X-Message-Number: 27974
Date: Mon, 22 May 2006 19:57:47 -0700 (PDT)
From: Doug Skrecky <>
Subject: dietary antioxidants retard cold storage injury

[There is accumulating evidence that standard transplantation
solutions are far from optimized. Indeed, it is somewhat pathetic
that even a minor green tea addition could exert such significant
benefits. I have a sneaking suspicion that an Iron Chef with a little
table sugar, some spices, and some green tea could mix together, in the
kitchen, a transplantation solution which could put expensive
patented solutions to shame.]

Transplantation. 2006 Jan 27;81(2):231-9.
Improved cold preservation of kidney tubular cells by means of
adding bioflavonoids to organ preservation solutions.
  BACKGROUND: Cold ischemia and reperfusion during renal
transplantation result in release of reactive oxygen species. The
aim of this study is to examine whether cold storage induced cell
injury can be ameliorated by adding flavonoids directly to
preservation solutions. METHODS: Cultured renal tubular epithelial
cells (LLC-PK1) were stored in University of Wisconsin (UW) or
Euro-Collins (EC) solution at 4 degrees C for 20 hours.
Preservation solutions were supplemented with various flavonoids.
After rewarming, structural and metabolic cell integrity was
measured by lactate dehydrogenase (LDH) release and MTT-test, and
lipid peroxidation was assessed from generation of thiobarbituric
acid-reactive substances (TBARS). RESULTS: Twenty hours of cold
storage resulted in a substantial loss of cell viability in both
preservation solutions (in EC: LDH release 92.4+/-2.7%; MTT-test
0.5+/-0.7%). Addition of luteolin, quercetin, kempferol, fisetin,
myricetin, morin, catechin, and silibinin significantly reduced
cell injury (for luteolin in EC: LDH release 2.4+/-1.6%; MTT-test
110.3+/-10.4%, P<0.01; TBARS-production (related to cold stored
control cells) 8.9+/-2.6%). No cytoprotection was found for
apigenin, naringenin, and rutin. Protective potency of flavonoids
depends on number of hydroxyl-substituents and lipophilicity of
the diphenylpyran compounds. CONCLUSION: Cold storage induced
injury of renal tubular cells was substantially ameliorated by
adding selected flavonoids directly to preservation solutions.

Transplantation. 2005 Dec 15;80(11):1556-9.
Beneficial effects of the bioflavonoids curcumin and quercetin on
early function in cadaveric renal transplantation: a randomized
placebo controlled trial.
  BACKGROUND: The bioflavonoids quercetin and curcumin are
renoprotective natural antioxidants. We wished to examine their
effects on early graft function (EF). METHODS: Between September
2002 and August 2004, 43 dialysis dependent cadaveric kidney
recipients were enrolled into a study using Oxy-Q which contains
480 mg of curcumin and 20 mg of quercetin, started after surgery
and taken for 1 month. They were randomized into three groups:
control (placebo), low dose (one capsule, one placebo) and high
dose (two capsules). Delayed graft function (DGF) was defined as
first week dialysis need and slow function (SGF) as Cr >2.5 mg/dl
by day 10. Category variables were compared by chi squared and
continuous variables by Kruskal-Wallis. RESULTS: There were four
withdrawals: one by patient choice and three for urine leak. The
control group had 2/14 patients with DGF vs. none in either
treatment group. Incidence of EF was control 43%, low dose 71%
and high dose 93% (P=0.013). Serum creatinine was significantly
lower at 2 days (control 7.6+/-2.1, low 5.4+/-0.6, high
3.96+/-.35 P=0.0001) and 30 days (control 1.82+/-.16,
low 1.65+/-.09, high 1.33 +/-.1, P=0.03). Acute rejection
incidence within 6 months was control 14.3%, low dose 14.3% and
high dose 0%. Tremor was detected in 13% of high dose patients
vs. 46% of others. Urinary HO-1 was higher in bioflavonoid
groups. CONCLUSION: Bioflavonoid therapy improved early graft
function. Acute rejection and neurotoxicity were lowest in the
high dose group. These bioflavonoids improve early outcomes in
cadaveric renal transplantation, possibly through HO-1 induction.

