Severe hepatotoxicity following ingestion of Herbalife nutritional supplements contaminated with Bacillus subtilis.

Abstract

BACKGROUND/AIMS: Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife preparations was reported but the underlying pathogenesis remained cryptic. METHODS: Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic, metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury were excluded. However, both patients reported long-term consumption of Herbalife products. All Herbalife products were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamination according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by sequencing the 16S rRNA and gyrB genes. RESULTS: Causality between consumption of Herbalife products and disease according to CIOMS was scored "probable" in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibrosis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing showed no drug hypersensitivity. However, samples of Herbalife products ingested by both patients showed growth of Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity. CONCLUSIONS: Two novel incidents of severe hepatic injury following intake of Herbalife products contaminated with Bacillus subtilis emphasize its potential hepatotoxicity.

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Severe hepatotoxicity following ingestion of Herbalife?
nutritional supplements contaminated with Bacillus subtilisq
Felix Stickel1,*, Sara Droz2, Eleonora Patsenker1, Katja Bo ¨gli-Stuber2,
Beat Aebi3, Stephen L. Leib2
1Institute of Clinical Pharmacology, University of Bern, Murtenstrasse 35, 3010 Bern, CH, Switzerland
2Institute for Infectious Diseases, University of Bern, Friedbu ¨hlstrasse 51, CH-3010 Bern, Switzerland
3Institute of Legal Medicine, University of Bern, Bu ¨hlstrasse 20, CH-3012 Bern, Switzerland
See Editorial, pages 13–16
Background/Aims:Nutritional supplements are widely used. Recently, liver injury after consumption of Herbalife?prep-
arations was reported but the underlying pathogenesis remained cryptic.
Methods:Two patients presented with cholestatic hepatitis and pruritus, and cirrhosis, respectively. Viral, alcoholic,
metabolic, autoimmune, neoplastic, vascular liver diseases and synthetic drugs as the precipitating causes of liver injury
were excluded. However, both patients reported long-term consumption of Herbalife?products. All Herbalife?products
were tested for contamination with drugs, pesticides, heavy metals, and softeners, and examined for microbial contamina-
tion according to standard laboratory procedures. Bacteria isolated from the samples were identified as Bacillus subtilis by
sequencing the 16S rRNA and gyrB genes.
Results:Causality between consumption of Herbalife?products and disease according to CIOMS was scored ‘‘proba-
ble” in both cases. Histology showed cholestatic and lobular/portal hepatitis with cirrhosis in one patient, and biliary fibro-
sis with ductopenia in the other. No contamination with chemicals or heavy metals was detected, and immunological testing
showed no drug hypersensitivity. However, samples of Herbalife?products ingested by both patients showed growth of
Bacillus subtilis of which culture supernatants showed dose- and time-dependent hepatotoxicity.
Conclusions:Two novel incidents of severe hepatic injury following intake of Herbalife?products contaminated with
Bacillus subtilis emphasize its potential hepatotoxicity.
? 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Drug-induced hepatitis; Food supplement; Herbal medicine; Herbalife; Weight reduction; Hepatotoxicity
1. Introduction
Widespread use of nutritional supplements contain-
ing vitamins, antioxidants, trace elements, proteins,
amino acids and herbal components has been recorded
both in the US [1] and Europe [2,3]. Consumers turn
to such products for presumed benefits regarding
enhanced physical performance during sports activities,
disease prophylaxis, improvement of nutritional status,
and, particularly, weight reduction [2–4]. Consequently,
dietary weight loss products have become a multi-billion
market [5], and although not recommended for losing
weight by nutrition expert panels [6,7], their long-term
use without medical consultation is frequent [8]. Strong
evidence supporting health advantages from these
weight loss remedies is lacking and the significant costs
0168-8278/$34.00 ? 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.jhep.2008.08.017
Received 19 June 2008; received in revised form 14 August 2008;
accepted 20 August 2008; available online 18 October 2008
Associate Editor: C.P. Day
qThe authors declare that they do not have anything to disclose
regarding funding from industries or conflict of interest with respect to
this manuscript.
*Corresponding author. Tel.: +41 31 632 8715; fax: +41 31 632 4997.
