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Hemochromatosis and fatty liver disease: Building evidence for insulin resistance in bottlenose dolphins (Tursiops Truncatus)


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Unlabelled: Hemochromatosis in bottlenose dolphins (Tursiops truncatus) is associated with high postprandial plasma insulin levels, suggestive of insulin resistance. In humans, insulin resistance is associated with liver pathologies, including excessive iron deposition and nonalcoholic fatty liver disease. Dolphin liver tissues, in addition to excessive iron storage, were evaluated for other pathologies supportive of underlying insulin resistance. Archived liver tissues collected postmortem during 1985-2010 from 18 dolphins (median age 27.9 yr, range 0.7-51.4) that were part of the Navy Marine Mammal Program's managed collection were assessed for the presence and severity of hemosiderin deposition, fatty liver disease, and hepatitis. Demographics, clinical pathology values, and percentage weight loss were compared among dolphins with and without these changes. Twelve (66.7%) dolphins had mild to moderate hemosiderin deposition, 7 (38.9%) had mild to severe fatty liver disease, and 11 (61.1%) had mild to moderate hepatitis. Of the 12 dolphins with hemosiderosis, deposition occurred in the Kupffer cells among 11 (91.7%). Dolphins with fatty liver disease were more likely to have higher postprandial serum hyperglycemia (>140 mg/dl), leukocytosis (>11,000 cells/microl), and hyperglobulinemia (>3.5 g/dl). Unlike in many nonhuman terrestrial animals, fatty liver disease was not associated with rapid weight loss or hypoglycemia. Interestingly, there were no significant associations among dolphins with hemosiderosis, fatty liver disease, and hepatitis. This study supports that both hemochromatosis and fatty liver disease were present in the dolphin study population, and histopathology and clinical pathology among these animals suggest a nonhereditary, metabolic etiology. Keywords: Bottlenose dolphin, fatty liver disease, hemochromatosis, hemosiderosis, hepatic lipidosis, hepatitis, Tursiops truncatus.
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Journal of Zoo and Wildlife Medicine 43(3): S35–S47, 2012
Copyright 2012 by American Association of Zoo Veterinarians
Steph anie Venn-Watson, D.V.M., M.P.H., Celeste Benh am, B.S., M.S., Kevin Carlin, B.A., M.P.H.,
Damian DeRienzo, M.D., and Judy St. Leger, D.V.M., Dipl. A.C.Z.P.
Abstract: Hemochromatosis in bottlenose dolphins (Tursiops truncatus) is associated with high postprandial
plasma insulin levels, suggestive of insulin resistance. In humans, insulin resistance is associated with liver
pathologies, including excessive iron deposition and nonalcoholic fatty liver disease. Dolphin liver tissues, in
addition to excessive iron storage, were evaluated for other pathologies supportive of underlying insulin resistance.
Archived liver tissues collected postmortem during 1985–2010 from 18 dolphins (median age 27.9 yr, range 0.7–
51.4) that were part of the Navy Marine Mammal Program’s managed collection were assessed for the presence and
severity of hemosiderin deposition, fatty liver disease, and hepatitis. Demographics, clinical pathology values, and
percentage weight loss were compared among dolphins with and without these changes. Twelve (66.7
) dolphins
had mild to moderate hemosiderin deposition, 7 (38.9
) had mild to severe fatty liver disease, and 11 (61.1
mild to moderate hepatitis. Of the 12 dolphins with hemosiderosis, deposition occurred in the Kupffer cells among
11 (91.7
). Dolphins with fatty liver disease were more likely to have higher postprandial serum hyperglycemia
(.140 mg/dl), leukocytosis (.11,000 cells/
ll), and hyperglobulinemia (.3.5 g/dl). Unlike in many nonhuman
terrestrial animals, fatty liver disease was not associated with rapid weight loss or hypoglycemia. Interestingly, there
were no significant associations among dolphins with hemosiderosis, fatty liver disease, and hepatitis. This study
supports that both hemochromatosis and fatty liver disease were present in the dolphin study population, and
histopathology and clinical pathology among these animals suggest a nonhereditary, metabolic etiology.
Keywords: Bottlenose dolphin, fatty liver disease, hemochromatosis, hemosiderosis, hepatic lipidosis,
hepatitis, Tursiops truncatus.
Hemochromatosis in bottlenose dolphins (Tur-
siops truncatus) is associated with high transferrin
saturation (83–85
), high serum iron (.300 lg/
dl), high serum aminotransferases, hyperglobulin-
emia, hypercholesterolemia, and hypertriglyceri-
Liver biopsies from three out of three
dolphins with hemoc hromatosis had excessive
hemosiderin deposition with or without hepatitis.
Dolphins are responsive to phlebotomy therapy
involving weekly removal of 7–17
of estimated
blood volume (1–3 L) for 22–30 wk, including
successful return of serum iron, aminotransferas-
es, and globulins back to normal values.
botomy does not, however, ameliorate high
cholesterol or triglyceride levels.
The underlying cause of hemochromatosis in
bottlenose dolphins remains unknown. In hu-
mans, increasing serum iron and excessive liver
iron has been associated with hyperinsulinemia
and insulin resistance.
Interestingly, dolphins
have metabolic states mimicking insulin resis-
tance and type 2 diabetes.
After ingesting a
high-protein meal, healthy dolphins can demon-
strate a prolonged fasting hyperglycemia and
moderately high fasting insulin levels.
it is hypothesized that a diabetes-like state may be
physiologic for dolphins ingesting very-low-car-
bohydrate diets, diseases associated with insulin
resistance in humans also exist in dolphins,
including urate nephrolithiasis and hemochroma-
Further, dolphins with hemochromatosis
have significantly higher 2-hr postprandial insulin
levels compared to dolphins without hemochro-
In humans, insulin resistance can be part of
metabolic syndrome, which consists of a combi-
nation of endogenous risk factors that increase
the risk of type 2 diabetes.
Risk factors for
metabolic or insulin-resistant syndromes include
age, physical inactivity, and abdominal obesi-
A predominant nding among people with
metabolic syndrome is nonalcoholic fatty liver
disease, which can progress to a severe disease
state called nonalcoholic steatohepatitis.
From the Nat ional Mari ne Mammal Foundation, San
Diego, California 92106, USA (Benham, Carlin, Venn-
Watson); the Naval Medical Center San Diego, San
Diego, California 92105, USA (DeRienzo); and Sea-
World San Diego, San Diego Cali fornia, 92016, USA (St.
Leger). Cor responden ce should be directed t o Dr. Venn-
Watson (
To better understand potential associations
among hemochromatosis, liver pat hology, and
insulin resistance, histology of all formalin-fixed
parafn-embedded postmortem nonneonatal dol-
phin liver tissues or slides that were available at
the Navy Marine Mammal Program (1985–2010)
was reviewed. Liver pathology ndings, including
hemosiderosis, fatty liver disease, and hepatitis,
were compared by dolphin demographics, weight
loss, and clinical pathology to determine potential
risk f actors for these histologic changes.
Nonneonatal dolphins from the Navy Marine
Mammal Program f rom which the Navy had
arch ived postmortem formalin-fixed blocks or
slides of liver tissue were evaluated and included
in the study. Eighteen dolphins that died during
1985–2010 met these criteria. Dolphins included
in this study were housed in San Diego, Califor-
nia. Dolphins lived in open-ocean, netted enclo-
sures and were fed a high-quality frozen–thawed
fish diet including capelin, herring, mackerel, and
squid. Dolphins were routinely provided antipar-
asitics (ivermectin, fenbendazole, and praziquan-
tel) prophylactically. The median age of dolphins
in the study at time of necropsy was 27.9 yr (range
0.7–51.4 yr), and eight (44.4
) were females. For
clinical pathology analyses, data throughout the
last 12 mo before death were available for 16
) dolphins.
His tologic evaluations of liver tissues were
conducted using hematoxylin and eosin–stained
slides made from parafn-embedded blocks. In-
formation entered into an electronic, standardized
database inc luded unique animal identifier, evi-
dence of hepa titis (yes/no), hepatitis severity
(mild, moderate, severe), hepatiti s dur ation
(acute, subacute, ch ronic), hepatitis infiltr ate
(eosinophilic, lymphocytic, neutrophilic, plasma-
cytic, other), hemosiderosis (yes/no), location of
hemosiderosis (Kupffer cells, hepatocytes, centri-
lobular), hemosiderosis severity (mild, moderate,
severe), fatty change (yes/no), fatty change de-
scriptor (lipid-type), fatty change severity (mild,
moderate, severe), fatty change location (centri-
lobular, hepatocellular), necrosis (yes/no), bro-
sis (yes/no), hematopoiesis (yes/no), cholestasis
(yes/no), and atrophy or degener ation (yes/no).
Lipid was confirmed among dolphins with evi-
dence of fatty change (lipid type) using oil red O
staining on paired frozen liver tissue archived at
808 C. All hematoxylin and eosin–stained slides
were graded by the same pathologist experienced
in viewing dolphin tissues; all oil red O–stained
frozen liver tissu es were graded by a second
pathologist who was familiar with viewing human
tissues with steatosis.
Blood samples were originally collected from
dolphins by venipuncture from either the periar-
terial venous rete in the caudal peduncle or a uke
blade. These methods for blood collection have
Table 1. Hemosiderosis, fatty change, hepatitis, and general cause of death by animal among 18 bottlenose
dolphins (Tursiops truncatus).
