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Institutes of 1 Pediatrics and 2 Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
a Author for correspondence. Fax +31.24.3540297; e-mail r.wevers{at}ckslkn.azn.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: cholesterol oxidase • inborn errors of metabolism • gas chromatography • dehydrocholesterol • enzymatic assays • analytical error
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7-reductase in liver microsomes
(9). The aim of our study was to evaluate the reliability of the standard enzymatic assay (based on cholesterol oxidase) for measuring plasma cholesterol in samples from SLO patients. Because the technique for measuring 7-DHC in plasma is not widely available, clinicians may wish to use the presence of low concentrations of plasma cholesterol in patients with SLO as a first step towards confirming the diagnosis. Furthermore, measurement of plasma cholesterol will play a role in the follow-up of therapy strategies with high-cholesterol diets. We also compared the plasma cholesterol results obtained with the cholesterol oxidase method with those of a gas-chromatographic (GC) technique.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) should not be considered as such.
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Enzymatic assays.
Plasma cholesterol was measured
at 30 °C with an enzymatic test on a Hitachi 747 analyzer
(Boehringer Mannheim, Mannheim, Germany) according to the
instructions of the manufacturer and with use of Boehringer
reagents (CHOD-PAP test; SYS-3:1127578/1489704). In this test,
cholesterol and cholesterol esters are converted by the sequential
action of cholesterol esterase and cholesterol oxidase. The
H2O2 formed in the latter reaction is
determined quantitatively in the last step by using a peroxidase that
converts phenol and 4-aminophenazone into
4-(p-benzoquinone-monoimino)-phenazone.
GC assays.
For GC determination of cholesterol and its
precursors (7-DHC and 8-DHC), we combined 60 µL of plasma with 7.58
nmol of 5ß-cholestane-3
-ol as internal standard (no. C5050;
Steraloids, Wilton, NH) in 1 mL of a solution of 0.32 mol/L KOH in 95%
ethanol. The CFAS-calibrator for cholesterol was used (no. 759350;
Boehringer Mannheim) in combination with two control sera (Precinorm
and Precipath; also from Boehringer Mannheim). After an incubation of
15 min at 55 °C, 1 mL of H2O and 4 mL of pentane were
added and mixed for 5 min. The steroids, which were extracted into the
pentane layer, were taken up by pipetting after centrifugation
(5000g • min). The pentane was then evaporated with
nitrogen at ~50 °C. To derivatize the steroids, we added 100 µL
of an equivolume solution of
N,O-bis(trimethylsilyl)trifluoroacetamide (no.
15238; Fluka, Buchs, Switzerland) and pyridine and incubated at
60 °C for 30 min. GC analysis was performed with a Hewlett-Packard
(Amstelveen, The Netherlands) Model 5890 GC and a 25 m x 0.25 mm
(i.d.) CP-Sil-19 CB column (film thickness 0.2 µm; Chrompack, Bergen
op Zoom, The Netherlands). The temperature program was started at
240 °C, increased to 300 °C at 5 °C/min, and held for 3 min at
300 °C. Temperatures of the injector and detector were 280 and
300 °C, respectively. Pure 7-DHC for calibration purposes was
purchased from Sigma Chemical Co., St. Louis, MO; no. D-3625); to
quantify 8-DHC, we used the calibration curve for 7-DHC because 8-DHC
for calibration was not available commercially.
GC-MS.
Mass spectra from peaks separated by GC were
obtained by using a VG Trio 2 quadrupole mass spectrometer (Micromass,
Altrincham, Cheshire, UK) coupled to the Hewlett-Packard 5890 GC. The
cholesterol peak was characterized by fragments at m/z 329,
353, and 458. 7-DHC and 8-DHC gave identical spectra, with
characteristic fragments at m/z 351 and 456.
Recovery studies.