E-mail address: felix.stickel@ikp.unibe.ch (F. Stickel).
www.elsevier.com/locate/jhep
Journal of Hepatology 50 (2009) 111–117

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of these products contrast sharply with their unproven
benefits [9,10]. Even more worrisome are recent reports
about adverse effects, particular liver injury, following
the intake of LipoKinetix [11], preparations containing
ephedrin [12], and green tea extracts [13,14] resulting
in acute and chronic liver injury.
Recently, three case series from Israel, Switzerland
and Spain analyzed incidents of severe liver injury after
the intake of several Herbalife?products [15–17].
Among a total of twenty-six cases of liver damage fol-
lowing Herbalife?intake, two patients developed ful-
minant hepatic failure requiring liver transplantation
after which one patient survived while the second died.
Causality of consumption of Herbalife?products and
disease was considered ‘‘certain” in six patients due to
a positive re-challenge reaction, and ‘‘probable” in 16
further patients applying the CIOMS and WHO score
[18,19]. However, causality scores serve as tentative
diagnostic tools to compensate for lacking specific
markers and are not a substitute for a clarification of
hepatotoxic mechanisms; in relation to Herbalife?this
remains cryptic.
Here, we describe two novel incidents of severe hepa-
tic injury subsequent to intake of Herbalife?products
contaminated with Bacillus subtilis of which bacterial
supernatant revealed a dose-dependent direct hepato-
toxicity in HepG2 cells.
2. Patients
2.1. Patient 1
A 78-year old man was referred to the Institute’s out-patient clinic
by his general practitioner presenting with nausea, painless jaundice,
light stools, dark brown urine lasting 4 weeks, weight loss of 2.5 kg,
and elevated serum levels of aspartate-aminotransferase (AST) of
2100 U/L (normal range 10–41), alanine-aminotransferase (ALT)
2339 U/L (5-41), alkaline phosphatase (AP) 168U/L (36–108), and
gamma-glutamyl-transpeptidase (c-GT) 111U/L (<60). Apart from
moderate pruritus during the last week prior to admission the patient
had no other skin-related symptoms. His medical history revealed arte-
rial hypertension for several years, implantation of a hip endoprosthe-
sis, appendectomy and cholecystectomy for cholecystolithiasis, but was
otherwise uneventful regarding liver illnesses. He reported alcohol con-
sumption between 60 and 80 g/week and denied the use of illicit drugs.
Laboratory tests showed elevated serum bilirubin 611 lmol/L (3–26),
decreased albumin 28 g/L (30–52), and prolonged INR 1.7. His blood
cell count showed normal red and white blood cells without eosino-
philia, and normal platelets. Serology and viral markers were all neg-
ative. Carbohydrate-deficient transferrin (CDT), serum iron, ferritin,
transferrin saturation, ceruloplasmin and alpha-fetoprotein were nor-
mal. However, autoantibodies were elevated with anti-nuclear antibod-
ies (ANA) at 1:1280 and anti-smooth muscle antibodies (SMA) at
1:320, but liver-kidney microsomal antibodies type 1 (LKM-1), anti-
mitochondrial antibodies (AMA), p-ANCA, soluble liver antigen
antibodies (SLA) and immunoglubulin subclasses G, M and A were
normal. Computed tomography (CT) revealed moderate ascites,
splenomegaly, and a cystic lesion located in the pancreatic head with-
out obstructive features on either the pancreatic or the bile duct. Two
follow-up CT scans revealed a stable lesion diagnosed as a cystic ade-
noma. Prescribed medication consisted of irbesartan/hydrochlorothia-
zide (300 mg/12.5 mg) and carvedilol (25 mg) for hypertension,
occasional omeprazole 20 mg for reflux symptoms, and zolpidem
10 mg as a sleeping aid all of which were started more than 12 months
previously. Upon direct questioning the patient admitted to the con-
sumption of a Herbalife?product (Herbalife?F1 Shake Strawberry
and Cappuccino) in the previous 3 years recommended by his daugh-
ter, a Herbalife?sales person. The Herbalife?Shake was stopped
whereas all other drugs were continued and was followed by an imme-
diate improvement of liver enzyme levels and synthetic function with-
out specific treatment. However, 8 weeks later the patient presented
again with elevated serum liver enzyme activities and impaired liver
function with coagulopathy and hypalbuminemia (Fig. 1). Treatment
with corticosteroids at 40 mg daily and ursodeoxycholic acid at
15 mg/kg body weight was initiated resulting in a rapid normalization
of abnormal liver laboratory including coagulation parameters and
albumin. Both drugs were stopped 3 months after normalization of
liver laboratory with no relapse after 10 months of follow-up. At the
second bout of hepatitis the patient underwent transjugular liver
biopsy and histology showed mixed lobular and portal/periportal hep-
atitis, no eosinophilia or plasma cells, marked cholestasis and partial
cirrhotic transformation compatible with toxic liver injury (Fig. 2A
and B). According to CIOMS, causality in this patient is ‘‘probable”
due to temporal relationship, response to dechallenge, exclusion of
other causes, and, although not part of the CIOMS criteria, compati-
ble liver histology.