Animal Hemosiderosis Fatty change Hepatitis
Acute or chronic disease
proceeding death
A None None Mild Chronic
B Mild None Mild Acute
C Mild None None Acute
D None None Mild Chronic
E Mild Moderate Moderate Chronic
F Mild None None Chronic
G Mild None Mild Chronic
H None Moderate Mild Acute
I Mild Mild None Acute
J None None Mild Chronic
K Mild Severe None Chronic
L None None None Acute
M Mild None None Chronic
N Mild None Mild Chronic
O None Severe Mild Chronic
P Mild None None Acute
Q Moderate Mild Mild Chronic
R Moderate Moderate Mild Chronic
Acute ¼ less than 2 wk before death.
been used by the Navy for over 30 yr, including for
establishing normal reference intervals of com-
plete blood cell counts and serum chemistries.
Although blood collection from the periarterial
venous rete has the potential for mixed arterial–
venous sampling, none of the blood values
reported in this study have demonstrated signif-
icant differences by sampling site (Navy Marine
Mammal Program, unpubl. data). Animals were
trained to present their tail for sampling, or
behavioral conditioning was used to collect sam-
ples out of the water with animals resting on a
foam mat during a routine physical exam. Sam-
ples were collected using either a 20-or 21-gauge
3.8-cm Vacutainert needle (Vacutainer Systems;
Becton Dickinson, Rutherford, New Jersey
07070, USA) or a 21-gauge 1.9-c m butterfly
needle attached to a Vacutainer holder. Blood
was collected into a Vacutainer serum separator
tube or a Vacutainer EDTA (K
) tube for ser um
chemistries and complete blood counts, respec-
Samples were c hilled for 30 min and centrifuged
within 2 hr. Centrifugation was performed at
1,006 g at 218C for 10 min. Fibrin clots were
removed and serum was transferred to a 5-ml
plastic submission tube. Whole blood was sub-
mitted in EDTA Vacutainer tubes. All samples
were sent on ice via courier to a reference
laboratory. Aut omated analyses were used by
Quest Diagnostic Labora tories, incl uding the
Coultert LH 1500 Series (Beckman Coulter,
Inc., Fullerton, California 32834-3100, USA) for
hematology and the Olympust AU600 (Olympus
America Inc., Center Valley, Pennsylvania 18034-
0610, USA) for seru m chemistry analysis. Fish-
erbrand Dispette 2t (Fisher Scientific, Waltham,
Massachusetts 02454, USA), correlating with the
Westergren method, was used to determine 60-
min erythrocyte sedimentation rates (ESRs) from
1 ml EDTA whole blood.
The following hematologic and serum biochem-
ical variables were measured and incorporated
into the retrospective study: total white blood cell
(WBC) count, hematocrit, hemoglobin, red blood
cell (RBC) count, RBC distribution width
(RDW), mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), MCH concen-
tration (MCHC), mean platelets, absolute neu-
trophils, absolute lymphocyt es, absolute
monocytes, absolute eosinophils, glucose, blood
urea nitrogen (BUN), creatinine, uric acid, sodi-
um (Na
), potassium (K
), chloride (Cl
), carbon
dioxide (CO
), total protein, globulins, iron,
alkaline phosphatase (ALP), lactate dehydroge-
nase, aspartate aminotransferase, alanine amino-
transferase, gamma-glutamyl transpeptidase,
creatine kinase, and 60-min ESR. Because of
known differences in clinical pathology values
among overnight fasted and nonfasted samples,
and the greatest interest in postprandial metabol-
ic changes, blood data in this study were limited to
nonfasted samples.
SAS Release 9.2 statistical software was used
for all statistical analyses (SAS, Inc., Cary, North
Carolina 27513, USA). Fisher’s exact tests were
conducted to compare the prevalence of hemo-
siderosis, fatty liver disease, and hepatitis by sex;
to determine associations among them; and to
compare their prevalence by the presence of
acute or chronic disease that led to death.
‘‘Acute’’ was defined as disease initiated within
2 wk of death, and ‘‘chronic’’ was defined as
disease initiated longer than 2 wk before death.
Kruskal-Wallis tests were used to measure dif-
ferences in age and percentage weight loss over
the last 6 mo among dolphins with and without
hemosiderosis, fatty liver disease, and hepatitis.
Table 2. Prevalence of various potential risk factors by the presence or absence of liver hemosiderosis, hepatic
lipidosis, and hepatitis among 18 bottlenose dolphins (Tursiops truncatus).
risk factors
Hemosiderosis Fatty change Hepatitis
(No. and
(No. and
(No. and
(No. and
(No. and
(No. and
Sex female 4/12 (33.3) 4/6 (66.7) 0.32 4/7 (57.1) 4/10 (40) 0.49 3/10 (30) 5/8 (62.5) 0.16
Hemosiderosis NA NA NA 6/12 (50) 6/12 (50) 0.50 5/11 (45.5) 6/11 (54.6) 0.63
Hepatitis 5/11 (45.5) 6/11 (54.6) 0.63 5/10 (50) 5/10 (50) 0.37 NA NA NA
Mean age (yr) 29.1 25.6 0.85 30.3 26.4 0.49 35.6 18.3 0.008
weight loss over
last 6 mo 4.7 5.1 1.0 6.6 4.3 0.77 6.1 3.2 0.81
NA ¼ Not applicable. Note: 6-mo weight data available for nine dolphins.
Because of relatively normal distribution curves
among nonfasted clinical pathology values, one-
way analyses of variance using a general linear
model (to account for varying sample numbers
by animal) were conducted to compare these
values among dolphins with and without hemo-
siderosis, fatty liver disease, and hepatitis, in-
cluding values collected from 12 mo up to 1 mo
before death and values collected during the last
month before death. The prevalence of selected
Table 3. Comparisons of nonfasted hematologic and serum biochemistry values (mean 6 SD) by presence or
absence of liver hemosiderosis, hepatic lipidosis, and hepatitis among blood samples collected from the last 12–1
mo before death (Tursiops truncatus).
Blood variables
Yes (n ¼ 135) No (n ¼ 60) P value
WBCs (cells/ll) 9,959 6 3,868 12,470 6 5,388 ,0.001
RBCs (3 10
/ll) 3.2 6 0.4 3.6 6 0.5 ,0.001
)416 6426 6 0.21
Hb (g/dl) 14 6 2156 2 ,0.001
MCH (pg) 43 6 3406 2 ,0.001
MCV (fl) 127 6 9 116 6 5 ,0.001
MCHC (g/dl) 34 6 1356 1 ,0.001
)156 3146 1 0.002
NRBC (3 10
/ll) 0.3 6 1.2 0.3 6 0.8 0.85
MPV (fl) 14 6 2146 2 0.41
Platelets (cells/
ll) 85,422 6 32,819 131,317 6 37,790 ,0.001
ll) 11,715 6 44,573 9,446 6 5,226 0.70
Lymphocytes (cells/
ll) 881 6 443 1,135 6 426 ,0.001
Monocytes (cells/
ll) 358 6 310 451 6 328 0.06
Eosinophils (cells/
ll) 830 6 527 1,439 6 858 0.004
Glucose (mg/dl) 106 6 21 149 6 54 ,0.001
BUN (mg/dl) 52 6 12 54 6 8 0.24
Creatinine (mg/dl) 1.6 6 0.5 1.2 6 0.3 ,0.001
BUN:creatinine 29 6 8446 11 ,0.001
(mEq/L) 155 6 2.8 152 6 4 ,0.001
(mEq/L) 3.8 6 0.3 2.7 6 0.3 0.04
(mEq/L) 120 6 4 116 6 6 ,0.001
Uric acid (mg/dl) 0.4 6 0.3 0.3 6 0.3 0.05
(mEq/L) 25 6 4256 3 0.50
Protein (g/dl) 7.1 6 0.6 7.8 6 1.6 ,0.001
Albumin (g/dl) 3.6 6 1.5 5.4 6 9.0 0.04
Globulins (g/dl) 2.8 6 0.3 3.9 6 1.8 ,0.001
Albumin:globulin 1.6 6 0.3 1.3 6 0.7 ,0.001
Calcium (mg/dl) 9.1 6 0.5 9.1 6 0.6 1.0
Bilirubin (mg/dl) 0.2 6 0.1 0.2 6 0.1 0.05
Inorganic phosphate (mg/dl) 4.7 6 0.7 5.1 6 0.8 ,0.001
ALP (U/L) 251 6 101 164 6 67 ,0.001
LDH (U/L) 539 6 765 439 6 96 0.32
AST (U/L) 271 6 182 185 6 39 ,0.001
ALT (U/L) 55 6 66 42 6 25 0.15
GGT (U/L) 65 6 65 47 6 39 0.05
Cholesterol (mg/dl) 208 6 48 246 6 71 ,0.001
Triglycerides (mg/dl) 118 6 86 106 6 64 0.36
CK (mU/ml) 130 6 65 109 6 38 0.03
Iron (
lg/dl) 192 6 79 131 6 53 ,0.001
ESR (mm/h) 11 6 10 21 6 28 ,0.001
Note: 16/18 study dolphins had blood values within the last yr before mortality. WBC, white blood cell; RBC, red blood cell; HCT,
hematocrit; Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular
hemoglobinconcentration; RDW, RBC distribution width; NRBC, nucleated red bloodcells; MPV, mean platelet volume;BUN, blood
urea nitrogen; Na
sodium; Kþ, potassium; Cl
chloride; CO
, carbon dioxide; ALP, alkaline phosphatase; LDH, lactate
dehydrogenase; AST, aspartate aminotransferase; ALT, alanine aminotransferase; G GT, gamma-glutamyl transpeptidase; CK,
creatine kinase; ESR, erythrocyte sedimentation rate.
clinically high or low values was compared
among dolphins with or without hemosiderosis
or fatty liver disease using a chi square test, or
Fisher’s exact test if categories had less than ve
samples. Clinical pathology reference intervals
have been previously determined for this study
One dolphin was removed from the
comparative hemosiderosis evaluations, because
this animal had confirmed hemochromatosis and
had been treated with phlebotomy; this dolphin
was included in the other evaluations and results.
