CLIN. CHEM. 37/5, 739-742 (1991)
Improved Preparation of Hepatic Microsomes for in Vitro Diagnosis of Inherited
Disorders of the Glucose-6-phosphatase System
Michael W. H. Coughtrie,1
James
N. R. Blair,2
Robert
Disruption of microsomal membranes after freezing liver
samples can undermine the reliabilityof in vitro enzymatic
diagnosis of the type 1 glycogen storage diseases. However, freezing of biopsy material is necessary if biopsy
samples are to be safely transported to the place of
assay. We have therefore examined several different
methods (each of which could easily be carried out in
routine hospital laboratories) of preparing and freezing
liver tissue before analysis for glucose-6-phosphatase
(EC 3.1.3.9) enzyme activity, and determination of microsomal intactness. Our study showed that homogenizing fresh liver, and centrifuging the homogenate at 10 000
x g for 10 mm at 4 #{176}C,
followed by freezing the resulting
supernatant material at -80 #{176}C,
provided the optimum
source of material for subsequent preparation of mlcrosomes for analysis of glucose-6-phosphatase activity.
We also demonstrated that 1-naphthol UDPglucuronosyltransferase (EC 2.4.1.17) activity could be used to assess
microsomal intactness in cases of type 1 a glycogen
storage disease, where mannose-6 phosphatase activity
cannot be used.
AddItional Keyphrases:
handling
.
glycogen storage disease
.
sample
enzyme activity
The in vitro diagnosis of rare inherited diseases usually necessitates
transport
of tissue from the site of
biopsy to a specialized center for analysis. The tissue can
be transported
either fresh (i.e., in wet ice, without
previous freezing) or frozen; if the transport times are
short (i.e., <1 h after biopsy) it is desirable to use fresh
tissue for enzymatic analysis. Such is not often the case,
however, so that usually the tissue has to be frozen to
allow transport to the site of analysis-even
though this
process
itself may result in inactivation
of enzyme
activities
or disruption of subcellular membranes (1,2).
Glucose-6-phosphatase
(EC 3.1.3.9) is a microsomal
enzyme system comprising the catalytic subunit of the
glucose-6-phosphatase
enzyme, a regulatory calciumbinding protein,
and three transport proteins (T1, T2,
and T3), which transport glucose 6-phosphate, phosphate, and glucose, respectively, across the endoplasmic
reticulum membrane (see references 3-5 for recent
of’ BiochemicalMedicine and2 Medicine, UniverDundee,Dundee DD1 9SY, Scotland,U.K.
3Department
of Child Life and Health, University of Edinburgh, Edinburgh EH3 9EF, U.K.
4Present address and address for correspondence: Department
of Obstetrics and Gynaecology,Centre for Research
into Human
Development,University of Dundee,Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland,U.K.
Received January 14, 1991; accepted March 6, 1991.
Departments
sity of
Hume,3 and Ann Burchell2’4
reviews). The inherited
metabolic
disorders
glycogen
storage diseases types la, 1aSP, ib, ic, and id are the
result of separate defects in these five proteins (3-5).
To determine the optimum conditions for freezing and
storing liver tissue samples to be used in the diagnosis of
these deficiencies in the glucose-6-phosphatase
system,
we assessed the effects of different storage and microsome preparation protocols on both microsomal
glucose-6-phosphatase
enzyme activity and microsomal intactness. We deliberately chose procedures that could be
performed in most hospital laboratories, and that do not
require the use of small-scale ultracentrifugation.