2.2. Patient 2
A 50-year old, female Herbalife?sales person presented with weak-
ness, painless jaundice and fluctuating lower abdominal pain of 4
weeks’ duration. She reported similar symptoms intermittently for sev-
eral years, but lately, pruritus had evolved the week before presenta-
tion. Her past medical history revealed a cholecystectomy in 1978
for cholelithiasis and vaginal hysterectomy for myoma of the uterus.
She denied any regular medication, alcohol consumption, drug abuse,
and had stopped smoking the previous year. She had moderate jaun-
dice, several spider angiomata on her shoulder, and her liver was ten-
der on palpation. Clinically, there was no ascites and the spleen was
not enlarged. Laboratory investigations revealed ALT levels of
128U/L, AST 153U/L, AP 597U/L, c-GT 810U/L, bilirubin
83 lmol/L, bile acids 51 lmol/L (upper limit of normal [ULN] <20),
normocytic anemia with hemoglobin of 99 g/L, elevated total choles-
terol of 6.77 mmol/L (ULN, <5.00), but normal INR and serum albu-
min. Anti-HAV IgG were positive but other viral markers including
anti-HBc, HBsAg, and anti-HCV were negative, as were ceruloplas-
min, ferritin, and CDT. ANA were found slightly elevated at 1:160,
but AMA-M2, SMA, SLA, p/c-ANCA tested negative and immuno-
globulins G and M were normal. The patient took no prescribed med-
ication, but repeated interrogation revealed that she had consumed
500
1000
1500
ALT/AST (U/L)
2000
INR
Albumin
(g/dL)
1.0
2.0
3.0
4.0
21/06/06 02.05.07
14.05.07
31.05.07
29/06/0702/08/07
17/09/07
09/11/07
Herbalife®(>3yrs)
LB
Fig. 1. Major liver laboratory results of case 1 show a biphasic course.
Along with elevation of transaminases, coagulopathy and hypalbumine-
mia reflecting impaired hepatic synthetic function is evident. Liver biopsy
(LB) was performed during the secound bout of hepatitis. (N, AST; O,
ALT; ?, albumin; w, INR).
112
F. Stickel et al./Journal of Hepatology 50 (2009) 111–117

Page 3

several Herbalife?products including RoseOx tablets, Complexe Mul-
tivitamine ´ tablets, Thermojetics granules, Tang Kuei Plus tablets, Vita-
min C tablets, Personalized Protein Powder Mix Formula 3, and
Herbalifeline omega 3 fatty acid capsules daily for more than one year.
Non-invasive transient elastography determined a liver stiffness of
19.2 kPa indicative of advanced fibrosis which was subsequently con-
firmed by a liver biopsy showing extensive bile duct lesions, periduct-
ular fibrosis, ductopenia, ductular proliferation with granulocytic
ductulitis/periductulitis, and partially complete fibrotic septa compat-
ible with drug-induced biliary injury (Fig. 2C and D). The patient
was strongly advised to stop all supplements and prescribed ursode-
oxycholic acid at 15 mg/kg body weight. After 6 months without con-
sumption of Herbalife?products clinical symptoms resided and a
normalization of AST/ALT levels and marked reduction of cholestasis
markers was recorded (Fig. 3).