P values less than or equal to 0.01 and 0.05 were
defi ned as significant for analyses of variance and
chi square or Fisher’s exact test, respectively.
A summary table is provided of individual
study dolphins, including severity of hemosider-
Table 3. Extended.
Fatty change Hepatitis
Yes (n ¼ 117) No (n ¼ 57) P value Yes ( n ¼ 167) No (n ¼ 31) P value
12,751 6 4,535 10,160 6 4,468 ,0.001 12,004 6 4,830 8,300 6 2,503 ,0.001
3.3 6 0.8 3.3 6 0.3 0.38 3.3 6 0.6 3.5 6 0.3 0.01
38 6 9426 4 0.002 40 6 7446 3 ,0.001
13 6 3146 1 0.02 14 6 3156 1 0.001
40 6 3436 3 ,0.001 42 6 3436 2 0.02
116 6 8 128 6 7 ,0.001 122 6 9 127 6 7 ,0.001
34 6 2346 1 0.20 34 6 1346 1 ,0.001
15 6 3146 3 0.26 16 6 2136 2 ,0.001
0.6 6 1.9 0.2 6 0.5 0.03 0.4 6 1.3 0.0 6 0.2 0.02
13 6 2146
1 0.15 14 6 2136 2 0.25
103,263 6 42,472 96,299 6 41,590 0.30 103,063 6 39,390 92,821 6 40,029 0.09
10,039 6 4,332 12,325 6 47,910 0.72 12,568 6 45,702 6,143 6 2,355 0.19
1,004 6 503 939 6 459 0.39 936 6 460 1,002 6 468 0.34
429 6 364 380 6 300 0.35 427 6 339 209 6 258 0.01
1,282 6 918 928 6 600 0.003 1,111 6 755 838 6 556 0.01
135 6 48 112 6 37 ,0.001 126 6 45 106 6 24 ,0.001
52 6 9556 12 0.24 55 6 12 49 6 8 ,0.001
1.1 6 0.3 1.6 6 0.4 ,0.001 1.4 6 0.5 1.6 6 0.3 0.008
50 6 12 34 6 9 ,0.001 38 6 11 28 6 11 ,0.001
152 6 5 155
6 3 ,0.001 154 6 4 155 6 3 0.002
3.6 6 0.4 3.8 6 0.3 ,0.001 3.7 6 0.3 3.8 6 0.3 0.17
116 6 6 119 6 4 ,0.001 118 6 5 119 6 3 0.14
0.4 6 0.3 0.4 6 0.3 0.28 0.4 6 0.3 0.3 6 0.2 0.53
25 6 3256 3 0.15 24 6 3276 3 ,0.001
8.1 6 1.4 7.0 6 0.7 ,0.001 7.7 6 1.1 6.7 6 0.6 ,0.001
4.1 6 6.6 4.2 6 0.5 0.95 4.4 6 0.4 3.8 6 8.2 0.43
5.0 6 1.4 2.7 6 0.5 ,0.001 3.6 6 1.3 2.4 6 0.4 ,0.001
0.9 6 0.5 1.7 6 0.3 ,0.001 1.4 6 0.5 1.9 6 0.4 ,0.001
9.2 6 0.5 9.0
6 0.7 0.04 9.1 6 0.6 9.0 6 0.3 0.09
0.2 6 0.1 0.2 6 0.1 0.74 0.2 6 0.1 0.2 6 0.1 0.02
4.7 6 0.6 4.8 6 0.8 0.32 4.8 6 0.8 5.0 6 0.7 0.16
163 6 87 228 6 79 ,0.001 197 6 84 277 6 110 ,0.001
705 6 1,115 424 6 177 ,0.001 525 6 772 476 6 216 0.62
278 6 272 228 6 59 0.06 249 6 189 236 6 61 0.61
75 6 94 42 6 18 ,0.001 57 6 67 37 6 13 0.02
73 6 94 58 6 33 0.13 64 6 70 51 6 27 0.15
230 6 82 223 6 44 0.54 227 6 65 205 6 33 0.09
108 6 66 132 6 92 0.11 121 6 84 84 6 44 0.02
142 6 73 102 6 43 ,
0.001 117 6 62 141 6 24 0.04
135 6 60 199 6 83 ,0.001 156 6 61 206 6 93 ,0.001
17 6 26 15 6 14 0.51 16 6 20 10 6 12 0.03
osis, fatty liver disease, and hepatitis, and
whether or not disease leading to death was
acute or chronic (Table 1). Twelve (66.7
dolphins had hemosiderin deposition in liver.
Among these, 10 (83.3
) and 2 (16.7
) were
characterized as mild or moderate respectively.
Of the 12 dolphi ns with hemo siderosis, 11
) had hemosiderin deposition in Kupffer
cells; 9 (75
) had deposition in both Kupffer
cells and hepatocytes. Only one dolphin had
hemosiderin deposition in hepatocytes only.
Age, sex, and weight were not significant predic-
Table 4. Last 30 d comparisons of nonfasted hematologic and serum biochemistry values (mean 6 SD) by
presence or absence of liver hemosiderosis, fatty change, and hepatitis among 13 bottlenose dolphins (Tursiops
Blood variables
Yes (n ¼ 53) No (n ¼ 13) P value
WBCs (cells/ll) 12,963 6 6,830 23,558 6 9,381 ,0.001
RBCs (3 10
/ll) 3.1 6 0.5 3.6 6 0.8 0.004
)396 7416 7 0.46
Hb (g/dl) 13 6 2156 3 0.04
MCH (pg) 42 6 346 2 0.19
MCV (fl) 127 6 9 115 6 8 ,0.001
MCHC (g/dl) 33 6 1366 1 ,0.001
)146 3156 3 0.68
NRBC (3 10
/ll) 0.8 6 2.8 0.2 6 0.4 0.44
MPV (fl) 14 6 4166 4 0.09
Platelets (cells/
ll) 63,113 6 32,880 149,923 6 85,494 ,0.001
Neutrophils (cells/
ll) 10,998 6 6,250 19,840 6 9,056 ,0.001
Lymphocytes (cells/
ll) 785 6 501 1,800 6 675 ,0.001
Monocytes (cells/
ll) 626 6 755 990 6 863 0.14
Eosinophils (cells/
ll) 434 6 456 704 6 775 0.11
Glucose (mg/dl) 115 6 26 236 6 86 ,0.001
BUN (mg/dl) 86 6 52 83 6 49 0.88
Creatinine (mg/dl) 3.7 6 3.7 1.3 6 0.7 0.03
BUN:creatinine 23 6 6626 13 ,0.001
(mEq/L) 157 6 6 148 6 5 ,0.001
(mEq/L) 4.0 6 0.6 4.6 6 1.4 0.02
(mEq/L) 120 6 9 110 6 5 ,0.001
Uric acid (mg/dl) 0.5 6 0.5 0.6 6 0.4 0.35
(mEq/L) 24 6 7206 6 0.10
Protein (g/dl) 7.1 6 1.4 9.6 6 1.9 ,0.001
Albumin (g/dl) 3.2 6 1.7 3.7 6 0.3 0.30
Globulins (g/dl) 3.2 6 0.8 5.9 6 1.9 ,0.001
Albumin:globulin 1.4 6 0.2 0.8 6 0.5 ,0.001
Calcium (mg/dl) 9.1 6 0.8 9.1 6 0.8 1.0
Bilirubin (mg/dl) 0.3 6 0.4 0.5 6 0.2 0.22
Inorganic phosphate (mg/dl) 5.6 6 2.1 5.1 6 1.2 0.39
ALP (U/L) 156 6 84 175 6 65 0.44
LDH (U/L) 1,330 6 2,329 2,617 6 2,353 0.08
AST (U/L) 832 6 1,173 1,800 6 2,180 0.03
ALT (U/L) 358 6 1,271 972 6 1,321 0.12
GGT (U/L) 157 6 117 112 6 88 0.20
Cholesterol (mg/dl) 219 6 89 257 6 42 0.15
Triglycerides (mg/dl) 212 6 132 346 6 269 0.03
CK (mU/ml) 311 6 1,078 332 6 454 0.95
Iron (
lg/dl) 225 6 203 187 6 98 0.52
ESR (mm/h) 46 6 34 70 6 42 0.07
Note: 13/18 studydolphins had bloodvalueswithin the last 30 d before mortality. WBC, white blood cell; RBC, red bloodcell; HCT,
hematocrit; Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular
hemoglobinconcentration; RDW, RBC distributionwidth; NRBC, ??; MPV, ??;BUN, bloodurea nitrogen; Na
sodium; K
, potassium;
chloride; CO
, carbon dioxide; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; ALT,
alanine aminotransferase; GGT, gamma-glutamyl transpeptidase; CK, creatine kinase; ESR, erythrocyte sedimentation rate.
tors of hemosiderosis (Table 2). The presence of
acute versus chronic disease leading to death was
not associated with the presence or absence of
hemosiderosis (4/6 [66.7
] acute disease animals
with hemosiderosis versus 8/12 [66.7
] chronic
disease animals with hemosiderosis; P ¼ 1.0).