Assessment of microsomal intactness
is essential
for
the in vitro enzymatic diagnosis of type 1 glycogen
storage diseases (1-5). However, determination of mannose-6-phosphatase
hydrolysis by the glucose-6-phosphatase enzyme that is commonly used for this assessment, cannot be applied in these cases because the
glucose-6-phosphatase
enzyme is deficient. We have,
therefore, observed the effects of these different preparation procedures on the activity of another, unrelated
endoplasmic reticulum membrane-associated
enzyme,
whose active site lies in the lumen of the organelle:
UDPglucuronosyltransferase
(EC 2.4.1.17) (for recent
reviews, see references 6, 7). We used a UDPglucuronosyltransferase substrate
(1-naphthol) that does not appear to easily access the active site in freshly prepared,
native microsomes-as
assessed by the parallel latencies of this enzyme activity and mannose-6-phosphatase
activity (8).
Both of these enzymes provide good model systems for
studying the effects of freezing. Because the enzymes
are latent, the measured enzyme activities and associated transport of substrates or products depend completely on the intactness of the endoplasmic reticulum
(microsomal) membrane (3, 6, 9).
Materials and Methods
Chemicals.
Glucose 6-phosphate (monosodium salt),
mannose 6-phosphate
(monosodium
salt), histone
2AS,
UDPglucuronic
acid (sodium salt), and Lubrol PX
nonionic detergent were purchased
from Sigma Chemical Co. Ltd., Poole, U.K. Sodium cacodylate, also from
Sigma, was recrystallized from ethanol (950 mLIL) before use (10). 1-Naphthol and sodium dodecyl sulfate
(specially purified for biochemical work) were from BDH
Ltd., Glasgow, U.K. All other reagents
were purchased
from commonly used local suppliers, and were of at least
analytical
grade.
assays.
Glucose-6-phosphatase
was assayed
at 30#{176}C
in microsomes that had been fully disrupted by
treatment with histone 2A8 as previously described (1,
Enzyme
CLINICAL CHEMISTRY, Vol. 37, No. 5, 1991 739
lytic potential of the enzyme.
All microsome preparations consist of a mixture
of
intact and disrupted membrane structures
(9); therefore, to make a more nearly accurate measurement
of
the enzyme activity, one must minimize the disrupted
component of the microsomal sample. This is particularly the case with glucose-6-phosphatase,
where it is
essential, in addition, to know the intactness of the
microsomal sample so that one can accurately diagnose
Tissue preparation.
Female
Sprague-Dawley
rats
deficiencies in the transport proteins that result in
(12-14
weeks old) were from the colony maintained in
glycogen storage diseases types ib, ic, and ld (3-5).
this Institute. Livers from these animals were divided
Microsomal
preparations freshly isolated (and immediinto 100- to 500-mg pieces, and the microsomal fractions
ately assayed) in our laboratory from nonfrozen samples
prepared as outlined in Figure 1.
of normal rat and human liver are routinely found to be
at least 90% intact (e.g., 1). However, for many frozen
Results and Discussion
human
tissue samples sent to our laboratory for diagOne of the principal
problems
encountered
when
nosis,
or
for frozen autopsy samples, the intactness is
trying to diagnose deficiencies in enzymes having their
usually
lower,
and extremely variable (2). Similar probactive sites in the lumen of the endoplasmic reticulum is
lems
are
encountered
with frozen rat liver samples
the phenomenon of latency. The substrates for and
(unpublished observations). However, we stress that the
products of the enzyme reactions have to cross the
use of frozen tissue is preferable to fresh tissue that has
endoplasmic reticulum membrane, and in many cases
spent many hours in transit stored in wet ice, which can
this transport
process is rate limiting. Removal of the
result in a high percentage of samples with extremely
membrane barrier, e.g., by full disruption with deterlow (<60%) intactness
(14), making diagnosis of type 1
gents, permits free access of substrates and free exit of
glycogen storage diseases unreliable.