3. Methods
3.1. Toxicological evaluation
A test for bulk toxicity was performed by extracting 100 mg of Her-
balife?F1 Shake Strawberry (see case 1) with 100 mL of analytical
grade methanol. This extract was directly injected into a gas chromato-
graph with mass spectrometric and nitrogen–phosphorus specific
detection as described [20]. Also, a part of this extract was evaporated
and derivatized with acetic acid anhydride, in order to protect ther-
mally instable compounds from decomposition with the gas chromato-
graphic inlet. In addition, 1 mL of the methanolic extract was
evaporated and extracted under basic conditions. The extract was
again injected with or without chemical derivatization. Subsequent
mass spectra interpretation was performed using MassLib with update
mass spectrometry libraries [21]. No evidence of bulk contamination or
bulk toxicity was found. Specific and sensitive analyses for traces of
pesticides and toxic metals were performed by the state food control
laboratory in Bern [Kantonales Labor, Bern] using standard mass
spectrometry procedures, but no contaminants were detected.
3.2. Testing for immunoallergic sensitization
Peripheral blood mononuclear cells (PBMC) were prepared over
Ficoll gradient density centrifugation and processed as described
[22]. The powder of Herbalife?– Shape Works Shake Formula 1
(Strawberry) was dissolved in RPMI-1640 medium and used in non-
toxic concentrations (1, 10 and 100 lg/mL) in cell cultures with the
PBMC. Stimulation was evaluated with3H-thymidine incorporation
after 6 days. The product was also dissolved in 5% petrolatum and
applied for epicutaneous patch tests in Finn-chambers for 24 h. Eval-
uation at 48 and 72 h did not register any reaction.
3.3. Testing for microbiological contamination
For microbiological analysis of Herbalife?products all samples
ingested by both patients and two sealed Herbalife?products (Shape
Works Shake Formula 1 Cappuccino) were processed and cultured
using standard laboratory procedures. DNA was extracted from cul-
ture by using the PrepMan?Ultra Sample Preparation Reagent
(Applied Biosystems, Foster City, CA, USA) according to the manu-
facturer’s instruction. Bacillus subtilis was identified by sequencing
the16S rRNA and gyrB genes, and analysis of the cellular fatty acids.
Amplification and sequencing of the 16S rRNA was performed by
using the MicroSeq?500 16S rDNA Bacterial Identification Kit
(Applied Biosystems) according to the manufacturer’s instructions. A
GeneAmp?PCR System 9700 (Applied Biosystems) was used for
amplification and cycle sequencing. Capillary electrophoresis of
sequencing products was performed using an ABI 310 Genetic Ana-
lyzer (Applied Biosystems). Further sequencing analysis was based
on the amplification and sequencing of the gyrB gene of Bacillus
spp. by using a BigDye?Terminator Sequencing Kit v1.1 (Applied
Biosystems) as described [23] using sequencing primers displayed in
Table 1. DNA sequences were assembled and analyzed with SeqMan
and MegAlign computer programs (DNASTAR, Madison, USA).
Comparison of DNA sequences and their corresponding amino acid
sequences with sequences in the GenBank database were performed
with BLAST [24].
Fig. 2. (A) H&E staining of the liver biopsy of case 1 reveals a ductular
reaction with increased cellularity of granulocytes and mononuclear cells,
in part focused on genuine bile ducts. Moreover, collagen is increased
with occurence of porto-portal septa. (B) Immuno-histochemical staining
with cytokeratin 19 highlights the ductular reaction in the portal tract.
(C) H&E staining of case 2 demonstrates a lobular and portal
inflammation with cholestatsis and (D) an increase of collagen in the
portal tracts and beginning septal demarcation.
08/09/06
19/06/07
03/08/07
18/09/07
13/12/07
50
100
150
200
200
400
600
800
AP/γ γ-GT (U/L)
ALT/AST (U/L)
Herbalife®(>12 mo)
LBUDCA
Fig. 3. Course of standard liver laboratory values of case 2. Cholestatic
hepatitis was present several months prior to recognition of Herbalife?as
the precipitating cause. Liver biopsy (LB) was performed at the peak of
laboratory abnormalities, followed by the administration of ursodeoxy-
cholic acid (UDCA) 6 weeks later. (N, AST; O, ALT; ?, alkaline
phosphatase [AP]; w, c-GT).
Table 1
Primers used for sequencing of the gyrB gene of Bacillus spp.