Gastroenterology. 2005 Aug;129(2):652-64.
Free radical-dependent dysfunction of small-for-size rat liver
grafts: prevention by plant polyphenols.
  BACKGROUND & AIMS: The mechanisms by which small-for-size liver
grafts decrease survival remain unclear. This study investigated
the role of free radicals in injury to small-for-size grafts.
METHODS: Rat liver explants were reduced in size ex vivo and
transplanted into recipients of the same or greater body weight,
resulting in a graft weight and standard liver weight of
approximately 50% and 25%, respectively. A polyphenol extract
from Camellia sinenesis (20 microg/mL) or an equivalent
concentration of epicatechin was added to the storage solution
and the lactated Ringer poststorage rinse solution. RESULTS: Serum
alanine aminotransferase release increased from approximately
60 U/L before implantation to 750, 1410, and 2520 U/L after
full-size, half-size, and quarter-size transplantation,
respectively. Total bilirubin increased slightly after
transplantation of full-size and half-size grafts but increased
104-fold in quarter-size grafts. In quarter-size grafts,
histological changes included necrosis, leukocyte infiltration,
and eosinophilic inclusion body formation. Polyphenol treatment
ameliorated these effects by > or =67%. Survival was 30% after
transplantation of small-for-size grafts. After polyphenol
treatment, survival increased to 70%. Free radicals in bile
assessed by spin trapping and 4-hydroxynonenal adducts measured
by immunohistochemistry were also greater in reduced-size grafts,
an effect ameliorated by polyphenols. Epicatechin, a major
polyphenol from Camellia sinenesis, also improved graft function
and decreased enzyme release, histopathologic changes, and free
radical formation. CONCLUSIONS: Increased formation of free
radicals occurs after transplantation of reduced-size livers,
which contributes to graft dysfunction and failure. Plant
polyphenols decrease liver graft injury and increase survival
of small-for-size liver grafts, most likely by scavenging free

Biotechnol Lett. 2005 May;27(9):655-60.
Protection of osteoblastic cells from freeze/thaw cycle-induced
oxidative stress by green tea polyphenol.
  Green tea polyphenol (GTP) together with dimethylsulphoxide
(DMSO) were added to a freezing solution of osteoblastic cells
(rat calvarial osteoblasts and human osteosarcoma cells) exposed
to repeated freeze/thaw cycles (FTC) to induce oxidative stress.
When cells were subjected to 3 FTCs, freezing medium containing
10% (v/v) DMSO and 500 mug GTP ml(-1) significantly (p<0.05)
suppressed cell detachment and growth inhibition by over 63% and
protected cell morphology. Furthermore, the alkaline phosphatase
activity of osteoblastic cells was appreciably maintained after 2
and 3 FTCs in this mixture. Polyphenols may thus be of use as a
cell cryopreservant and be advantageous in such fields as cell
transplantation and tissue engineering.

Free Radic Biol Med. 2004 May 15;36(10):1248-58.
Polyphenols from Camellia sinenesis prevent primary graft failure
after transplantation of ethanol-induced fatty livers from rats.
  Fatty liver caused by ethanol decreases survival after liver
transplantation in rats. This study investigated if antioxidant
polyphenols from Camellia sinenesis (green tea) prevent failure
of fatty grafts from ethanol-treated rats. Donor rats were given
ethanol intragastrically (6 g/kg). After 20 h, livers were
explanted and stored in University of Wisconsin solution for 24 h.
Prior to implantation, the explanted grafts were rinsed with
lactated Ringer's solution containing 0 to 60 microg/ml
polyphenols. Alanine aminotransferase (ALT) release after liver
transplantation was 4.5-fold higher in recipients receiving
ethanol-induced fatty grafts than in those receiving normal
grafts. Liver grafts from ethanol-treated donors also developed
severe focal necrosis. Graft survival was 11% in the ethanol
group versus 88% for normal grafts. Polyphenol treatment at
60 microg/ml blunted ALT release by 66%, decreased necrotic areas
by 84%, and increased survival to 75%. Ethanol increased
alpha-(4-pyridyl-1-oxide)-N-tert.-butylnitrone free radical
adducts in bile by 2.5-fold, as measured by electron spin
resonance spectroscopy, and caused accumulation of
4-hydroxynonenal in liver sections, effects blunted by
polyphenols. Epicatechin gallate, a major polyphenol from C.
sinenesis, also decreased enzyme release, minimized pathological
changes, and decreased free radical adduct formation. In
conclusion, polyphenols scavenged free radicals in
ethanol-induced fatty livers and decreased injury after liver