Within 1 yr, as well as within 1 mo before
mortality, dolphins with hemosiderosis were more
likely to have lower WBC counts, RBC counts,
MCHC, platelets, lymphocytes, serum glucose,
BUN-to-creatinine ratio, protein, and globulins
com pared to dolphins without hemosiderosis.
Dolphins with hemosiderosis were more likely to
have higher MC V, Na
, and albumin-to-
globulin ratios compared to dolphins without
hemosiderosis (Tables 3 and 4). Upon evaluating
clinically relevant values, dolphins with hemosid-
erosis were more likely to have lymphopen ia
Table 4. Extended.
Fatty change Hepatitis
Yes (n ¼ 18) No (n ¼ 46) P value Yes ( n ¼ 41) No (n ¼ 25) P value
20,128 6 8.736 13,222 6 7,755 0.003 18,788 6 7,442 8,920 6 6,212 ,0.001
3.4 6 0.8 3.1 6 0.5 0.11 3.2 6 0.6 3.2 6 0.6 0.95
37 6 8406 7 0.17 38 6 6426 8 0.05
13 6 3136 2 0.63 13 6 2146 3 0.47
39 6 3436 1 ,0.001 41 6 3436 1 0.05
112 6 9 129 6 5 ,0.001 120 6 9 131 6 7 ,0.001
35 6 2336 2 0.007 34 6 1336 2 ,0.001
15 6 2146 3 0.47 16 6 2126 3 ,0.001
0.8 6 3.1 0.6 6 2.3 0.74 1.0 6 3.1 0.1 6 0.3 0.19
15 6 4136 4 0.13 15 6 4136
4 0.10
89,611 6 49,402 73,587 6 62,511 0.33 84,220 6 39,208 71,520 6 81,758 0.40
17,560 6 8,220 10,976 6 6,835 0.002 16,171 6 7,025 7,112 6 4,929 ,0.001
1,263 6 708 889 6 646 0.05 1,167 6 578 685 6 716 0.004
613 6 480 750 6 887 0.54 842 6 821 461 6 670 0.06
693 6 641 404 6 485 0.05 610 6 598 285 6 345 0.02
202 6 93 115 6 26 ,0.001 153 6 74 111 6 28 0.01
65 6 17 94 6 58 0.04 99 6 52 59 6 38 0.001
1.1 6 0.3 4.1 6 3.8 0.002 4.1 6 4.0 1.6 6 0.5 0.004
59 6 10 28 6 17 ,0.001 34 6 18 52 6 34 0.10
151 6 6 157 6 6 0.001 156 6 8 154 6 3 0.18
4.1 6 0.7 4.2 6
0.9 0.73 4.2 6 0.7 4.0 6 1.0 0.31
115 6 9 119 6 10 0.10 121 6 10 112 6 5 ,0.001
0.6 6 0.4 0.5 6 0.5 0.32 0.5 6 0.5 0.6 6 0.4 0.90
20 6 4246 7 0.03 20 6 5306 6 ,0.001
9.0 6 1.9 7.3 6 1.5 ,0.001 8.2 6 1.9 6.5 6 0.7 ,0.001
3.8 6 0.4 3.1 6 1.7 0.17 4.2 6 0.9 1.9 6 1.3 ,0.001
6.8 6 0.6 3.2 6 0.8 ,0.001 4.3 6 1.8 2.2 6 0.6 0.03
0.5 6 0.0 1.4 6 0.2 ,0.001 1.1 6 0.4 1.9 6 0.3 ,0.001
8.9 6 0.7 9.1 6 0.9 0.47 8.7 6 0.5 9.2 6 0.9 0.01
0.3 6 0.2 0.3 6 0.5 0.81 0.3 6 0.5 0.3 6
0.1 0.78
5.5 6 1.6 5.5 6 2.2 0.94 5.6 6 2.0 4.4 6 1.1 0.15
151 6 53 159 6 89 0.71 134 6 55 209 6 99 ,0.001
2,055 6 2,095 1,450 6 2,505 0.37 1,482 6 2,747 1,769 6 1,438 0.64
1,392 6 1,933 912 6 1,249 0.24 910 6 1,639 1,231 6 1,015 0.40
759 6 1,167 389 6 1,360 0.31 572 6 1,596 298 6 258 0.41
179 6 151 144 6 96 0.28 122 6 107 194 6 108 0.01
206 6 73 250 6 81 0.07 231 6 83 211 6 67 0.60
298 6 256 234 6 127 0.26 263 6 190 125 6 89 0.12
191 6 93 410 6 1,230 0.46 287 6 974 581 6 690 0.52
189 6 122 232 6 210 0.42 163 6 110 337 6 257 ,0.001
42 6 45 55 6 32 0.22 54 6
38 43 6 32 0.29
(,400 cells/ll) compared to those without hemo-
siderosis (17/134 [12.7
] versus 2/60 [3.3
]; P ¼
Upon examining hematoxylin and e osin–
stained slides, seven (38.9
) dolphins had mor-
phologic change suggestive of fatty liver disease
(Fig. 1). Of these, four (57.1
) and three (42.9
were characterized as mild or moderate, respec-
tively. Lipid was confirmed among all of these
animals with oil red O staining, and based upon
the staining results, cases were recategorized as
mild (n ¼ 1), moderate (n ¼ 4), and severe (n ¼ 2)
steatosis (Fig. 2). Five (71.4
) dolphins had fatty
liver disease that was hepatocellular, and two
) had fatty change that was both centrilob-
ular and hepatocellular. Five (71.4
) and two
) dolphins were characterized as having
fatty liver disease that was multifocal or diffuse,
weight loss were not significant predictors of fatty
liver disease (Table 2). The presence of acute
versus chronic disease leading to death was not
associated with the presence or absence of fatty
liver disease (2/6 [33.3
] acute-disease animals
had fatty liver disease, where as 5/11 [45.5
chronic-disease animals had fatty liver disease; P
¼ 0.84).
Within 1 yr, as well as within 1 mo before
mortality, dolphins with fatty liver disease were
more likely to have lower MCH, MCV, creatinine,
and Na
and higher WBC counts, serum BUN-to-
creatinine ratios, glucose, proteins, and globulins
compared to dolphins without fatty change (Ta-
bles 3 and 4). Upon assessing clinically relevant
values, blood samples from dolphins with fatty
liver disease during the last 12–1 mo before death
were more likely to have postprandial hypergly-
cemia (.140 mg/dl) compared to dolphins that
did not have fatty liver disease (.3.5 g/dl) (18/58
] versus 15/115 [13
]; P ¼ 0.006). These
dolphins were more likely to have leukocytosis
(WBC count .11,000 cells/
ll) (66.7
and 34.2
respectively; P , 0.001) and hyperglobulinemia
(27/31 [87.1
] and 0/65 [0
], respectively; P ,
0.001) compared to dolphins without fatty liver
Eleven (61.1
) dolphins had hepatitis. Of these,
10 (90.9
) and 1 (9.1
) were characterized as
mild and moderate, respectively; the duration of
hepatitis was acute (5 [45.5
]), or subacute (6
]). Aside from one dolphin with concurrent
disseminated Cryptococcus infection and apopto-
sis and/or suspected viral inclusion, there was no
overt evidence of pathogens in other liver tissue.
This dolphin had neither hemosiderosis nor fatty
liver disease.
Inflammatory infiltrates of dolphins with hepa-
titis included lymphocytes (8 [72.7
]), plasma-
cytes (7 [63.6
]), and neutrophils (7 [45.5
Inflammation location was described as random
(7 [63.6
]), periportal (4 [36.4
]), sinusoidal (2
]), and centrilobular (1 [9.1
]). Sex and
percentage weight loss were not significant predic-
tors of hepatitis (Table 2). Dolphins with hepatitis
were more likely to be older compared to those
that did not have hepatitis (mean age with hepatitis
¼35.6 yr, mean age without hepatitis ¼18.3 yr; P ¼
0.008). Having acute versus chronic disease lead-
ing to death was not associated with the presence
or absence of hepatitis (2/6 [33.3
] acute disease
with hemosiderosis versus 8/12 [66.7
] chronic
disease with hemosiderosis; P ¼ 0.32).