SequencePrimer
gyrB_Bsub_SF_2
gyrB_Bsub_SF_3
gyrB_Bsub_SR_2
gyrB_Bsub_SR_3
gyrB_Bsub_SR_4
50-TGA AGA GCC GAT TTA CAT TGA AGG-30
50-AAA GGT TTA ATG GCG GCA AGA G-30
50-GTC TGT CGC GTC CTT GTT T-30
50-CGC TTA GGT TTG GAT CAT TTT CTT-30
50-GCA GAT CGT AAT CAT ACT CGG TT-30
F. Stickel et al./Journal of Hepatology 50 (2009) 111–117
113

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3.4. Cell culture experiments
Cellular toxicity of bacterial supernatants was assayed using
HepG2 cells (ATCC, Rockville, MD, USA) grown in DMEM supple-
mented with 10% fetal calf serum (FCS), 200 IU/mL penicillin, 200 lg/
mL streptomycin (all from Biochrom, Berlin, Germany). To test for
direct cytotoxicity of bacterial supernatants, lactate dehydrogenase
(LDH) leakage was measured in the supernatants with an autoanalyzer
(Olympus Autoanalyzer AU 2700, Kobe, Japan). Total LDH activities
were determined by sonicating a parallel cell monolayer and LDH
leakage was expressed as the percentage of LDH in the medium rela-
tive to the total LDH content. The release of LDH into the medium
from cells reflects cytolysis.
4. Results
Toxicology screening of the Herbalife?F1 Shake
revealed no relevant contamination with pesticides,
heavy metals, antibiotics, alkyl phosphates, and soften-
ers which were either not detected or below the thresh-
olds that are considered safe (data not shown).
Immunoallergic activation towards the used Herba-
life?products was not detectable neither by skin hyper-
sensitivity testing nor
stimulation indicative of drug-induced hypersensitivity.
Herbalife?F1 Shake and Personalized Protein Pow-
der Mix Formula 3 were subjected to a standard micro-
biology screening. Four samples of Herbalife?products,
namely two of seven ingested by the female patient and
the only sample ingested by the male patient as well as
one sample of a sealed batch of Shape Works Shake
Formula 1 Cappuccino showed growth of Gram posi-
tive rods after 48 h of incubation. Bacteria from three
out of four were subsequently identified by sequencing
the 16S rRNA gene as Bacillus spp. (one product sample
ingested by the female patient also harboured Paeniba-
cillus spp.). Bacillus spp. was analyzed to the species
by assayinglymphocyte
level by perfoming gyrB gene sequencing and identified
as Bacillus subtilis (Table 2).
Bacterial supernatants were collected and used in
incremental dilutions for cell toxicity assays. As shown
in Fig. 4, bacterial supernatants from cultures of B. sub-
tilis caused a dose-dependent increase of LDH leakage
from HepG2 cells into the culture media.
5. Discussion
The consumption of remedies containing certain
‘‘active” nutrients, herbals and combinations of tradi-
tional medicines is rising steadily, partly due to easy
access to commercial products [25]. Concerns over their
safety were raised after incidents of adverse hepatic reac-
tions had been recorded in Europe and the US [11–
14,26]. Recently, severe liver injury associated with the
consumption of Herbalife?products was described
ranging from reversible cholestatic hepatitis to acute
Table 2
Herbalife?products tested for bacterial contamination.
ProductBacterial Culture
gyrB gene sequencing
Patient 1
Herbalife – Shape Works
Shake Formula 1 (Strawberry)
Herbalife – Shape Works
Shake Formula 1 (Cappuccino)
Herbalife – Shape Works
Shake Formula 1 (Cappucino)
Bacillus subtilisBacillus subtilis
Brevibacillus parabrevis
Not assayed
No growth–
Patient 2
Herbalife – Personalized
Bacillus subtilis
Paenibacillus polymyxa
Bacillus subtilis
Paenibacillus polymyxa
Protein Powder Formula 3
Vitamin C tablets
Bacillus subtilis
Bacillus cereus
No growth
No growth
No growth
No growth
No growth
Bacillus subtilis
Not assayed
Tang Kuei Plus tablets
RoseOx tablets
Multivitamin tablets
Thermojetics granules
Herbalifeline omega 3 fatty acids capsules
0
50
100
150
200
250
300
350
400
450
0%
B. subt. 1:10
B. subt. 1:5B. subt. 1:2 B. subt. 1:1
LDH leakage
(U/L)
Fig. 4. LDH leakage from HepG2 after incubation of 24 h with serial
dilution of bacterial supernatant from Bacillus subtilis cultures.