To test whether 7-DHC contributes to
the result of the enzymatic cholesterol assay, we performed recovery
experiments for cholesterol and 7-DHC in a human serum matrix and in
human serum albumin solution (Sigma, no. A 16535; 50 g/L in 0.15 mol/L
NaCl). The sterols, which had been dissolved to 33 mmol/L solution in
ethanol, were added to both matrices to yield final concentrations of
1, 2, and 3 mmol/L. Complete solubilization was obtained by adding
Nonidet P-40 (no. N6507; Sigma) to a final concentration of 100 mL/L.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows the plasma cholesterol concentrations obtained with the
cholesterol oxidase method for our diseased control group and the SLO
patients. For the group of SLO patients as a whole, the cholesterol
concentrations generally were below the concentration range found in
the control group. However, there was an overlap between values for the
two groups. One SLO patient (case 8) had a plasma cholesterol
concentration of 3.8 mmol/L with this test, which would be interpreted
as a low but normal value for an adult. The values found for the SLO
children (cases 1–3) were close to the range found in our diseased
control group; their diagnosis could easily have been missed if no
further data had been available.
GC analysis of cholesterol, 7-DHC, and 8-DHC.
The gas
chromatograms of the plasma samples from a patient in the diseased
control group and from two patients with SLO all showed peaks for the
internal standard, cholesterol, 8-DHC, and 7-DHC (Fig. 1
). Identification of the compounds was based on their retention
times and on mass spectra (not shown). In agreement with the data from
Axelson (10), we observed that 7-DHC occurs in trace
amounts in normal human plasma. Using calibration curves for
cholesterol, 7-DHC, and the internal standard, we confirmed the
linearity of the method for the concentration range used in this study
(data not shown).
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Plasma concentrations of cholesterol measured with GC vary
considerably among SLO patients (20–2030 µmol/L) and also show an
overlap in concentrations between the SLO group and the diseased
control group, similar to the overlap observed in the enzymatic
cholesterol assay. In all patients with SLO, the concentrations of
7-DHC and 8-DHC were clearly increased (GC results, Table 1
). These
data lead to a straightforward diagnosis for all SLO cases in this
study. The ratio of (7-DHC + 8-DHC)/cholesterol may correlate with the
severity of the disease. Patients 1 and 2, who had a severe form of the
disease (type 1), gave higher values for this ratio than did the rest
of the SLO group.
The correlation between the enzymatic assay and the GC assay results
for plasma cholesterol was good in the diseased control group
(Fig. 2
), the Passing and Bablok ([11]) regression line
(and 95% confidence intervals) being y = 1.02
(0.95–1.08) x - 0.080 (-0.31 to 0.20). In the SLO
group, the results of both methods for the plasma samples all deviated
from that correlation line, to yield y = 0.68
(0.37–1.15) x - 0.211 (-0.84 to 0.44). Because the
cholesterol concentrations found for the SLO group were systematically
higher by the enzymatic test than by GC (Table 1
), we hypothesized that
7-DHC and 8-DHC might contribute to the results of the enzymatic
cholesterol assay; consequently, we performed recovery experiments with
cholesterol and 7-DHC.
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Recovery experiments.
Cholesterol recovery in the
enzymatic assay was complete, the mean recovery in the serum matrix
being 108% (n = 9, range 100–120%) and the mean in the albumin
matrix being 94% (n = 9, range 90–100%). Most of the added
7-DHC was also measured: mean in serum matrix, 61% (n = 6, range
5–70%); mean in albumin matrix, 71% (n = 6, range 70–75%).
Possibly, the known instability of 7-DHC contributed to the partial
recovery of this compound.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because patients with SLO are often severely affected at birth,
reference values for plasma cholesterol in the first weeks of life are
of special interest. Lane and McConathy (12) observed that
cholesterol at day 28 postpartum ordinarily is in the lower
5th percentile of the adult population values; at day 3
postpartum, values were even lower (12). Age-related
reference values are thus invaluable for the correct interpretation of
plasma cholesterol concentrations in children. As Table 1
makes clear,
SLO patients measured with the enzymatic assay may give plasma
cholesterol values close to the lower limit of the range of the
diseased control group or even within this range. In one of our adult
patients (case 8), the plasma cholesterol concentration of 3.8 mmol/L
would be interpreted as normal. We were able to better discriminate
between both groups by assessing the cholesterol values measured by GC.
However, even with this assay, the result for cholesterol could easily
be misinterpreted. We conclude that the diagnosis of SLO cannot be
excluded definitely on the basis of the plasma cholesterol results.
