X-Message-Number: 30271
Date: Mon, 7 Jan 2008 11:26:09 -0800 (PST)
From: 
Subject: sodium mediates some toxic effects

[Replacing sodium chloride with choline reduces toxicity. IMHO, it is
rather amazing that sodium chloride is still a standard additive in
2008, despite good evidence of toxicity being demonstrated back in 1998.]

Anim Reprod Sci. 2008 Feb;103(3-4):239-48. Epub 2007 Jan 8.
Improved cryopreservation of bovine preimplantation embryos cultured in
chemically defined medium.
  Lim KT, Jang G, Ko KH, Lee WW, Park HJ, Kim JJ, Kang SK, Lee BC.
Department of Theriogenology and Biotechnology, College of Veterinary
Medicine, Seoul National University, San56-1, Shillim-Dong, Kwanak-Gu, Seoul
151-742, South Korea; Embyro Research Center, Seoul Dairy Corporation,
Gyeonggi 476-851, South Korea.
  The aim of this study was to examine the effects of modifications to a
standard slow freezing protocol on the viability of in vitro produced bovine
embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen,
and presumptive zygotes cultured in defined two-step culture media. The
standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10%
fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A
preliminary trial showed that in vitro produced embryos cryopreserved in
this medium had a survival rate of 74.6% at 24h and 53.5% at 48h post-thaw.
Experiment 1 studied the effects of omitting the sucrose supplement or
replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%,
25% or 50%) or total (100%) replacement of sodium chloride with choline
chloride in the cryopreservation medium were examined (the medium with 100%
replacement was designated CJ1). The effects of replacing the 10% FBS with
0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment
3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer
of in vitro produced embryos cryopreserved in the standard freezing medium
were compared with those of in vitro and in vivo produced embryos
cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of
the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw
survival rates at 24h (71.3% versus 53.5 and 51.7%; P<0.05), 48h (51.1%
versus 45.3 and 40.2%), and 72h (34.0% versus 24.4 and 23.0%) than 0.1M
xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed
that embryos cryopreserved in the standard medium had poorer survival rates
at 24h (72.8% versus 86.5%; P<0.05), 48h (53.1% versus 66.3%) or 72h (28.4%
versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of
embryos frozen in medium supplemented with Albumax I was better than that
for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48h
(87.3% versus 76.9 and 70.9%; P<0.05), and 72h (70.4% versus 49.1 and 46 4%;
P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos
cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher
pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus
14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than
similar embryos frozen in the standard medium. However, in vivo produced
embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates
at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and
calving rate (43.7%; P<0.05). It was concluded that modification of the
freezing medium by addition of lipid-rich BSA and replacing sodium chloride
with choline chloride improves the post-thaw survival of in vitro produced
embryos, and their viability post-transfer.
PMID: 17321080

Cryobiology. 2007 Nov 17 [Epub ahead of print]
Reduction of cryoprotectant toxicity in cells in suspension by use of a
sodium-free vehicle solution.
  Wusteman M, Rauen U, Simmonds J, Hunds N, Pegg DE. Medical Cryobiology
Unit, Biology Department, University of York, YO10 5YW, UKPropane-1,2-diol
(PD) possesses physico-chemical properties that make it a useful biological
vitrifying agent but, although of relatively low toxicity, it still has
substantial damaging effects on cells. This study aimed to identify possible
toxic mechanisms using primary cell cultures from vascular tissue: these
were exposed to the cryoprotectant at room temperature to avoid any
possibility of hypothermic injury. Toxicity was evaluated by measuring the
ability of the cells to divide in culture after exposure to the
cryoprotectant. A variety of interventions, which addressed either possible
consequences of PD exposure, or known mediators of other types of cell
injury, were utilized in an attempt to inhibit PD toxicity. Some comparative
studies with dimethyl sulphoxide (Me(2)SO) exposure were also made.
Replacing sodium in the vehicle solution with choline was the only
intervention that reduced PD toxicity. It did so both in smooth muscle
cells, where the loss of functional capacity was reduced from 56% to 13%,
and in endothelial cells. where the reduction was from 40% to18%. Similar
observations were also made in smooth muscle cells exposed to Me(2)SO. We
failed to find evidence for a role of pH regulation, for oxidative injury
and/or an involvement of redox-active iron as a mediator of the injury. The
results strongly suggest that the influx of sodium into the cell provides
one mechanism whereby both PD and Me(2)SO exert their toxic effects. We
suggest that the use of choline-based vehicle solutions in cryopreservation
would be beneficial.
PMID: 18160065

[snip: "promotion of intracellular acidification likely mediates
cytoprotection in low-Na+ buffer."]