products, resulting in the expression of the true cataFor obvious ethical and practical reasons, we used rat
liver
for this study. Table 1 demonstrates that freezing
LWER
liver samples, either in liquid nitrogen or at -80 #{176}C
(Figure 1, cases D and E), results in a decrease in
microsomal intactness as assessed by mannose-6-phosHomogenize
Homogenize
Homogenize
Freeze (-80#{176}C)Freeze (liq N2)
phatase
activity. This is confirmed by the decreased
latency (as assessed by the decrease in activation of the
enzyme by detergent) of 1-naphthol UDPglucuronosyll0000g spin
I0000g spin
l0000g spin
Store
Store
transferase
activity in the same microsomal
samples,
prepared from frozen liver (see Figure 2). Conversely,
the 1-naphthol UDPglucuronosyltransferase
detergent
Freeze (-80#{176}C)
Freeze (Uq N2)
Thaw
Thaw
activation curves for the microsomal samples prepared
from frozen 10000 x g supernates (Figure 1, cases B
and C) were similar to those of freshly prepared and
Store
Store
I0000g spin
10000g spin
assayed rat liver microsomes
(Figure 2), a finding also
supported by the measurements of microsomal intactness (Table 1). The intactness (Table 1) was decreased
Thaw
Thaw
slightly in these samples prepared from frozen 10 000 x
g supernates,
but the data suggest that the preparation
of liver 10
x g supernates before freezing (either in
Prepare Mics Prepare Mica
Prepare Mics
Prepare Mica
Prepare Mica
liquid nitrogen or at -80 #{176}C)
eventually results in
microsomal preparations that more faithfully represent
E
the freshly isolated/assayed situation.
A
D
B
C
Therefore, in suspected
cases of glycogen storage
Fig. 1. SchematIcrepresentation
ofsamplestorageand preparation
techniques
disease types ib, ic, and id (transport protein defects),
In all cases, liver was homogenized by using 10 strokes
of a motor-driven
where the degree of microsomal
intactness
determines
Teflon/glass homogenizerat 4#{176}
in homogenization buffer [per lIter.250 mmol
the accuracy of the diagnosis, we strongly recommend
of sucrose and 5 mmd of 4-(2-liydro ethyl)-1-piperazlneethanesuftonic acid,
pH7.4) togivea 10% homogenate. Homogenates were centrifuged at 10 000
that, if fresh tissue cannot be used, the biopsy material
x g for 10 mmat 4#{176}C,
and the pellets discarded. To preparemicrosomes, we
prepared in the form of a 10000
centrifugedthe 10000 x gsupemates at 105000 x gfor6Omlnat4#{176}C, should be immediately
resuspendedtheresultingpellets inhomogenization
buffer,using 20 strokes of
x g supernate (as described in Figure 1, case C), and
a hand-held Teflon/glass homogenizer, in a final volume (In mIlliliters)equal to
then frozen (preferably at -80 #{176}C)
before transport in
the original weight(in grams) of tissue, and assayed for gluoose-6-phoephatase and UDPglucuronosyltransferase activities immediately. Uq N2
lkluid
solid CO2 to the site of assay.
nitrogen; mica
microsomes; store
storage at -80#{176}C
for one to four
To establish which preparation procedure gave better
weeks; thaw Immersion in homogenizationbuffer at 30#{176}C
for 4 mm, followed
results when glycogen storage disease types la and
by Immediate placement on Ice
11), and microsomal
intactness
mining mannose-6-phosphatase
was estimated by deteractivity (1,9). UDPg1ucuronosyltransferase
activity towards 1-naphthol was
measured
at 37#{176}C
by the radioisotopic method of Otani
et al. (12), in the presence of a range of concentrations of
Lubrol PX (6,7), to determine the latency of the enzyme
activities
under different conditions of freezing.
Protein determination.
Microsomal protein content
was estimated
by the method of Peterson (13).