114
F. Stickel et al./Journal of Hepatology 50 (2009) 111–117

Page 5

liver failure requiring liver transplantation, and death
from post-transplant complications [15–17]. The details
provided in the Israeli, Swiss and Spanish series leave lit-
tle doubt over consumption of (a) Herbalife?product(s)
as the precipitating cause of liver damage in at least
some of the patients, but it remains entirely speculative
what mechanism might have precipitated the reported
incidents. As in the case of patient 2 of our report, most
previously documented cases occurred in individuals
who consumed several Herbalife?
makes it difficult to identify the toxic product or,
let alone, substance. In this study we were able to
retrieve all Herbalife?preparations that patients had
consumed for a detailed scrutiny for toxic, immunoaller-
gic, and infectious etiologies. No toxins could be found
in any of the 10 Herbalife?products tested, and no sen-
sitization towards the ingested preparations. However,
four batches of Herbalife?products revealed bacterial
contamination with Gram positive rods identified as B.
subtilis of which the bacterial supernatant caused dose-
dependent increase of LDH leakage in HepG2 cells.
Causality of Herbalife?products as the precipitating
factor of liver damage was assessed according to
CIOMS and scored ‘‘probable” in both cases due to
exclusion of other causes and immediate resolution of
liver damage after dechallenge [18].
Adulteration of nutritional and herbal supplements
with bacterial pathogens of which some may produce
hepatotoxins has been described [27,28]. In fact, micro-
bial contamination can be quite extensive as demon-
strated by a recent FDA-initiated investigation of
commercial ginseng supplements in the US detecting a
high concentration of yeasts and fungi including Asper-
gillus flavus well above the microbial limits established
by the US Pharmacopeia [29]. These investigators also
detected Bacillus spp. forming highly processing-resis-
tant endospores which cannot be easily eliminated by
standard decontamination. In our report, detailed char-
acterization of bacterial cultures revealed growth of
Bacillus subtilis. It is unknown whether Bacillus subtilis
colonies can be routinely detected in sealed Herbalife?
products, or rather reflect contamination during usage.
However, one unopened batch of Herbalife?F1 Shake
also revealed growth of B. subtilis which raises concerns
over improper production, handling, packaging, and/or
storage. B. subtilis is not generally considered a strong
human pathogen [30], but B. subtilis-related food poi-
sonings and an isolated case of cholangitis in a patient
receiving immunosuppression after kidney transplanta-
tion have been described [31]. Better known to poten-
tially precipitate fatal human disease is B. anthracis
causing anthrax and B. cereus as the cause of two types
of food poisoning, the emetic and diarrheal syndromes.
Mahler et al. described two dramatic cases of B. cereus-
related food poisoning with fulminant liver failure after
the ingestion of reheated pasta sauce contaminated with
products which
B. cereus [32]. Indeed, B. cereus was also detected in a
single batch of Herbalife Vitamin C tablets ingested by
patient 2 who presented with chronic liver injury unlike
the two patients reported by Mahler et al. [32] who
developed fulminant liver failure, and only B. subtilis
was detected in Herbalife?batches from both patients.
So, we considered B. cereus as the less likely cause of
liver injury. Nevertheless, the documentation of two
pathogenic species in the tested Herbalife?products fur-
ther underscores the need for their bacteriologic testing.
Lack of standardization, quality and safety short-
comings of herbal and nutritional supplements are
major concerns of advocates of a tighter regulation of
these products since several reports depicting contami-
nation with heavy/toxic metals, pesticides, and drugs
including antibiotics, non-steroidal anti-inflammatory
drugs and anabolic steroids [28,33,34]. However, screen-
ing of Herbalife?products ingested by our two patients
detected no chemical contamination.
Both patients in this report had remarkably advanced
liver injury with incomplete cirrhosis in the male patient,
and ductopenia and partially complete fibrotic septa in
the female patient. The prevailing histological pattern
of liver injury in the previous case series on Herba-
life?-related liver injury was acute and chronic cytolytic
or cholestatic hepatitis, but established liver cirrhosis
was also noted in one patient [15,16]. So, advanced
fibrosis could relate to long-term intake of Herbalife?
products, possibly by a yet unknown profibrotic compo-
nent, and acute decompensation may be related to
contamination with B. subtilis. The natural course of
drug-induced liver injury was assessed in previous retro-
spective studies which demonstrated that patients are
more prone to chronicity despite withdrawal of the pre-
cipitating agent if fibrosis and/or cholestatic injury was
present on histology than patients who present with
acute cytolytic liver injury [35–37].