J Hepatol. 2006 May;44(5):894-901. Epub 2005 Sep 15.
Role of pH in protection by low sodium against hypoxic injury in isolated
perfused rat livers.
  Vairetti M, Richelmi P, Bert  F, Currin RT, Lemasters JJ, Imberti R.
Department of Internal Medicine and Therapeutics, University of Pavia,
Pavia, Italy.
  BACKGROUND/AIMS: The purpose of the present study was to characterize the
role of Na+, pH and cellular swelling in the pathogenesis of hypoxic injury
to rat livers. METHODS AND RESULTS: When livers were perfused with hypoxic
Krebs-Henseleit bicarbonate buffer (KHB) containing 143 mM Na+, release of
LDH began after 30 min and was maximal after 60 min. In livers perfused with
choline-substituted low-Na+ KHB (25 mM Na+), LDH release began after 60 min
and peaked after 120 min or longer. Supplementation of KHB with mannitol, a
permeant sugar with antioxidant properties, suppressed LDH release, whereas
sucrose, an impermeant disaccharide, did not afford protection. At the end
of hypoxic perfusions with KHB and low-Na+ KHB, liver weight was not
different, whereas mannitol but not sucrose increased liver weight after
hypoxia. At pH 7.4, monensin, a Na+-H+ ionophore, reversed protection
against hypoxia by low-Na+ KHB (10 mM Na+) but had no effect at pH 6.8. As
measured directly by confocal microscopy of
biscarboxyethylcarboxyfluorescein fluorescence, pH was lower during
perfusion with low-Na+ KHB than KHB. CONCLUSIONS: Cytoprotection by low Na+
was not mediated by prevention of Na+-dependent tissue swelling. Rather,
promotion of intracellular acidification likely mediates cytoprotection in
low-Na+ buffer.
PMID: 16313996

 J Cell Physiol. 2005 Aug;204(2):508-21.
Regulatory and necrotic volume increase in boar spermatozoa.
  Petrunkina AM, Jebe E, T pfer-Petersen E. Institute for Reproductive
Medicine, School of Veterinary Medicine Hannover, Foundation, Hannover,
Germany.
  Spermatozoa of many species initially respond to hypotonicity as perfect
osmometers. Thereafter they undergo a regulatory process resulting in a
decrease in cell volume, similar to that reported for somatic cells.
Regulatory volume increase (RVI), a complementary process which is assumed
to occur following initial shrinkage of sperm volume after exposure to a
hypertonic medium, has not yet been described in detail for spermatozoa. In
this study, we investigated whether spermatozoa are able to regulate their
volume after hypertonic stress and whether this ability is maintained in
preserved sperm. Cell volume changes were recorded using electronic cell
sizing. Sperm response to the ion channels blockers quinidine, tamoxifen,
and dydeoxyforskolin, and to protein kinase/phosphatase inhibitors
lavendustin, staurosporine, and vanadate was studied to investigate possible
mechanisms of RVI. Annexin V staining was used in combination with propidium
iodide to determine whether hypertonic stress may induce apoptosis. Overall
protein tyrosine phosphorylation under hypertonic conditions was measured
via flow cytometry using antiphosphotyrosine antibody. Spermatozoa exposed
to hypertonic stress initially responded with an abundant subpopulation
according to the perfect osmometer model and recovered their volume from
this shrinkage after 20 min. RVI was inhibited by quinidine and tamoxifen,
which indicates the involvement of the important cellular ions sodium and
chloride in this process. Volume regulatory ability was essentially
maintained during storage of liquid semen. However, the response of the
sperm population was heterogeneous. A second population raised, containing
spermatozoa with larger volumes, which demonstrated irregularities in the
volume response with respect to osmotic challenge, ion channel blockers, and
storage. Under hypertonic conditions, both protein kinase inhibitors (PKI)
led to increased isotonic volumes and to elevated initial relative volumes
and subsequent volume decrease. RVI was inhibited by the vanadate.
Hypertonic stress did not result in an increase in early apoptotic cells,
but produced a shift toward late necrotic cells. Substitution of sodium and
chloride by choline and sulfate resulted in decreased isotonic volume of
sperm treated with lavendustin. Tyrosine phosphorylation levels were reduced
after 20 min under hypertonic conditions. It was concluded that RVI is
regulated via a protein tyrosine kinase-dependent pathway, and that
dephosphorylation occurs when volume regulation is required. The necrotic
volume increase (NVI) is associated with the accumulation of sodium and
chloride following uncontrolled opening of the channels. The ability to
regulate volume after exposure to hypertonic conditions is important for
sperm functionality and can have practical applications in spermatological
diagnostics and cryopreservation. (c) 2005 Wiley-Liss, Inc.
PMID: 15744775