44
4
1 1
44
444
44
44
1
1
+
000
=
=
=
=
740 CLINICAL CHEMISTRY, Vol. 37, No. 5, 1991
pected, we recommend that, if freshly prepared
microsomes cannot be used, biopsy material be prepared as
the 10000 x g supernate,
followed by freezing at
-80 #{176}C,
and transported to the site of assay in solid CO2
(Figure 1, case C). This protocol is likely to produce a
more nearly accurate diagnosis of these protein defects,
because it yields enzyme activity
values that most
closely reflect the values obtained with freshly isolated
and assayed
0.2
0.4
0.3
DETERGENT/PROTEIN
RATIO
0.5
(mg/mg)
Fig. 2. Latency of hepaticmicrosomal1-naphtholUoPglucuronosyltransferaseactivity after variousmicrosomepreparationprocedures
Microsome preparations isolatedas described in Fig. 1 were assayed for
1-naphthol UDPglucuronosyltransferaseactivity in the presence of various
concentrations of the detergent Lubrol PX. Data are expressed as fold
activation relative to native (i.e., non-detergent-treated) microsomes, and are
the meanofduplicatedeterminationsperformed on either two orthree different
microsomalsamples. The mean native enzyme activities (expressed as U/g of
microsomalprotein) for the different methods of preparationwere A (0)30.5;
B (U) 24.5; C () 31.0; 0(0) 21.3; E (#{149})
27.3
Table 1. Effects of Microsome Preparation Procedures
on Hepatic Giucose-6-phosphatase Enzyme Activity
and Microsomal Intactness
Mean ± SEN
Treatment
A
B
C
0
MlcrosomalIntactno&
93.2 ± 0.4
86.5±1.0
87.5 ± 1.0
78.2±1.0
(n
7), %
G+PUI actYc
100
72.5 ± 2.5
81.5 ± 4.2
62.2 ± 4.9
53.4 ± 3.8
E
77.9±2.6
Treatments A-E are as descnbed In Fig. 1.
b Determined by measurement
of the low-K,,, mannose-6-phosphatase
activity, which is not expressed in intact microsomes (see ref. .
colt
ppttase
(0-6-Pase) enzyme activity measured in mla
ciosomes fully disrupted with histone 2A, expressed as a percentage ofthe value
(0.31,SEM0.03, U/mg ofmicroscmal protein) obtained with the freshly prepared
and assayed microeomes (treatment A) isolated from the same liver sample.
1aSP were suspected,
we measured the activity of the
glucose-6-phosphatase
enzyme in fully disrupted
microsomes prepared by the various procedures outlined in
Figure 1 (Table 1). The results show that microsomes
prepared from frozen liver samples (Figure 1, cases D
and E) resulted in an unacceptable loss (almost 50%) of
glucose-6-phosphatase
enzyme activity. Glucose-6-phosphatase
activity
in microsomes
prepared from frozen
10000 x g supernates
(Figure 1, cases B and C) was
higher than that in cases D and E (Figure 1); however,
it was still considerably lower than glucose-6-phosphatase activity in freshly prepared microsomes. Therefore,
where glycogen storage diseases la and 1aSP are sus-
microsomes.
The data presented here strongly indicate that the use
of frozen liver tissue should, wherever possible, be
avoided for the preparation of microsomes to be used in
diagnosis of glycogen storage diseases types la, 1aSP, ib,
ic, and id. Frozen tissue results in microsomal preparations with poor intactness
and with highly inactivated
glucose-6-phosphatase
enzyme. In cases where type la
glycogen storage disease is suspected,
and therefore
where mannose-6-phosphatase activity cannot be used to
measure microsomal
intactness
(because by definition it
is absent or defective), we recommend
that 1-naphthol
UDPglucuronosyltransferase
activity, rather than mannose-6-phosphat.ase activity, be measured to estimate the
intactness of the hepatic microsomal membranes.
This work was supported by grants from the Scottish Hospitals
EndowmentResearch Trust and the Lister Institute for Preventive
Medicine (A.B.), the Scottish Home and Health Department (A.B.
and R.H.), and the Medical Research Council (M.W.H.C).
M.W.H.C. is a Caledonian Research Foundation Research Fellow
and A.B. is a Lister Institute Research Fellow.
References
1. Burchell A, Hume R, Burchell B. A new microtechnique for the
analysisof the human hepatic microsomal glucose-6-phosphatase
system. Clin Chim Acta 1988;173:183-92.