Within 1 yr, as well as within 1 mo before
mortality, dolphins with hepatitis were more likely
to have lower MCV, serum albumin-to-globulin
ratios, ALP, iron, Ca
, and CO
and higher WBC
counts, MCHC, RDW, lymphocytes, serum cre-
atinine, Cl
albumin compared to dolphins without hepatitis
(Tables 3 and 4). Other abnormalities reported in
liver tissue among the 18 study dolphins included
fibrosis (3 [16.7
]), cholestasis (7 [38.9
]), and
necrosis or degeneration (6 [33.3
Although the study dolphins had any combina-
tion of hemosiderosis, fatty liver disease, and
hepatitis, dolphins with any one of these changes
were no more or less likely to have either of the
other changes (Table 2).
Extensive discussion regarding chronic hepati-
tis has been provided previously for this dolphin
As such, the discussion focuses on
hemosiderosis, hemochromatosis, and fatty liver.
Of 18 dolphins in the study, 12 (66.7
) had mild
or moderate hemosiderin deposition in liver
tissue. In humans, the most important cause of
hemosiderosis is hereditar y hemochrom atosis;
most often, this is caused by a point mutation in
the HFE gene that enables increased uptake of
di etary iron from the gut.
In addition to
hereditary hemochromatosis, hemosiderosis can
occur because of hemolytic anemia, anemia of
chronic disease, dietary iron overload, chronic
viral infection, or fatty liver disease.
animals known to have hemochromatosis include
a variety of captive birds, black rhinos, Egyptian
fruit bats, lemurs, northern fur seals, tapirs, and
Salers cattle.
Multiple dolphins from the Navy have hemo-
chromatosis, documented by progres sively in-
creasing and high serum iron (.300
lg/dl) over
9–19 yr, transferrin saturation ranging from 83
to 85
, excessive hemosiderin deposition in the
liver, and favorable response to phlebotomy
Dolphins with untreated hemochro-
matosis have phasic increases in serum amino-
transferases and are more likely to have
hyperglobulinemia, hype rcholesterolemia, and
hypertriglyceridemia compared to healthy con-
An increased immune response could be
due to direct trauma of iron in liver tissue or
underlying viral infections, but r esol ution of
hyperglobulinemia with phlebotomy sup ports
that direct insult by iron on tissues is likely the
primary issue.
In the current study, dolphins
Figure 1. Hematoxylin and eosin–stained liver
tissu es (3200 magnificati on) from three bottlenose
dolphins (Tursiops truncatus) representing (a) normal
hepatic cells and arc hitecture without excess lipid
accumulation and (b) mild diffuse hemosiderosis with
minimal hepatic lipidosis. Scattered inflammatory cells
are noted within sinusoids, and (c) moderate to severe
diffuse hepatic lipidosis and disorganization of paren-
chyma. There are large, round, clear vacuoles within
the cytoplasm of many hepatocytes.
Figure 2. Oil red O–stained frozen liver tissues
from three bottlenose dolphins (Tursiops truncatus)
paired in the same order as presented in Figure 1.
These images represent cases of (a) mild (3400), (b)
moder ate (3200), and (c) severe (3400) fatty liver
disease with or without hemosiderin deposition.
with mild to moderate hemosiderin deposition
were more likely to have lower globulins com-
pared to dolphins without hemosiderosis. The
discrepancy may be due to the need for enough
iron deposition to occur to stimulate an immune
response; in this study, no animals had severe
In humans, hypertriglyceridemia and dyslipide-
mia have been associated with excessive iron
storage, metabolic syndrome, and insulin resis-
Insulin resistance and hyperinsulinemia
are also risk factors for excessive iron and iron
overload in humans.
Interestingly, Navy dol-
phins with hemochromatosis and associated hy-
perlipidemia were more likely to have high 2-hr
postprandial insulin compared to dolphins with-
out hemochromatos is (25 versus 8
Among three dolphins with hemochromatosis
treated with phlebotomy (1–3 L blood per wk for
20–22 wk), all abnormal blood variables returned
to within normal ranges, with the exception of
serum lipids; these remained high.
treatment of human patients with hereditary
hemochromatosis and hyperlipidemia had con-
current decreases in serum ferritin and triglycer-
ide levels; glucose and c holesterol levels, however,
were not affected.
Mutations in the HFE gene
have been linked with hemochromatosis and have
been associated with primary hypertriglyceride-
mia, indicating th at there may be combined
metabolic and heritable risk factors for hemo-
chromatosis in humans.
In the current study,
dolphins with mild or moderate hemosiderosis
were no more likely to have dyslipidemia, changes
in liver enyzmes, or serum iron, indicating that
these dolphins did not have disease significant
enough to be clinically relevant. Similar multivar-
iate associations among hyperlipidemia and ex-
cessive iron storage need to be further assessed in
The location of iron deposition within the liver
can provide valuable clues to its etiology. Of 12
dolphins with hemosiderosis, 91.7
, and
occurred in Kupffer cells, Kupffer cells and
hepatocytes, and hepatocytes alone, respectively.
Kupffer cells are macrophages that are part of the
liver’s reticuloendothelial system.
In most hu-
mans with HFE hemochromatosis, iron deposi-
tion is primarily in hepatocytes and typically does
not involve Kupffe r cells.
Kupffer cell iron
deposition has been associated with one heredi-
tary hemochromatosis disorder, ferroportin dis-
ease, but this condition in humans is not
responsive to phlebotomy treatments.
diseases associated with Kupffer cell hemosiderin
deposition include chronic viral hepatitis B and C,
blood tr ansfusions, hemolytic anemia, and ane-
mia of chronic disease.
In this study, there were
no dif ferences in hematocrit or hemoglobin when
comparing dolphins with and without hemosider-
in deposition, indicating that iron deposition was
not due to hematologic disorders. Further, there
were no significant differences in inflammatory
indicators (higher WBC counts, serum globulins,
and ESR) among dolphins with and without
hemosiderin deposition, suggesting that infec-
tious disease may not be associated with mild to
moderate hemosiderosis.
Iron sequestration may occur in response to
bacterial infections, leading to hemosiderosis. In
this study, WBC counts were more likely to be
lower among dolphins with hemosiderosis, and
there were no significant differences in neutrophil
counts, indicating that concur rent bacterial infec-
tion was not associated with hemosiderosis. To
better understand the cause of mild to moderate
hemosiderosis in dolphin livers, future studies
should assess causes of chronic viral hepatitis that
do not necessarily elicit an inflammatory re-
sponse, as well as c ontinue to evaluate the
potential relationship between metabolic disease
and hemochromatosis.
Mild to severe fatty liver disease was present in
seven (38.9
) dolphins in this study. Dolphins
with fatty liver disease were more likely to have
postprandial hyperglycemia (.140 mg/dl), leuko-
cytosis (.11,000 cells/
ll), and hyperglobulinemia
(.3.5 g/dl) compared to dolphins without fatty
liver di sease throughout the last year before
death. In nonhuman terrestrial animals, excessive
fat deposition in liver tissue is often called hepatic
lipidosis, whereas in humans it is called f atty liver
disease. Here, we discuss both syndromes.
In general, hepatic lipidosis occurs when mo-
bilized lipids in the liver exceed the amount of
lipids leaving the liver. Hepatic lipidosis can be
found in many mammals, including cats, cattle,
and miniature horses.
Obesity and nega-
tive energy balances, especially those that result in
hypoglycemia, are often associated with hepatic
Higher risk conditions in nonhuman
terr estrial mamm als inc lude anorexia, rapi d
weight loss, lactation, and physical stress.
Hepatic lipidosis can be a fatal disease associated
with vomiting, anorexia, lethargy, weight loss,
hepatomegaly, hepatic encephalopathy, hyperbili-
rubinemia, and increased aminotransferases.
In the present study, dolphins with fatty liver
disease did not have a significant difference in
percentage weight loss prior to death compared to
dolphins without fatty liver disease. Further,
dolphins with fatty liver disease had significantly
higher, not lower, serum glucose compared to
those without fatty change. These ndings suggest
that mild to moderate fatty liver found in this
study’s dolphins is not likely due to rapid weight
loss or hypoglycemia.
Among humans, excessive fat depo sition in
liver is called fatty liver disease and is character-
ized as nonalcoholic or alcoholic.
holic fatty liver disease that progresses to a severe
disease state is called nonalcoholic steatohepati-
tis. Among humans, nonalcoholic fatty liver
disease is a key component of metabolic syn-
drome, which also includes insulin resistance,
high blood pressure, obesity, high triglycerides,
and high fasting glucose.
Metabolic syndrome,
in turn, is a risk f actor for type 2 diabetes and
cardiovascular disease.
Nonalcoholic fatty liver
disease is commonly reported among people with
type 2 diabetes, and insulin resistance may play a
role in poor outcomes (cirrhosis and mortality)
among patients with nonalcoholic f atty liver
Dolphins in the current study with fatty liver
disease were more likely to have postprandial
hyperglycemia compared to d olphins without
fatty change. A natural type 2 diabetes–like
metabolism among dolphins, including sustained
postprandial hyperglycemia and moderately high
insulin levels, was previously repor ted.
resistance may be a natur al condition for dol-
phins, and it is hypothesized that insulin resis-
tance may have evolved in dolphins to support
glucose needs in the face of a very-high-protein,
very-low-carbohydrate diet.
A similar hypothe-
sis has been made for humans, in which insulin
resistance may have evolved during the Ice Age to
enable humans to maintain adequate blood glu-
cose levels while feeding on primarily high-
protein diets.