Hum Reprod. 2002 Dec;17(12):3149-52.
Birth of two babies using oocytes that were cryopreserved in a choline-based
freezing medium.
  Quintans CJ, Donaldson MJ, Bertolino MV, Pasqualini RS. Halitus Instituto
M dico, Affiliated to the University of Buenos Aires, Marcelo T de Alvear
2084 (C1122AAF), Buenos Aires, Argentina.
  BACKGROUND: Oocyte cryopreservation may have significant potential for
assisted reproductive technology. However, to date, successful results have
been limited. We report a preliminary series of IVF outcomes after
fertilization of oocytes that were frozen in a low-sodium medium. METHODS:
In this retrospective analysis, 12 patients (21-41 years old), who underwent
IVF in a fertility clinic affiliated to the University of Buenos Aires, had
oocytes cryopreserved in a modified phosphate buffered saline medium, in
which sodium chloride was replaced by choline chloride. A slow-freezing,
rapid-thawing protocol was used and oocytes were inseminated by ICSI.
Outcome measures included oocyte survival, fertilization, implantation and
pregnancy rates. RESULTS: Median oocyte survival was 63%. Median
fertilization rate was 59%. Overall implantation rate was 25%. Six clinical
pregnancies were achieved; two of these pregnancies went to term resulting
in the birth of two babies. CONCLUSIONS: To the best of our knowledge, these
are the first pregnancies and normal births using oocytes that were
cryopreserved in a choline-based medium. The small sample size prevents us
from concluding that freezing in a low-sodium medium is superior to using a
conventional one.
PMID: 12456615

Cryobiology. 1998 Dec;37(4):346-54.
Cryopreservation of unfertilized mouse oocytes: the effect of replacing
sodium with choline in the freezing medium.
  Stachecki JJ, Cohen J, Willadsen SM. Institute of Reproductive Medicine
and Science of Saint Barnabas Medical Center, 101 Old Short Hills Road,
Suite 501, West Orange, New Jersey, 07052, USA.
  Although embryo cryopreservation has become commonplace in many species,
effective methods are not available for routine freezing of unfertilized
eggs. Cryopreservation-induced damage may be caused by the high
concentration of sodium ions in conventional freezing media. This study
investigates the effect of a newly developed low-sodium choline-based medium
(CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive
cryopreservation and develop to the blastocyst stage in vitro. Specifically,
the effects of cooling to subzero temperatures, thawing rate, LN2 plunge
temperature, and equilibration with a low-sodium medium prior to freezing
are examined. In contrast to cooling to 23, 0, or -7.0 degreesC in a
sodium-based freezing medium (ETFM), cooling in CJ2 had no significant
negative effect on oocyte survival or development. Oocytes frozen in CJ2
survived plunging into LN2 from -10, -20, or -33 degreesC at significantly
higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M
PrOH, 0.1 M sucrose, -0.3 C/min, plunging at -33 degreesC) rapid thawing by
direct submersion in 30 degreesC water was more detrimental to oocyte
survival than holding in air for 30 or 120 s prior to transfer to water.
Equilibration of unfertilized oocytes with a low-sodium medium prior to
cryopreservation in CJ2 significantly increased survival and blastocyst
development. These results demonstrate that the high concentration of sodium
in conventional freezing media is detrimental to oocyte cryopreservation and
show that choline is a promising replacement. Reducing the sodium content of
the freezing medium to a very low level or eliminating sodium altogether may
allow oocytes and other cells to be frozen more effectively. Copyright 1998
Academic Press.
PMID: 9917351

Biol Reprod. 1998 Aug;59(2):395-400.
Detrimental effects of sodium during mouse oocyte cryopreservation.
Stachecki JJ, Cohen J, Willadsen S.Institute of Reproductive Medicine and
Science of Saint Barnabas Medical Center, West Orange, New Jersey 07052,
USA. james
  Cryopreservation is an established way of storing embryos, but effective
methods are not available for freezing eggs. Most freezing damage is caused
by high solute concentration (solution effects) and intracellular ice.
Sodium salts are the major components of cryopreservation media, and the
main contributor to the solution effects. The present experiments examine
the effect of substituting choline for sodium as the major extracellular
cation in the cryopreservation of mouse eggs. The effects of serum and
various cryoprotectants were also examined. Survival, fertilization, and
development were inversely related to the concentration of sodium in the
freezing medium. Oocytes frozen in a choline-based medium had the highest (p
< 0.001) survival and development rates. The absence of serum during thawing
inhibited fertilization, whereas exposure to serum or opening the zona
allowed fertilization to reach the control level. Dimethyl sulfoxide was as
effective as 1,2 propanediol for obtaining high survival and fertilization
rates. These results support the hypothesis that the high concentration of
sodium in conventional freezing media is detrimental to cells and show that
choline is a promising replacement for sodium. Reducing or eliminating
sodium may allow oocytes and other cells to be frozen more efficiently.
PMID: 9687313

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