Maternal medicine

DOI: 10.1111/j.1471-0528.2011.02960.x
www.bjog.org

Prediction of pre-eclampsia in early pregnancy

by estimating the spot urinary albumin:
creatinine ratio using high-performance liquid
chromatography
S Baweja,a A Kent,a R Masterson,b S Roberts,c LP McMahona
a

Department of Renal Medicine, Monash University, Box Hill, Victoria, Australia b Department of Nephrology, Royal Melbourne Hospital,
Parkville, Victoria, Australia c Division of Obstetrics, Sunshine Hospital, St Albans, Victoria, Australia
Correspondence: Professor LP McMahon, Department of Renal Medicine, Monash University, 2nd Floor, 5 Arnold Street, Box Hill, Victoria,
Australia. Email lawrence.mcmahon@easternhealth.org.au
Accepted 21 February 2011. Published Online 12 April 2011.

Objective To establish whether a spot urinary albumin: creatinine

ratio (ACR) measured before 20 weeks of gestation can predict

subsequent pre-eclampsia when urinary albumin is measured by
high-performance liquid chromatography (HPLC).
Design Prospective exploratory study.
Setting Antenatal clinic in a tertiary teaching hospital, Victoria,

Australia.
Population A cohort of 265 women with a singleton pregnancy,

normal renal function, and no evident proteinuria, attending

antenatal clinics between 12 and 20 weeks of gestation.
Methods The ACR was determined from a mid-stream urine

(MSU) sample taken between 17 and 20 weeks of gestation. Intact

urinary albumin was determined by HPLC; creatinine was
measured by modified Jaffes method.
Outcome measures Pre-eclampsia (primary); gestational

hypertension, small for gestational age (SGA), gestational

diabetes mellitus, gestational age at delivery, and prematurity
(secondary).

Results The median ACR was 28 mg/mmol (IQR 1646 mg/

mmol). Women who subsequently developed pre-eclampsia had a
significantly higher ACR (median 50 mg/mmol; IQR 3390 mg/
mmol) compared with women suffering from gestational
hypertension (median 27 mg/mmol; IQR 835 mg/mmol), and
compared with unaffected women (median 28 mg/mmol; IQR
1646 mg/mmol). Mothers of SGA infants also had a significantly
higher median ACR. By ROC analysis, the optimum ACR to predict
pre-eclampsia was 35.5 mg/mmol: the relative risk of developing
pre-eclampsia in women with a urinary ACR 35.5 mg/mmol was
7.8 times more than in those with a urinary ACR < 35.5 mg/mmol.
Conclusions When urinary albumin is measured by HPLC, spot

urinary ACR values are higher in early uncomplicated pregnancy

compared with previously reported conventional methods. When
measured early in the second trimester, an ACR 35.5 mg/mmol
predicted pre-eclampsia well before the onset of clinical
manifestations.
Keywords High-performance liquid chromatography, pre-eclamp-

sia, pregnancy, proteinuria.

Please cite this paper as: Baweja S, Kent A, Masterson R, Roberts S, McMahon L. Prediction of pre-eclampsia in early pregnancy by estimating the spot
urinary albumin: creatinine ratio using high-performance liquid chromatography. BJOG 2011;118:11261132.

Introduction
Pre-eclampsia remains a major cause of perinatal and
maternal morbidity and mortality, and is one of the most
common medical complications of pregnancy.13 Although
there is no established preventative therapy, there is potential gain in being able to identify the women and fetuses at
risk, so that appropriate monitoring can ensue, as well as

1126

some evidence to support the prophylactic benefit of the

early introduction of aspirin,4,5 calcium (in women at high
risk and low baseline calcium intake),6 and/or possibly heparin (in high-risk women with prothrombotic conditions).79 Although there is currently no established
preventative therapy, early diagnosis is helpful for research
studies, and may in the future be beneficial if new preventative therapies are discovered.

2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology 2011 RCOG

Proteinuria in early pregnancy study

The pathophysiological events resulting in pre-eclampsia

begin early in gestation, and precede the onset of the clinical features.10 One of the early pathophysiological hallmarks is endothelial cell damage.11,12 Microalbuminuria is
a marker of endothelial dysfunction and, in the general
population, is associated with hypertension, obesity, diabetes, and covert renal disease, and also with an increased
risk for myocardial infarction, stroke, and premature
death.13,14 The risk rises with the urinary albumin concentration, even within the so-called normal range.15 Microalbuminuria might be used as an early marker of endothelial
dysfunction in pre-eclampsia, before the onset of the overt
syndrome, as it is likely that overt proteinuria is preceded
by a microalbuminuric phase. Previous studies examining
the effectiveness of such a screening procedure have had
variable results.1618 Although the 24-hour collection of
urine is the gold standard for quantifying urinary albumin
excretion, it is cumbersome, and results in a delay of at
least a 24 hours in diagnosis.19 Therefore, the spot urinary
protein: creatinine ratio, or urinary albumin: creatinine
ratio (ACR), has been advocated as an alternative.19,20
Urinary albumin is usually measured by an immunochemical method such as immunonephelometry, immunoturbidimetry, enzyme-linked immunosorbent assays
(ELISA), or radioimmunodiffusion. Recently, a number of
studies have used high-performance liquid chromatography
(HPLC) for urinary albumin measurement in different
populations, and demonstrated that the level of albumin
detected in the urine by HPLC, when compared with conventional assays, is significantly greater because HPLC is able
to measure both immunoreactive and immunounreactive
intact albumin.2427
However, none of the previous studies have used HPLC
to measure urinary albumin in pregnant women. In this
study we wished to establish prospectively whether a spot
urinary ACR measured before 20 weeks of gestation can
predict the development of pre-eclampsia when urinary
albumin is measured by HPLC.

