming previous reports of more
advanced fibrosis in men (42). To our knowledge, this is the first
study that shows a significant association between plasma zinc
concentrations and liver disease or FIB-4 index scores over time in
adults with HIV or HIV/HCV coinfection. Previously, we showed a
significant difference between zinc concentrations in people with
HIV and HIV/HCV coinfection; however, to our knowledge, a
relation between zinc status and a measure of liver fibrosis has
not been previously shown, which may have been due to a much
smaller sample size (14). In this study, we also showed that zinc
TABLE 2 Associations between plasma zinc and FIB-4 at baseline and longitudinally in the entire cohort and separated by sex among
HIV-monoinfected and HIV/HCV-coinfected groups
1
Entire cohort Women Men
Baseline multivariate
analysis
2
Longitudinal multivariate
3
analysis
Baseline multivariate
analysis
4
Longitudinal multivariate
analysis
5
Baseline multivariate
analysis
4
Longitudinal multivariate
analysis
5
b P b P b P b P b P b P
Cohort
Zinc, mg/L 20.63 6 0.25 0.011 20.37 6 0.17 0.033 20.53 6 0.27 0.054 20.24 6 0.16 0.126 20.76 6 0.35 0.033 20.43 6 0.24 0.073
Zinc deficiency
6
20.44 6 0.14 0.002 0.38 6 0.12 0.001 20.34 6 0.16 0.043 0.08 6 0.10 0.002 20.53 6 0.20 0.008 0.52 6 0.17 0.002
HIV monoinfected
Zinc, mg/L 20.01 6 0.202 0.98 20.06 6 0.16 0.70 20.06 6 0.17 0.72 20.1 1 6 0.10 0.29 0.05 6 0.29 0.87 20.07 6 0.22 0.76
Zinc deficiency
6
20.01 6 0.125 0.97 0.27 6 0.13 0.038 20.01 6 0.11 0.96 0.07 6 0.07 0.34 20.02 6 0.18 0.93 0.33 6 0.18 0.066
HIV/HCV coinfected
Zinc, mg/L 22.38 6 0.72 0.001 21.79 6 0.58 0.003 21.17 6 0.82 0.16 20.36 6 0.56 0.53 23.62 6 1.17 0.003 23.23 6 0.94 0.001
Zinc deficiency
6
20.70 6 0.32 0.031 0.53 6 0.26 0.045 20.44 6 0.38 0.25 20.03 6 0.25 0.92 20.84 6 0.48 0.086 0.90 6 0.42 0.036
1
Values are means 6 SEs, n = 487. FIB-4 was calculated with the use of the following formula: [age (years) 3 AST (U/L)]/[PLT (10
9
cells/L) 3 ALT
1/2
(U/L)]. ALT, alanine
aminotransferase; AST, aspartate aminotransferase; AUDIT, Adult Use Disorders Identification Test; FIB-4, fibrosis-4; HCV, hepatitis C virus; PLT, platelet count.
2
Analyses conducted with the use of linear and logistic regression. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
3
Analysis conducted with the use of mixed models. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
4
Analyses conducted with the use of linear and logistic regression. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
5
Analysis conducted with the use of mixed models. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
6
Defined as plasma zinc concentration ,0.75 mg/L.
Zinc is associated with liver disease 559
concentrations were lower in the HIV/HCV group and were
associated with faster liver disease progression than in the HIV
group.
Our data, consistent with our previous studies and those of
others in people living with HIV (11–13), demonstrated a high
prevalence of zinc deficiency (70.2%) or plasma zinc <0.75 mg/L
in HIV-infected adults (Table 1). Those who were in the
HIV/HCV coinfection group had an even higher prevalence of
zinc deficiency, 82.3%. We also demonstrated that there were
lower plasma zinc concentrations in HIV/HCV coinfection than
in HIV monoinfection (14). Lower plasma zinc concentrations
were also found in HCV-infected participants compared with
healthy controls in a study by Guo et al. (43). Moreover, low
plasma zinc concentrations were found in liver biopsy specimens
from patients with alcoholic liver disease and those with chronic
active hepatitis (44). In addition, a relation was previously found
between lower blood zinc concentrations and an increased
severity of liver fibrosis determined with liver biopsy in a cross-
sectional study (45).
HCV infection in an HIV-infected patient is associated with
accelerated progression to hepatic fibrosis compared with in
individuals with HCV infection alone (46). ROSs are key
contributors to hepatic fibrosis in HCVand HIV/HCV coinfection
(47). We previously showed that HIV/HCV-coinfected adults have
higher levels of oxidative stress, including mtDNA 8-oxo-dG, than
do those who are HIV monoinfected (14, 39). In this study, 8-oxo-
dG was measured in the mitochondria as a measure of oxidative
stress because mitochondrial damage is increased during HIV
replication (48), and the mtDNA-to–nuclear DNA ratio is signif-
icantly lower in patients with selective antiretroviral combinations,
suggesting a more rapid decrease of mtDNA than nuclear DNA.
This condition is supported by the lack of protected introns in
mtDNA compared with nuclear DNA (49).