Exp Neurol. 2003 Dec;184(2):688-96.
Successful storage of peripheral nerve before transplantation
using green tea polyphenol: an experimental study in rats.
  Green tea polyphenol is known to act as a buffer, reducing
biological responses to oxidative stress. Several effects of
polyphenol have been reported, such as protection of tissue from
ischemia, antineoplasmic and anti-inflammatory effects, and
suppression of arteriosclerosis. In this study, we investigated
whether peripheral nerve segments could be kept viable in a
polyphenol solution for 1 month. Sciatic nerve segments, 20 mm
long, were harvested from Lewis rats and treated in three
different ways before transplanting to recipient Lewis rats to
bridge sciatic nerve gaps created by removal of 15-mm-long nerve
segments. Group F: nerve segments were transplanted immediately
after harvesting. Group P: nerve segments were transplanted after
they had been stored in Dulbecco's Modified Eagle's Medium (DMEM)
containing polyphenol for 7 days at 4 degrees C and then in DMEM
for 21 days at 4 degrees C. Group M: nerve segments were stored
in DMEM solution alone for 28 days at 4 degrees C. Viability of
the nerve segments was assessed by vital staining
(calcein-AM/ethidium homodimer), by electron microscopy and by
genomic studies before transplantation. Nerve regeneration was
evaluated using electrophysiological and morphological studies 12
and 24 weeks after transplantation. Neural cell viability of the
preserved nerve segments was confirmed in group P, in which the
nerve regeneration was similar to that in group F and superior to
that in group M. Peripheral nerve segments can be successfully
preserved for 1 month using green tea polyphenol.

Kidney Int. 2003 Feb;63(2):554-63.
Bioflavonoids attenuate renal proximal tubular cell injury during
cold preservation in Euro-Collins and University of Wisconsin
  BACKGROUND: Cold ischemia and reperfusion during kidney
transplantation are associated with release of free oxygen
radicals and damage of renal tubular cells. Bioflavonoids may
diminish cold storage-induced injury due to antioxidant and
iron chelating activities. This study was designed to delineate
the renoprotective mechanisms of bioflavonoids and to define the
structural features conferring cytoprotection from cold injury.
METHODS: LLC-PK1 cells were preincubated for three hours with
bioflavonoids and cold stored in University of Wisconsin (UW)- or
Euro-Collins (EC)-solution for 20 hours. After rewarming, cell
viability was assessed by the lactate dehydrogenase (LDH) release,
MTT-test, and amino acid transport activity. Lipid peroxidation
was assessed from the generation of thiobarbituric acid-reactive
substances. RESULTS: Twenty-hours of cold storage of LLC-PK1 cells
resulted in a substantial loss of cell integrity that was more
pronounced in the EC (LDH release, 93.6 +/- 1.6%) than the
UW solution (67.2 +/- 6.9%; P < 0.0001). Pretreatment with
quercetin significantly enhanced cell survival (LDH release,
5.4 +/- 2.7% for UW and 8.4 +/- 4.2% for EC) in a concentration
dependent manner. Structure-activity studies revealed similar
renoprotection for kaempferol, luteolin and fisetin, unlike
myricetin, morin, apigenin, naringenin, catechin, silibinin and
rutin. Lipid peroxidation was reduced (UW alone, 2.7 +/- 1.2 vs.
UW+quercetin 0.5 +/- 0.2 nmol/mg protein, P < 0.01), and
l-threonine uptake completely sustained by pretreatment with
quercetin, kaempferol, luteolin, and fisetin. However,
renoprotection by fisetin was rapidly lost during rewarming.
Protective properties of bioflavonoids were governed by the
number and arrangement of hydroxyl substitutes,
electron-delocalization, sterical planarity, and lipophilicity
of the basic diphenylpyran skeleton. CONCLUSION: Cold
storage-induced renal tubular cell injury is ameliorated by
bioflavonoids. Renoprotective effects of bioflavonoids are defined
by structure, suggesting that flavonoids are incorporated into
membrane lipid bilayers and interfere with membrane lipid

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