Methods
This was a prospective exploratory study of women with
singleton pregnancies attending an antenatal clinic at a tertiary teaching hospital in Victoria, Australia, from March
2006 to December 2007. All women attending clinics were
asked to participate in the study at the time of booking
(between 12 and 20 weeks of gestation), and were recruited
if they agreed. The study was approved by the regional ethics committee, and written informed consent was obtained
from all participating women. Inclusion criteria were
women over 18 years of age, singleton pregnancy,
20 weeks of gestation at the time of recruitment, and nil
proteinuria upon measurement with a dipstick. Women

with haematuria, dipstick-positive proteinuria, ongoing urinary tract infection, multiple pregnancy, acute renal failure,
chronic kidney disease (CKD), assisted reproduction, or a
poor obstetric history were excluded. However, women
with a past history of urinary tract infection, renal failure,
haematuria, or proteinuria were included if there was no
evidence of current disease, if the urine was dipstick-negative for proteinuria, and if the serum creatinine level was
within the normal range. CKD was diagnosed if the patient
had a personal history of CKD, if there was evidence of
impaired renal function, or if the patient was under a
nephrologists care. Chronic hypertension was diagnosed if
the patient was already taking anti-hypertensive medication
or displayed hypertension before 20 weeks of gestation.
Data regarding demographic profile, blood pressure, body
mass index (BMI), and medical and family history (history
of chronic hypertension, diabetes mellitus, and/or CKD)
were recorded. Obstetric history documented gravidity,
parity, past history of pre-eclampsia, prematurity, small for
gestational age (SGA), miscarriage, and family history of
pre-eclampsia.
Between 17 and 20 weeks of gestation, all women were
given a sterile urine container without preservative and,
after instruction, a mid-stream urine sample was collected.
Routine microscopic and dipstick examination of urine was
performed, and within 3060 minutes of collection the
sample was stored at 20C for ACR analysis at the end of
the study. Participants were then followed until delivery.
The primary outcome measure was pre-eclampsia, defined
according to standard clinical criteria.25 Secondary outcome
measures included gestational hypertension (GH), SGA,
gestational diabetes mellitus (GDM), gestational age, and
prematurity, and a normal range estimate of urinary ACR
was established.
The definition of GH was a blood pressure of 140/
90 mmHg occurring after 20 weeks of gestation in a previously normotensive woman.28 GDM was defined as a positive 50-g oral glucose challenge test [with a venous plasma
glucose level 1 hour after glucose challenge of at least
7.8 mmol/l (140 mg/dl)], and a positive 75-g oral glucosetolerance test at 2428 weeks of gestation [with venous
plasma glucose levels of <7.8 mmol/l after an overnight fast
and 7.811.0 mmol/l (198 mg/dl) at 2 hours).26 SGA was
diagnosed when the birthweight was below the tenth centile
for the gestational age,27 and prematurity was defined as
birth on or before the end of the last day of the 37th week
(259th day) following the onset of the mothers last menstrual period, or by initial ultrasound estimation.28
Total intact urinary albumin (immunoreactive plus immunounreactive) was determined by analysing urine samples
using a Hewlett Packard 1100 HPLC system with UVvisible
detector (Hewlett Packard, Waldbronn, Germany). Aliquots
of urine (25 ll) were injected onto a Zorbax Bio series

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Baweja et al.

preparative GF-250 column. The mobile phase was phosphate-buffered saline run at a flow rate of 0.5 or 2.0 ml/
minute in a gradient system. The urinary albumin peak
was identified to within 2% of the elution time of the
monomer albumin standard. Urine creatinine was measured by modified kinetic Jaffe reaction without deproteinization using COBAS INTEGRA systems.
Statistical analysis was performed by pasw statistics
v18 software. As this is the first exploratory study to use
this technique in pregnancy, a meaningful power calculation could not be performed. Comparison of spot urinary
ACR and birthweight between unaffected, gestational
hypertensive, and pre-eclamptic groups were performed by
KruskalWallis test or MannWhitney U-test. Comparison
of the mean of the continuous data (normally distributed)
was performed by analysis of variants (anova), and chisquare analysis was used to compare categorical data
between groups. For post hoc analysis of normally-distributed continuous data, Bonferonis test was applied. Sensitivity, specificity, positive predictive value (PPV), and
negative predictive value (NPV) at different values of ACR

for predicting pre-eclampsia were calculated from the receiver operating curve (ROC).