The control samples gave results by both the enzymatic and the GC
method for cholesterol that correlated well (Table 1
and Fig. 1
). For
SLO patients, however, obvious differences between the techniques were
apparent, the cholesterol concentrations measured with the enzymatic
assay being invariably higher than the GC results. As Table 1
shows,
the summed GC data for cholesterol, 7-DHC, and 8-DHC correlated well
with the enzymatically determined cholesterol concentration. This
suggests that 7-DHC and 8-DHC may contribute to the plasma cholesterol
results measured by the enzymatic cholesterol assay. We partly
confirmed this by adding 7-DHC to solutions of albumin and to plasma
samples. The majority (61–71%) of 7-DHC added was measured as
cholesterol by the enzymatic cholesterol test, in both the serum matrix
and the human albumin matrix. The results for cholesterol measured by
GC were not influenced by these additions (data not shown). Apparently
the enzymatic cholesterol test cannot discriminate between the various
steroids. This may be due to aspecific conversion of 7-DHC and 8-DHC by
cholesterol oxidase. Using a sterol monolayer system, Slotte has
described that cholesterol oxidase from Streptomyces
cinnamomeus oxidizes 7-DHC at a rate 5.1-fold slower than it
oxidizes cholesterol (14). We expect that similar results
will be found for other commercially available reagents for plasma
cholesterol determinations that make use of cholesterol oxidase. Our
results indicate that the enzymatic test for measuring plasma
cholesterol gives falsely high results for SLO plasma samples.
The plasma cholesterol concentration in our youngest patient with SLO
was 20 µmol/L, whereas the (7-DHC + 8-DHC)/cholesterol ratio for
this patient was by far the greatest we saw. This is, to our knowledge,
the lowest plasma cholesterol ever reported in humans. Clinically, the
affected girl had a very severe form of the disease (type I), with
major malformations; the patient died at age 5 weeks. As also described
by Tint et al. [15], we observed that the plasma
cholesterol concentration in older SLO patients is generally higher
than in young, more severely affected patients. In our study the lowest
and the highest cholesterol values we saw differed by 20-fold. Those
for 7-DHC differ by only 6-fold, and the concentrations of 7-DHC and
8-DHC are not significantly different between younger and older
patients in our study. The (7-DHC + 8-DHC)/cholesterol ratio, however,
is clearly higher in the young patients (cases 1 and 2 in Table 1
).
The differences in plasma cholesterol concentrations of SLO patients at different ages might result from a higher dietary intake of cholesterol in older, clinically more mildly affected patients. Kelley et al. have suggested (16) that higher cholesterol concentrations correlate more with the length of survival and the amount of dietary cholesterol than with clinical severity. Another explanation for higher cholesterol concentrations in older SLO patients could be the residual activity of the cholesterol biosynthesis pathway in older patients, as was suggested by Tint et al. (15). This would also explain the milder course of the disease in this group. At present, it is unknown whether the shortage of cholesterol and the abundance of cholesterol precursors both contribute to the development of the clinical signs and symptoms of SLO. As with cholesterol oxidase, other enzymes involved in cholesterol-converting pathways may also accept a 7-dehydro or 8-dehydro variant of their normal substrates. This would give rise to unexpected intermediates with unpredictable functional characteristics. This concept may give further impetus to attempts to understand the importance of cholesterol precursors in the development of the clinical picture.
Summarizing, we conclude that the enzymatic test for measuring plasma cholesterol gives unreliable results in SLO patients. For diagnosis of patients clinically suspected to have the disease, measurements of plasma cholesterol (by any method) should not be used. Obviously, quantification of plasma 7-DHC and 8-DHC is the method of choice here (4)(5)(15). For follow-up studies of SLO patients receiving high-cholesterol dietary treatment, the enzymatic test for measuring plasma cholesterol is also not suitable. Instead, the method of choice for this purpose may be GC, which provides reliable quantitative data for plasma cholesterol, 7-DHC, and 8-DHC.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7-reductase activity in liver microsomes from Smith–Lemli–Opitz homozygotes. J Clin Invest 1995;96:1779-1785.
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) and
diseased control patients (
).