Several studies have provided evidence that plasma concentra-
tions of zinc are correlated with copper and zinc superoxide
dismutase (SOD) activity (50–52). A study in transgenic mice
suggested that the antioxidant enzyme mitochondrial manganese
SOD and the cytosolic isoenzyme copper and zinc SOD work in
TABLE 3 Associations between plasma zinc and mtDNA 8-oxo-dG at baseline and longitudinally in the entire cohort and separated by
sex among HIV-monoinfected and HIV/HCV-coinfected groups
1
Entire cohort Women Men
Baseline multivariate
analysis
2
Longitudinal multivariate
analysis
3
Baseline multivariate
analysis
4
Longitudinal multivariate
analysis
5,6
Baseline multivariate
analysis
4
Longitudinal multivariate
analysis
5,7
b P b P b P b P b P b P
Cohort
Zinc, mg/L 20.20 6 0.05 ,0.001 20.04 6 0.03 0.12 20.14 6 0.09 0.11 20.06 6 0.06 0.37 20.21 6 0.07 0.003 20.03 6 0.03 0.22
Zinc deficiency
8
20.09 6 0.03 0.011 0.05 6 0.02 0.044 20.05 6 0.06 0.34 0.05 6 0.04 0.27 20.11 6 0.04 0.017 0.04 6 0.03 0.16
HIV monoinfected
Zinc, mg/L 20.12 6 0.05 0.009 20.03 6 0.03 0.19 20.11 6 0.08 0.15 20.05 6 0.06 0.44 20.12 6 0.06 0.037 20.03 6 0.03 0.32
Zinc deficiency
8
20.05 6 0.03 0.08 0.03 6 0.02 0.20 20.03 6 0.05 0.53 0.03 6 0.04 0.47 20.06 6 0.04 0.11 0.02 6 0.03 0.34
HIV/HCV coinfected
Zinc, mg/L 20.53 6 0.30 0.08 20.15 6 0.11 0.16 20.07 6 0.41 0.87 — — 21.11 6 0.49 0.037 20.47 6 0.21 0.032
Zinc deficiency
8
20.13 6 0.13 0.32 0.04 6 0.05 0.44 20.18 6 0.20 0.37 — — 20.18 6 0.20 0.37 — —
1
Values are means 6 SEs, n = 487. AUDIT, Adult Use Disorders Identification Test; HCV, hepatitis C virus; mtDNA, mitochondrial DNA; 8-oxo-dG, 8-hydroxyguanosine.
2
Analyses conducted with the use of linear and logistic regression. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
3
Analysis conducted with the use of mixed models. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
4
Analyses conducted with the use of linear and logistic regression. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
5
Analysis conducted with the use of mixed models. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
6
Because of small sample size, longitudinal models in women in the HIV/HCV group did not converge.
7
Because of small sample size, longitudinal models in men in the HIV/HCV group with zinc deficiency did not converge.
8
Defined as plasma zinc concentration ,0.75 mg/L.
TABLE 4 Associations between FIB-4 and mtDNA 8-oxo-dG at baseline and longitudinally in the entire cohort and separated by sex
among HIV-monoinfected and HIV/HCV-coinfected groups
1
FIB-4 score
2
Entire cohort Women Men
Baseline multivariate
analysis
3
Longitudinal multivariate
analysis
4
Baseline multivariate
analysis
5
Longitudinal multivariate
analysis
6
Baseline multivariate
analysis
5
Longitudinal multivariate
analysis
6
b P b P b P b P b P b P
Cohort 0.42 6 0.17 0.017 0.60 6 0.17 ,0.001 0.20 6 0.19 0.29 0.08 6 0.12 0.51 0.76 6 0.26 0.004 0.75 6 0.23 0.001
HIV monoinfected 0.15 6 0.20 0.47 20.14 6 0.22 0.53 0.20 6 0.20 0.33 0.11 6 0.12 0.38 20.60 6 0.36 0.094 20.32 6 0.32 0.32
HIV/HCV coinfected 0.59 6 0.35 0.10 0.61 6 0.26 0.023 20.56 6 0.45 0.23 20.52 6 0.31 0.11 0.88 6 0.36 0.022 0.83 6 0.35 0.023
1
Values are means 6 SEs, n = 487. ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUDIT, Adult Use Disorders Identification Test; FIB-4, fibrosis-4; HCV,
hepatitis C virus; mtDNA, mitochondrial DNA; PLT, platelet count; 8-oxo-dG, 8-hydroxyguanosine.
2
Calculated with the use of the following formula: [age (years) 3 AST (U/L)]/[PLT (10
9
cells/L) 3 ALT
1/2
(U/L)].
3
Analyses conducted with the use of linear and logistic regression. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
4
Analysis conducted with the use of mixed models. All models were adjusted for sex, race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
5
Analyses conducted with the use of linear and logistic regression. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
6
Analysis conducted with the use of mixed models. All models were adjusted for race, BMI, CD4 cell count, HIV viral load, AUDIT, and tobacco use.
560 Martinez et al.