Although insulin resistance and sustained post-
prandial hyperglycemia may be a natural and
healthy state for dolphins, diseases that have been
associated with insulin resistance in humans have
also been found in dolphins. Examples include
hemochromatosis, urate nephrolithiasis, chronic
inflammation, and now, fatty liver disease.
this study, dolphins with fatty change were more
likely to have leukocytosis and hyperglobulinemia
throughout the last year of life compared to those
without fatty change, indicating that chronic
inflammation was present among dolphins with
mild to moderate fatty liver disease. There is a
compelling theory that links insulin resistance,
type 2 diabetes, chronic inflammation, and nonal-
coholic fatty liver disease among humans. It is
believed that obesity leads to high plasma free
fatty acids, which cause insulin resistance in
skeletal muscle and liver.
Resulting insulin resis-
tance causes type 2 diabetes, low-grade inflam-
mation, and nonalcoholic fatty liver disease.
There are few studies regarding free fatty acids
in marine mammals, though high levels of omega-
3 fat ty acids have been reported in marin e
mammal lipids.
Given this model in humans,
future studies in dolphins should include com-
parative measurements of plasma free fatty acids
with fatty changes in skeletal muscle and liver.
A limit ation in this study is the lac k of
independence among samples used for clinical
pathology comparisons; for example, dolphins
with or without hemosiderosis may or may not
have had concurrent fatty change and hepatitis.
Although there were no significant associations
among dolphins with or without hemosiderosis,
fatty liver disease, and hepatitis, future studies
should involve a larger population of dolphins,
ideally without concurrent liver pathologies.
In conclusion, this study continues to support
an insulin resistant–like syndrome in dolphins,
supported by a high prevalence of liver-associated
hemosiderosis involving Kupffer cell deposition
and fatty liver disease associated with postpran-
dial hyperglycemia and chronic inflammation.
Future research is needed to better understand
detrimental changes in dolphin metabolism; how
to detect, treat, and prevent these changes; and
the comparative value of the dolphin metabolism
for human diseases, including type 2 diabetes and
insulin resistance.
Acknowledgments: The authors thank Dr. Marc
Montminy from the Salk Institute for Biological
Studies for his valuable input for the discussion;
Ms. Risa Daniels at the National Marine Mammal
Foundation for entering histopathologic data; the
clinical veterinarians, especially Dr. Eric Jensen
and Dr. Cynthia Smith, and veterinary technicians
at the Navy Ma rine Mamm al Prog ram and
National Marine Mammal Foundation for collec-
tion of high quality clinical samples; as well as Ms.
Erika Nilson at SeaWorld San Diego for her
assistance. This study was kindly supported by the
Biosciences Division of the Space and Naval
Warfare Systems Center Pacific.
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Received for publication 1 July 2011
... Bottlenose dolphins (Tursiops truncatus) are long-lived, large-brained mammals that can develop comorbidities of aging similar to humans, including chronic inflammation, elevated cholesterol, elevated insulin, and fatty liver disease [1][2][3]. Obesity and lower physical activity, two key risk factors for these comorbidities in humans, do not appear to be primary drivers for development of these conditions in dolphins [4,5]. While dolphin diets are primarily limited to fish, different wild and managed dolphin populations eat different species of fish on different schedules, which, in turn, vary in nutritional content by species and season [4,5]. ...
... Bottlenose dolphins, like humans, can develop chronic conditions that progress with advancing age [1][2][3][4][5]. We demonstrate in this prospective interventional study with 30 dolphins that a modified fish diet can attenuate comorbidities of aging, namely anemia, hypercholesterolemia and hyperinsulinemia. ...
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Bottlenose dolphins (Tursiops truncatus) are long-lived mammals that can develop chronic aging-associated conditions similar to humans, including metabolic syndrome. Initial studies suggest that these conditions may be attenuated in dolphins using a modified fish diet. Serum metabolomics, fatty acid panels, and blood-based health indices were compared between 20 dolphins on a modified, 50% wild-type diet (50% mullet, 25% capelin, and 25% squid and/or herring) and 10 dolphins on a baseline diet (75% capelin and 25% squid and/or herring). Blood samples were collected at Months 0, 1, 3 and 6. Dolphins on the modified diet had lower insulin (7.5 ± 4.0 and 14.8 ± 14.0 μIU/ml, P = 0.039), lower cholesterol (160 ± 26 and 186 ± 24 mg/dl, P = 0.015) and higher hematocrit (46 ± 3 and 44 ± 3%, P = 0.043) by Month 1 compared to controls. Dolphins with anemia (hemoglobin ≤ 12.5 g/dl, n = 6) or low-normal hemoglobin (12.5–13.5 g/dl, n = 3) before placed on the modified diet had normal hemoglobin concentrations (> 13.5 g/dl) by Month 3. The modified diet caused a significant shift in the metabolome, which included 664 known metabolites. Thirty prioritized metabolites at Months 1 and 3 were 100% predictive of dolphins on the modified diet. Among 25 prioritized lipids, 10 (40%) contained odd-chain saturated fatty acids (OCFAs); C15:0 was the highest-prioritized OCFA. Increased dietary intake of C15:0 (from 1.3 ± 0.4 to 4.5 ± 1.1 g/day) resulted in increased erythrocyte C15:0 concentrations (from 1.5 ± 0.3 to 5.8 ± 0.8 μg/ml, P < 0.0001), which independently predicted raised hemoglobin. Further, increasing age was associated with declining serum C15:0 (R² = 0.14, P = 0.04). While higher circulating OCFAs have been previously associated with lower risks of cardiometabolic diseases in humans, further studies are warranted to assess potential active roles of OCFAs, including C15:0, in attenuating anemia.
... In terms of medical issues, there are several parallels with elephants. Nutrition/ metabolism disorders (e.g., insulin resistance, fatty liver disease, hemochromatosis, and hypocitraturia; Mazzaro et al. 2012;Venn-Watson et al. 2012, 2013Zuckerman and Assimos 2009) are often linked to the captive diet (Rosen and Worthy 2018). Additionally, digestive and gastrointestinal disturbances (e.g., gastritis, ulcerations, and torsion) pose significant and sometimes fatal problems for captive cetaceans (Stoskopf 2015). ...
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The present review assesses the potential neural impact of impoverished, captive environments on large-brained mammals, with a focus on elephants and cetaceans. These species share several characteristics, including being large, wide-ranging, long-lived, cognitively sophisticated, highly social, and large-brained mammals. Although the impact of the captive environment on physical and behavioral health has been well-documented, relatively little attention has been paid to the brain itself. Here, we explore the potential neural consequences of living in captive environments, with a focus on three levels: (1) The effects of environmental impoverishment/enrichment on the brain, emphasizing the negative neural consequences of the captive/impoverished environment; (2) the neural consequences of stress on the brain, with an emphasis on corticolimbic structures; and (3) the neural underpinnings of stereotypies, often observed in captive animals, underscoring dysregulation of the basal ganglia and associated circuitry. To this end, we provide a substantive hypothesis about the negative impact of captivity on the brains of large mammals (e.g., cetaceans and elephants) and how these neural consequences are related to documented evidence for compromised physical and psychological well-being.
... However, this effect of age was not detected in one managed bottlenose dolphin population (Table 5). Hypothesized reasons for the difference in this one population included older age, overnight fasting, larger meal sizes with higher purine loads, deviations from circadian rhythms, genetics, and/or insulin resistance (Venn-Watson et al. 2012;Venn-Watson et al. 2013). ...
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Background: A comprehensive evaluation of the effects of gender, age, and season on blood analytes in a robust population size of ex situ bottlenose dolphins (Tursiops spp.) has not been investigated to date. Aim: To define the variation in hematological and biochemical analytes of dolphins due to gender, age, and season. Methods: 1,426 blood samples collected from 156 clinically normal dolphins consisting of 59 males and 97 females in which 37 analytes were measured were retrospectively identified. The dolphins were categorized by age, gender, and season, and categories were compared. Results: 23 (64%) analytes differed by age. The number of differences between adjacent age groups decreased with advancing age. MPV, glucose, BUN, globulins, GGT and Cl progressively increased with age, whereas Abs lymphs, total protein, ALP, CK and Ca progressively decreased with age. Three (8%) of analytes differed between gender, whereas 16 (44%) analytes differed by season. Female dolphins had higher median iron (33 µmol/L) than male dolphins (25 µmol/L). Female dolphins also had higher Abs lymphs and MCHC, but Abs lymphs and MCHC also differed between age and season, respectively. Gender inconsistently and relatively infrequently influences analytes. Delphinids of advancing age experience immune senescence and decreasing renal perfusion or clearance. Conclusions: These results demonstrate the importance of considering the influences of gender, age, and season on blood data, provide a baseline for accurate interpretation of clinicopathological analytes of delphinids in managed care, and will be useful for investigations into health, disease, and stressors of wild delphinids.
... Interestingly, in these aquatic mammals the glucose homeostasis are similar, in process, to humans. Studies have revealed that bottlenose dolphins (Tursiops truncatus) have sustained postprandial hyperglycemia and hyperinsulinemia, dyslipidemia, and fatty liver disease, which is similar to human diabetes (8)(9)(10). ...