Elevated autoantibodies were found in two patients of
the Israeli series, in none from Switzerland, and were
unreported in the Spanish series. It can be argued that
autoantibodies in case 1 indicate autoimmune hepatitis
(AIH), particularly, since the patient rapidly responded
to steroid treatment. Unlike with genuine AIH, in this
case steroid treatment could be ceased after 3 months
without adding another immunosuppressant, and the
patient has remained in remission without specific treat-
ment since. Also, a favourable response to steroids is
compatiblewithAIH,however,notdiagnostic.Toscreen
for the possibility of underlying AIH as the underlying
cause of hepatitis, the patient was subjected to a diagnos-
tic score recently developed and validated by the Interna-
tional Autoimmune Hepatitis Group which assigns 1 or 2
points for positive ANA/SMA, elevated IgG levels, com-
patible or typical liver histology, and the exclusion of
viral hepatitis, respectively [38]. It was proposed that
the diagnosis of AIH is probable at >6 points, and
F. Stickel et al./Journal of Hepatology 50 (2009) 111–117
115

Page 6

definite at >7 points. Applying these criteria, case 1
scored 5 points which does not rule out AIH. However,
absence of certain characteristic features suggesting
AIH such as elevated immunoglobulins type G or plasma
cellular infiltrates and interface lesions on histology,
patient‘s advanced age and male gender, and the pattern
of therapeutic response weaken the suspicion of typical
AIH. In our view, a more likely explanation for the
patient‘s presentation is drug-induced autoimmunity,
particularly, since elevated autoantibody titres are fre-
quently observed both along with synthetic [39] and her-
baldrugs[40,41].Conceptually,metabolitesderivedfrom
the metabolism of xenobiotics may bind to cellular pro-
teins or macromolecules, leading either to a direct toxic
effect on hepatocytes or the formation of protein adducts
recognized by the immune system as neoantigens. Lym-
phocyteactivationmaythenstimulateautoantibodypro-
duction and cell-mediated immune responses [42].
We found no evidence for immunoallergic sensitiza-
tion as both lymphocyte stimulation test and epicutane-
ous patch test resulted negative. The lymphocyte
transformation test is able to detect drug specific T cells
with a high sensitivity and specificity in allergic reactions
to drugs involving the skin and the liver, especially in
recently introduced drugs [22,43]. However, in cases in
which immunoallergic reactions are restricted to the
liver, its sensititvity is low even with optimized assays
[44]. So, a negative test cannot rule out involvement of
a particular drug, while a positive test is reliable prove
of a sensitization as false-positive results are rare.
These two novel cases on Herbalife?-related hepatic
damage add to the growing body of scientific evidence
of nutritional supplements as a rare, but worrisome
cause of severe adverse hepatic reactions considering
the widespread use of ‘‘neutraceuticals” by individuals
practising self-medication. So, similar incidents may be
observed as the awareness of physicians and consumers
towards their potential hazards increases.
Until recently, nutritional supplements and func-
tional food preparations were exempt from strict licens-
ing regulations but, in our view, these well-documented
incidents of adverse hepatic reactions call for caution
and safety actions from health authorities. In this
regard, the European Union has set forth legislative
measures relevant to the distribution of nutritional sup-
plements and functional foods that are outlined in the
European Commission 2000 White Paper on Food
Safety. Among others, it foresaw the establishment of
a General Food Law Regulation, laying down the prin-
ciples of food law and the creation of an independent
Food Authority endowed with the task of giving scien-
tific advice on issues based upon risk assessment, man-
agementandcommunication
acknowledges the fact that botanical and nutritional
supplements harbour specific problems because of their
complex composition particularly with respect to quality
[45,46].Legislation
aspects. Also, guidelines for conducting in vitro and
in vivo studies and their relevance for clinical safety data
are defined. Apparently, these initiatives are necessary
since efficacy and safety of dietary supplements and her-
bals is poorly documented, and the awareness of con-
sumers and health professionals towards nutritional
supplements as a potential source of health damage is
low [4,47].
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