2. Burchell A, Bell JE, Busuttil A, Hume R. The hepatic microsomal glucose-6-phosphatase system and sudden infant death syndrome. Lancet 1989;ii:291-4.
3. Burchell A. The molecular pathology of glucose-6-phosphatase
[Review]. FASEB J 1990;4:2978-88.
4. Burchell A, Waddell ID. Genetic deficiencies of the microsomal
glucose-6-phosphatase
system. In: Randle PJ, Bell J, ScottJ, eds.
Genetics and human nutrition. London: Libbey & Co., 1990:93110.
5. Burchell A, Waddell ID. The molecular basis of the hepatic
microsomal glucose-6-phosphatase
system[Review]. Biochim Biophys Acta 1991; in press.
6. Burchell B, Coughtrie MWH. UDP-glucuronoayltransferases
[Review]. Pharmacol Ther 1989;43:261-89.
7. Mulder GJ, Coughtrie MWH, BurchellB. Glucuronidation.
In:
Mulder GJ, ed. Conjugation reactions in drug metabolism. London:
Taylor and Francis, 1990:51-105.
8. Scragg I, Anon WJ, Burchell B. Microsomalmembrane integrity and expression of UDP-glucuronosyltransferaseactivity in
response to UDP-N-acetylglucosamine. In: Advances in glucuromde conjugation. Lancaster, U.K.: MTP Press, 1985:390-1.
9. Anion WJ, Lange AJ, Ballas LM. Quantitative aspects of
relationship between glucoae-6-phosphate
transport and hydrolysis for liver microsomal glucoee-6-phosphatase system. J Biol
Chem1976;251:6784-90.
10. Wallin BK, Anion WJ. Evaluation of the rate-determining
steps and the relative magnitude of the individual rate constants
for the hydrolytic and synthetic activities of the catalytic component of liver microsomal glucose-6-phosphatase.
J Biol Chem
1973;248:2380.-6.
11. Blair JNR, Burchell A. The mechanism of histone activation
of the hepatic microsomal glucose-6-phosphatase
system;a novel
methodto assay glucose-6-phoaphatase
activity. Biochim Biophys
Acta 1988;964:161-7.
CLINICAL CHEMISTRY, Vol. 37, No. 5, 1991 741
12. Otani G, Abou-el-Makarem MM, Bock KW. UDP-glucuronosyltransferase
in perfusedrat liver and in microsomes.Ill. Effects
of galactosamine and carbon tetrachloride on the glucuromdation of 1-naphthol and bilirubin. Biochem Pharmacol 1976;25:
1293-7.
13. Peterson GL. A simplification of the protein assay method of
Lowry et al., which is more generally applicable. Anal Biochem
1977;83:346-56.
14.. Shin YS. Diagnosis of glycogenstorage disease. J Inherited
Metab Dis 1990;13:419-34.
CLIN. CHEM. 37/5, 742-747 (1991)
Immunoturbidimetric Method for Routine Determinations of Apolipoproteins A-I and B
Domenico Brustolin,1
Giovanni Berti’
Mauro Maierna,’ Francesco
Aguzzi,2 Francesco
A simple immunoturbidimetric method for quantifying apolipoproteins (apo) A-i and B in serum or plasma is
described. A special reagent formulation, including large
amounts of suitable detergents, obviates the need for a
sample blank even with grossly lipemic specimens. The
assay is rapid, easily automated, and thus convenient for
routinework. For both apo A-I and apo B, the assay range
is about 0.2-3.5 g/L. The performance characteristics
were assessed with discrete (Optimate#{174}
and Olli CD) and
centrifugal analyzers (Cobas Fara and IL Monarch 2000).
