CHEMISTRY >
25
Cholesterol &
triglycerides
Ultrasensitive enzymatic cholesterol
and triglyceride profiles of density
gradient lipoprotein fractions
8th International Symposium on Atherosclerosis,
Roma, International Atherosclerosis Society (eds), CIC
Edizioni Internazionali, (Roma). (1988).
John D. Belcher and Jack O. Egan.
University of Minnesota, Minneapolis, Minnesota USA.
Summary. Microassays were developed
for the measurement of total cholesterol
(TC) and triglyceride (TG) by enzy-
matic methods. The assays are linear
between 1 and 100 mg/dL with a sensi-
tivity of 1 mg/dL for both TC and TG.
The assay uses 30 µL of sample or stan-
dard which is added to each well fol-
lowed by 150 µL of either TC or TG
enzymatic reagent. The microplates
were incubated at room temperature for
15 minutes and read in a VMax micro-
plate reader at 490 nm with a 650 nm
reference. The mean accuracy of the
assay was -0.5% and -0.7% for TC and
TG, respectively, and the within- and
between-assay coefficients of variation
were below 3%.
Citrate & glucose
consumption
Mechanism of citrate metabolism in
Lactococcus lactis: resistance against
lactate toxicity at low pH
J. Bacteriol. 181: 145157 (1999).
Christian Magni
1,2
, Diego de Mendoza
2
, Wil N.
Konings
1
, and Juke S. Lolkema
1
.
1
Department of Microbiology, Groningen Biotech-
nology and Biomolecular Sciences Institute, Univer-
sity of Groningen, 9751 NN Haren, The
Netherlands.
2
Programa Multidisciplinario de Biología Experimen-
tal (PROMUBIE-CONICET) and Departamento de
Microbiología, Facultad de Ciencias Bioquímicas y
Farmaceuticas, Universidad Nacional de Rosario,
2000 Rosario, Argentina.
Methods: measurement of citrate and
glucose consumption rates. Cells were
harvested and resuspended to an OD
660
of 6 in 50 mM potassium phosphate
buffer (pH 5.5). Citrate utilization was
initiated by the addition of 500 µL of
cells to 1,500 µL of buffer containing
2 mM citrate. When indicated, glucose
and lactate were included at concentra-
tions of 0.5 and 2 mM, respectively.
Glucose utilization was initiated by
addition of 50 µL of cells to 1,950 µL of
buffer containing 0.5 mM glucose and
2 mM citrate when indicated. At the
indicated times, 250 µL of the cell sus-
pension was centrifuged for 15 seconds
in an Eppendorf tabletop centrifuge
operating at maximal speed, and a sam-
ple of the supernatant was stored in liq-
uid nitrogen until analysis. The
concentrations of citrate, pyruvate, and
glucose in the supernatants were mea-
sured by commercially available enzyme
kits for citrate and D-glucose (Boe-
hringer). The protocol for the measure-
ment of citrate was modified slightly to
allow the measurement of pyruvate (and
oxaloacetate) at the same time, as
described before. Both protocols were
modified for use in 96-well plates as fol-
lows. For the citrate assay, 100 µL of the
chemical measurements: absorbance