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Insulin and glucagon are hormones secreted by pancreatic β and α cells, respectively, which together regulate glucose homeostasis. Dysregulation of insulin or glucagon can result in loss of blood glucose control, characterized by hyperglycemia or hypoglycemia. To better understand the endocrine physiology of cetaceans, we cloned and characterized the insulin and glucagon genes from pygmy sperm whale (Kogia breviceps). We obtained the complete coding sequences of the preproinsulin and preproglucagon genes, which encodes the preproinsulin protein of 110 amino acid (aa) residues and encodes the preproglucagon protein of 179 aa residues, respectively. Sequence comparison and phylogenetic analyses demonstrate that protein structures were similar to other mammalian orthologs. Immunohistochemistry and immunofluorescence staining using insulin, glucagon, and somatostatin antibodies allowed analysis of pygmy sperm whale islet distribution, architecture, and composition. Our results showed the pygmy sperm whale islet was irregularly shaped and randomly distributed throughout the pancreas. The architecture of α, β, and δ cells of the pygmy sperm whale was similar to that of artiodactyls species. This is the first report about insulin and glucagon genes in cetaceans, which provides new information about the structural conservation of the insulin and glucagon genes. Furthermore, offers novel information on the properties of endocrine cells in cetacean for further studies.
... Lipid extraction of tissues was necessary prior to d 13 C analysis to ensure normalized comparison of SI values among age groups and tissue types, consistent with a previous study (Giménez et al. 2017). Higher lipid content and corresponding C:N in liver of an animal that was diagnosed with severe hepatic lipidosis (a common result of poor nutrition or starvation) demonstrates the potential for elemental analyses to also contribute to the identification of metabolic stress (Center et al. 1993, Choudhury and Sanyal 2004, Venn-Watson et al. 2012). The C:N, therefore, may be useful to corroborate possible metabolic stress in carcasses of any age that are too decomposed for confident histological evaluation alone. ...
... The seal was sent to the Department of Pathology, University of Veterinary Medicine Hannover, Germany, where a full necropsy was performed. 11 The animal weighed 53.7 kg and was considered to be in a poor nutritional condition. Mild to severe acute congestion was found in the meninges, nasal cavity, lung, spleen, liver, and kidneys. ...
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Iron overload has been described in various wild species. The majority of cases involve captive animals, often associated with increased dietary iron uptake. Here a case of idiopathic iron overload in a female adult harbor seal under human care is presented. The animal displayed a progressive anorexia, apathy, and increased serum iron levels. Radiographs showed radiopaque foreign bodies in the stomach. The seal died during an elective laparotomy. Twenty-five coins and two metal rings were removed from the stomach. Histopathologic examination revealed iron storage without cellular damage in liver, spleen, kidney, and pulmonary and mesenteric lymph nodes. Atomic absorption spectrophotometry analysis for iron revealed values thirty times above the reference ranges in spleen and liver; however, the coins only contain minor levels (parts per million) of iron. The etiology of the iron overload in this animal remains unclear. A multifactorial process cannot be excluded.
... Interest has also increased in metabolic conditions that occur in aging bottlenose dolphins that parallel those in humans. Bottlenose dolphins can develop a subclinical metabolic syndrome, with elevated insulin, triglycerides, glucose, and ferritin, accompanied by fatty liver disease (Venn-Watson et al., 2012;Venn-Watson et al., 2011;Venn-Watson et al., 2013). Further, current models, based on data derived from studies in other species, suggest that metabolic perturbations arise from disrupted immune homeostasis in sites such as adipose tissue and liver, with activation status of leukocytes in these sites contributing to the disease pathology (Olefsky and Glass, 2010;Osborn and Olefsky, 2012). ...
The slow progress in understanding immunotoxic effects of environmental contaminants and their influence on disease susceptibility in whales is largely due to the limited information available on the immune systems and immune function of species included in the Cetancodontamorpha clade. Studies in species in the other major clades included in the Artiodactylamorpha, Ruminantiamorpha, Suinamorpha, and Camelidamorpha have revealed the immune systems are similar, but not identical. The present study was undertaken to expand the available monoclonal antibody reagents needed to gain insight into the composition, function, and evolution of the immune system in Cetancodontamorpha, using the dolphin (Tursiops truncates) as a model cetacean species. Screening of a set of mAbs that recognize highly conserved epitopes expressed on the major histocompatibility complex (MHC) and leukocyte differentiation molecules (LDMs) in cattle by flow cytometry revealed some of the mAbs recognize epitopes conserved on dolphin orthologues of MHC class I, MHC class II, CD11a, CD14, CD16, CD18, CD163 and CD172a. Comparison of the amino acid sequences of dolphin and bovine orthologues revealed limited changes in sequence have occurred during speciation, suggesting an approach for developing cross-reactive mAbs for use in cetacean research.
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While it is believed that humans age at different rates, a lack of robust longitudinal human studies using consensus biomarkers meant to capture aging rates has hindered an understanding of the degree to which individuals vary in their rates of aging. Because bottlenose dolphins are long-lived mammals that develop comorbidities of aging similar to humans, we analyzed data from a well-controlled, 25-y longitudinal cohort of 144 US Navy dolphins housed in the same oceanic environment. Our analysis focused on 44 clinically relevant hematologic and clinical chemistry measures recorded during routine blood draws throughout the dolphins’ lifetimes. Using stepwise regression and general linear models that accommodate correlations between measures obtained on individual dolphins, we demonstrate that, in a manner similar to humans, dolphins exhibit independent and linear age-related declines in four of these measures: hemoglobin, alkaline phosphatase, platelets, and lymphocytes. Using linear regressions and analyses of covariance with post hoc Tukey–Kramer tests to compare slopes (i.e., linear age-related rates) of our four aging rate biomarkers among 34 individual dolphins aging from 10 y to up to 40 y old, we could identify slow and accelerated agers and differentiate subgroups that were more or less likely to develop anemia and lymphopenia. This study successfully documents aging rate differences over the lifetime of long-lived individuals in a controlled environment. Our study suggests that nonenvironmental factors influencing aging rate biomarkers, including declining hemoglobin and anemia, may be targeted to delay the effects of aging in a compelling model of human biology.
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Dietary odd-chain saturated fatty acids (OCFAs) are present in trace levels in dairy fat and some fish and plants. Higher circulating concentrations of OCFAs, pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0), are associated with lower risks of cardiometabolic diseases, and higher dietary intake of OCFAs is associated with lower mortality. Population-wide circulating OCFA levels, however, have been declining over recent years. Here, we show C15:0 as an active dietary fatty acid that attenuates inflammation, anemia, dyslipidemia, and fibrosis in vivo, potentially by binding to key metabolic regulators and repairing mitochondrial function. This is the first demonstration of C15:0’s direct role in attenuating multiple comorbidities using relevant physiological mechanisms at established circulating concentrations. Pairing our findings with evidence that (1) C15:0 is not readily made endogenously, (2) lower C15:0 dietary intake and blood concentrations are associated with higher mortality and a poorer physiological state, and (3) C15:0 has demonstrated activities and efficacy that parallel associated health benefits in humans, we propose C15:0 as a potential essential fatty acid. Further studies are needed to evaluate the potential impact of decades of reduced intake of OCFA-containing foods as contributors to C15:0 deficiencies and susceptibilities to chronic disease.
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The aim of this study was to determine if ferritin is a reliable biomarker of iron overload disorder (IOD) progression and hemochromatosis in the Sumatran rhinoceros (Dicerorhinus sumatrensis) by developing a species-specific ferritin assay and testing historically banked samples collected from rhinos that did and did not die of hemochromatosis. Ferritin extracted from Sumatran rhino liver tissue was used to generate antibodies for the Enzyme Immunoassay. Historically banked Sumatran rhino serum samples (n = 298) obtained from six rhinos in US zoos (n = 290); five rhinos at the Sumatran Rhino Conservation Centre in Sungai Dusun, Malaysia (n = 5); and two rhinos in Sabah, Malaysia (n = 3) were analyzed for ferritin concentrations. Across all US zoo samples, serum ferritin concentrations ranged from 348 to 7,071 ng/ml, with individual means ranging from 1,267 (n = 25) to 2,604 ng/ml (n = 36). The ferritin profiles were dynamic, and all rhinos exhibited spikes in ferritin above baseline during the sampling period. The rhino with the highest mean ferritin concentration did not die of hemochromatosis and exhibited only mild hemosiderosis postmortem. A reproductive female exhibited decreases and increases in serum ferritin concurrent with pregnant and nonpregnant states, respectively. Mean (±SD) serum ferritin concentration for Sumatran rhinos in Malaysia was high (4,904 ± 4,828 ng/ml) compared to that for US zoo rhinos (1,835 ± 495 ng/ml). However, those in Sabah had lower ferritin concentrations (1,025 ± 52.7 ng/ml) compared to those in Sungai Dusun (6,456 ± 4,941 ng/ml). In conclusion, Sumatran rhino serum ferritin concentrations are dynamic, and increases often are not associated with illness or hemochromatosis. Neither a specific pattern nor the individual's overall mean ferritin concentration can be used to accurately assess IOD progression or diagnose hemochromatosis in this rhino species.