Average analytical recovery was 101.5% for apo A-I and
99.4% for apo B. Dilution tests showed found/expected
ratios of 101.2% (apo A-I) and 101.0% (apo B). Overall
precision (CV) ranged from 1.4% to 3.3% for apo A-I and
from 1.1% to 8.3% for apo B. Comparisons with commercially available rate nephelometry, radial immunodiffusion, and immunoturbidimetric methods gave good correlations (r 0.938).
Using the immunoturbidimetric
method, we also established the relationships between
apolipoproteins and lipids and determined the reference
intervals. We conclude that the proposed method is
suitable for routine use in clinical laboratories.
Additional Keyphrases:
“kit” methods
-
intermethod compari-
son
Clinical
interest in measuring
apolipoproteins
has
grown after the recognition
of their physiological importance and utility in assessing an individual’s atherosclerotic risk. Some studies suggest that apolipoprotein
(ape) A-I and ape B-the major proteins of high-density
lipoproteins (HDL) and low-density lipoproteins (LDL),
respectively-are
better
blood markers
for coronary
‘Research & Development Laboratory and Scientific Department, Bayer Diagnostici SpA, 20040 Cavenago Brianza, Milan,
Italy.
2Clinical Chemistry and MicrobiologyLaboratory, Hospital of
Broni and Stradella, 27049 Stradella, Pavia, Italy.
3Clinical Biochemistry and Hematology Laboratory, Ca
Granda-Niguarda
Hospital, 20162 Milan, Italy.
Presented in part at the XIVth International
Congress of Clinical Chemistry, San Francisco, CA, 1990 (abstract; Clin Chem
1990; 36:966)
Received September 4, 1990; accepted March 11, 1991.
742 CLINICAL CHEMISTRY, Vol. 37, No. 5, 1991
Zoppi,3 Giordano Tarenghi,’ and
disease than are the traditional lipid markers
(total cholesterol,
LDL cholesterol,
HDL cholesterol,
and triglycerides).4 Some authors today consider the ape
A-I/ape B ratio an even more effective indicator than the
heart
individual
apelipeproteins
(1-3).
To favor the extension of ape A-I and ape B assays
from research
or specialized centers to routine clinical
laboratories,
we developed an immunoturbidimetric
method that is simple to use, yet reliable. Based on the
formation
of immunocomplexes
in the presence of Polyethylene Glycol 6000, it overcomes many of the technical drawbacks shown by other methods, such as long
incubation times and the need for special equipment
and skilled personnel. Like other methods we have
described (4, 5), this assay also is particularly suited to
routine work: the test time is short (10-20 mm for
manual assays and 5 mm for fully automated assays);
no sample blank is needed, even for grossly lipemic
samples; the measurement range is broad (from -0.2 to
3.5
for both apelipeproteins); and automation is
easy. Here we describe characteristics and performances
of this method.
g/L
Materials and Methods
Immunoturbidimetnc Method
Reagents. The following reagents are available as kits
(Sera-Pak#{174}
Immuno) from Bayer Diagnostici SpA, Milan, Italy.
1) Antibody reagent: Specific anti-human
ape A-I or
ape B antiserum (from goat) diluted in buffer (Tris, 0.05
mol/L, pH 8.0) containing,
per liter, 50 g of Polyethylene
Glycol 6000, 1 g of surfactant
(Nonfix/95-96; D.A.C.
Industrie Chimiche SpA, Milan, Italy), and 1 g of
sodium azide. The reagent is ready for use and stable for
at least one year at 2-8 #{176}C.
2) Diluent: Tris buffer (0.01 mol/L, pH 8.0) containing, per liter, 9 g of sodium chloride, 1 g of bovine serum
albumin, 22 g of surfactants
(Adekatol SO-105, 10 g,
and Adekatol SO-160, 12 g; Asahi Denka Kogyo Co.,
Tokyo, Japan), and 1 g of sodium azide.
3) Calibrator: Pooled human serum (with 1 g of
4Nonstandard
abbreviations: apo, apolipoprotein; HDL, highdensity lipoprotein;and LDL, low-densitylipoprotein.