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We postulate a critical role for the quantity and quality of dietary carbohydrate in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM). Our primate ancestors ate a high-carbohydrate diet and the brain and reproductive tissues evolved a specific requirement for glucose as a source of fuel. But the Ice Ages which dominated the last two million years of human evolution brought a low-carbohydrate, high-protein diet. Certain metabolic adaptations were therefore necessary to accommodate the low glucose intake. Studies in both humans and experimental animals indicate that the adaptive (phenotypic) response to low-carbohydrate intake is insulin resistance. This provides the clue that insulin resistance is the mechanism for coping with a shortage of dietary glucose. We propose that the low-carbohydrate carnivorous diet would have disadvantaged reproduction in insulin-sensitive individuals and positively selected for individuals with insulin resistance. Natural selection would therefore result in a high proportion of people with genetically-determined insulin resistance. Other factors, such as geographic isolation, have contributed to further increases in the prevalence of the genotype in some population groups. Europeans may have a low incidence of diabetes because they were among the first to adopt agriculture and their diet has been high in carbohydrate for 10,000 years. The selection pressure for insulin resistance (i.e., a low-carbohydrate diet) was therefore relaxed much sooner in Caucasians than in other populations. Hence the prevalence of genes producing insulin resistance should be lower in the European population and any other group exposed to high-carbohydrate intake for a sufficiently long period of time.
Context The Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (ATP III) highlights the importance of treating patients with the metabolic syndrome to prevent cardiovascular disease. Limited information is available about the prevalence of the metabolic syndrome in the United States, however.Objective To estimate the prevalence of the metabolic syndrome in the United States as defined by the ATP III report.Design, Setting, and Participants Analysis of data on 8814 men and women aged 20 years or older from the Third National Health and Nutrition Examination Survey (1988-1994), a cross-sectional health survey of a nationally representative sample of the noninstitutionalized civilian US population.Main Outcome Measures Prevalence of the metabolic syndrome as defined by ATP III (≥3 of the following abnormalities): waist circumference greater than 102 cm in men and 88 cm in women; serum triglycerides level of at least 150 mg/dL (1.69 mmol/L); high-density lipoprotein cholesterol level of less than 40 mg/dL (1.04 mmol/L) in men and 50 mg/dL (1.29 mmol/L) in women; blood pressure of at least 130/85 mm Hg; or serum glucose level of at least 110 mg/dL (6.1 mmol/L).Results The unadjusted and age-adjusted prevalences of the metabolic syndrome were 21.8% and 23.7%, respectively. The prevalence increased from 6.7% among participants aged 20 through 29 years to 43.5% and 42.0% for participants aged 60 through 69 years and aged at least 70 years, respectively. Mexican Americans had the highest age-adjusted prevalence of the metabolic syndrome (31.9%). The age-adjusted prevalence was similar for men (24.0%) and women (23.4%). However, among African Americans, women had about a 57% higher prevalence than men did and among Mexican Americans, women had about a 26% higher prevalence than men did. Using 2000 census data, about 47 million US residents have the metabolic syndrome.Conclusions These results from a representative sample of US adults show that the metabolic syndrome is highly prevalent. The large numbers of US residents with the metabolic syndrome may have important implications for the health care sector.
Context: Most cases of primary hypertriglyceridemia (HTG) are caused by the interaction of unknown polygenes and environmental factors. Elevated iron storage is associated with metabolic syndrome, diabetes, and obesity, and all of them are associated with HTG. Objective: The aim of the study was to analyze whether HFE mutations causing hereditary hemochromatosis (HH) are associated with primary HTG. Design: Genetic predisposition to HH was analyzed in a case-control study. Setting: The study was conducted at University Hospital Lipid Clinic. Participants: We studied two groups: 1) the HTG group, composed of 208 patients; and 2) the control group, composed of 215 normolipemic subjects and 161 familial hypercholesterolemia patients. Intervention: Two HFE mutations (C282Y and H63D) were analyzed. Main Outcome Measure: We measured HH genetic predisposition difference between groups. Results: HH genetic predisposition was 5.9 and 4.4 times higher in the HTG group than in the normolipemic (P = 0.02) and FH (P = 0.05) subjects, respectively. There were 35 cases (16.8%) of iron overload in the primary HTG group, 14 (6.5%) and nine (5.6%) in the normolipidemic and FH groups, respectively. A higher HH genetic predisposition and a different prevalence of iron overload in subjects with HH genetic predisposition among groups contributed to this higher prevalence. None of the four cases with the HFE genotype associated with high risk of HH in the control groups presented iron overload; however, in eight of 11 subjects (72.7%) with primary HTG and HH genetic predisposition, the iron overload was present. Conclusion: Mutations in HFE gene, favoring iron overload and causing HH, could play an important role in the development of several phenotypes of primary HTG.
Over the past 3 decades, increasing incidence of hepatic iron storage disease has been documented in captive birds, particularly associated with frugivorous species of the families Paradiseadae (birds of paradise), Ramphastidae (toucans), and Sturnidae (starlings). Hematology and serum biochemical analyses are of little diagnostic value for assessment of iron storage disease in birds. A liver biopsy is needed to confirm the condition antemortem; enlargement of the liver, heart, or spleen is often seen radiographically. Both a genetic predisposition for altered regulation of intestinal iron uptake in sensitive species, as well as dietary factors influencing iron metabolism, are considered important in understanding the development and management of this condition. Physiological mechanisms that may have evolved to compensate for a low bioavailability of dietary iron in situ appear to contribute to iron storage disease in managed feeding programs. Although dietary iron requirements of frugivorous species remain unknown, many practical diets contain higher iron concentrations of greater bioavailability compared with domestic poultry recommendations (50–120 mg/kg) due to specific ingredients and/or nutrient interactions in mixed diets=mthe latter including effects of other minerals, ascorbic acid, polyphenols, and specific sugars. Treatment of the disease includes phlebotomy, targeted iron chelation therapy, and diet alteration. Current recommendations for managing this multifactorial issue include: analysis of dietary iron and vitamin C concentrations, particularly those of iron-sensitive species; formulation of low iron diets; minimization or elimination of animal proteins in diets of frugivorous birds; ascorbic acid levels < 100 mg/kg and/or the feeding of vitamin C-containing foods separately. Further study of possible interactions among dietary sugars, minerals, natural chelators (for example tannins, fiber, phytates) and iron metabolism in avian species is encouraged.
Iron storage disease (hemochromatosis) has been reported in many species of both captive and free-ranging animals. In this study we examined the relationship between this disease and concentrations of iron analytes in aquarium-held northern fur seals (Callorhinus ursinus). Sera were analyzed for iron, total iron-binding capacity (TIBC), ferritin, ceruloplasmin, and haptoglobin concentrations in a retrospective study that included samples taken over a 14-year period. The animals ranged in age from <1 year to an estimated 23 years. Serum ferritin was measured using an enzyme-linked immunosorbent assay (ELISA) for canine sera. The results from this assay are the first reported for any pinniped. Serum iron concentrations in presumed healthy animals ranged from 37 to 196 µg/dl, and TIBC ranged from 136 to 484 µg/dl. The transferrin saturation percentage differed significantly between male (41%) and female (63%) adult fur seals, as did the ferritin levels (54 ng/ml for males vs. 500 ng/ml for females). There was a trend toward increased serum ferritin and percent transferrin saturation with age, especially in females. The data also showed a relationship between serum iron and transferrin saturation among eight mother–pup pairs, which suggests that pups may develop increased iron levels due to placental transfer of iron and/or transfer of iron through the milk from iron-overloaded females. Diet was considered as a factor in the development of hemochromatosis in at least three geriatric female northern fur seals, and their diets were analyzed for iron concentrations. On the basis of these results, the diets were altered by replacing a portion of the high-iron-content fish (herring) with a lower-iron-content item (squid), and discontinuing iron and vitamin C supplementation (via a multivitamin tablet). Sera were analyzed before, and 1 and 4 years after the dietary changes were implemented. Paired t-tests showed no significant changes in the iron analytes from pre- to post-diet-change samples, which indicates that it may be too late to affect iron levels by diet alone in older animals with a chronic history of elevated iron levels. Zoo Biol 23:205–218, 2004. © 2004 Wiley-Liss, Inc.
In the late 1960s, pathologists at the San Diego Zoo began to notice iron storage in the internal organs of captive lemurs. Hemosiderin was found in liver, spleen, lymph nodes, duodenum, and occasionally other organs. This was most pronounced in Lemur macaco, least pronounced in Lemur catta, with the severity in Lemur variegatus variegatus and Lemur variegatus ruber falling somewhere in between. Since 1968, 20 of 29 necropsied lemurs had hemosiderosis, three with hepatomas, three with cholangiomas, and one with a metastatic pheochromocytoma. In a preliminary attempt to compare their iron absorption, five black-and-white ruffed lemur (Lemur variegatus variegatus) weanlings and five rhesus monkey weanlings were each given ∼500 nmol/kg FeCI3 (27.9 μg iron/kg) containing 5 μCi of 59Fe in 0.1 M HCl via a nasogastric tube. Retained 59Fe was measured by whole body gamma counting and found to be roughly the same in the two groups, possibly because insufficient quantities of iron were administered and/or excessive iron absorption becomes apparent only in older animals. Our conclusions were the following: (1) Hemosiderosis in lemurs is associated with tissue damage similar to that seen in humans with idiopathic hemochromatosis. (2) Because iron deposits occur both in parenchymal cells and reticuloendothelial cells, hemosiderosis in lemurs is probably not a model for idiopathic hemochromatosis in a pure sense.