Results
After obtaining informed consent, 295 women were
enrolled at between 12 and 20 weeks of gestation. Thirty
(10.1%) women were excluded subsequently because of
missing outcome data (miscarriage, transferral to another
facility, withdrawal of consent, or misplaced urine sample
during transport); therefore, 265 women were included in
the final analysis (Tables 1 and 2).
The mean maternal age was 29.5 4.7 years and the
mean BMI was 26.9 6.2 kg/m2. Most women were of
Australian (44.5%) or European (33.2%) descent, and
91 (34.3%) were primigravid. Six (2.3%) developed preeclampsia, and 11 (4.2%) developed GH. The remainder
did not develop hypertensive complications during followup. Nine babies (3.4%) were SGA at birth, three of which
were born to mothers who had pre-eclampsia. Twenty-one
(7.9%) women developed GDM. Fifteen women (5.75%)

Table 1. Baseline clinical parameters and demographic profile

Age (years)
Ethnicity, n (%)
Australian
European
Asian
African
ATSI
Others
BMI, kg/m2
Primigravida, n (%)
SBP, mmHg
DBP, mmHg
MAP, mmHg
DM, n (%)
CHT, n (%)
PH renal disease, n (%)
PH PE, n (%)
FH DM, n (%)
FH HT, n (%)
FH CKD, n (%)
GDM, n (%)

Unaffected
(n = 248)

Gestational
hypertension
(n = 11)

Pre-eclampsia
(n = 6)

SGA
(n = 9)

P*

29.5 4.7

31.1 5.8

26.3 4.9

27.6 5.3

0.142

111 (44.8)
84 (33.9)
40 (16.1)
6 (2.4)
1 (0.4)
6 (2.4)
26.8 6.1
82 (33.0)
109 10.7
65 8
101 10
15 (6.0)
19 (7.7)
9 (3.6)
5 (2.0)
101 (40.7)
96 (38.7)
10 (4.0)
20 (8.15)

5 (45.5)
1 (9.1)
3 (27.3)
1 (9.1)
1 (9.1)
0
27.8 5.9
5 (45.4)
120 13
71 8
111 12
0
2 (18.2)
1 (9.1)
0
6 (54.5)
7 (63.6)
1 (9.1)
1 (9.1)

3 (50.0)
0
2 (33.3)
1 (16.7)
0
0
30.3 10.2
4 (66.7)
116 21
67 12
106 18
0
2 (33.3)
0
0
3 (50.0)
4 (66.7)
2 (33.3)
0

4 (44.4)
4 (44.4)
0
1 (11.1)
0
0
26.4 7.1
4 (44.4)
108 18
64 10
100 16
0
1 (11.1)
0
0
4 (44.4)
6 (66.7)
2 (22.2)
0

0.023

0.334
0.98
0.001**
0.054**
0.008**
0.580
0.046
0.004
0.840
0.612
0.110
0.004
0.762

ATSI, Australian Torres Strait islanders; CHT, chronic hypertension; DBP, Diastolic blood pressure; FH, family history; MAP, mean arterial pressure;
PE, pre-eclampsia; PH, past history; SBP, systolic blood pressure.
Data are means SDs, except as indicated.
*Reflects comparison between unaffected, gestational hypertension, and pre-eclampsia groups. (The SGA group was comprised of patients from
the unaffected and pre-eclampsia groups).
**Significant difference only between gestational hypertension and unaffected group.

1128

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Proteinuria in early pregnancy study

Table 2. Spot urinary ACR, fetal complications, and outcome measures

Unaffected
(n = 248)

ACR (mg/mmol)
Gestational age at delivery
Mode of delivery
Normal vaginal delivery
Caesarean section
Birthweight (g)***
Prematurity, n (%)

Gestational
hypertension
(n = 11)

Pre-eclampsia
(n = 6)

SGA
(n = 9)

P*

28 (1646)
39.2 1.9

27 (835)
38.4 1.7

50 (3390)
36.7 2.4

55 (3276)
37.4 1.8

0.042
0.003**

179 (72.2)
69 (27.8)
3420 (30903740)
9 (3.6)

8 (72.7)
3 (27.3)
3270 (30383700)
1 (9.1)

2 (33.3)
4 (66.7)
2270 (13783420)
4 (66.7)

5 (55.6)
4 (44.4)
2250 (17312490)
3 (37.5)

0.115
0.024
<0.001

*Reflects the comparison between unaffected, gestational hypertension, and pre-eclampsia groups (as per Table 1).
**Significant difference between pre-eclampsia and other groups.
***Median and IQR.

had pregestational diabetes, 23 women (8.7%) had chronic

hypertension, and ten women (3.8%) had a past history of
renal or urinary tract disease. Of these, only two of the
women with hypertension developed pre-eclampsia. A family history of diabetes, hypertension, and CKD was present
in 110 (41.7%), 107 (40.5%), and 13 (4.9%) women,
respectively. Four of six women who developed preeclampsia were nulliparous; the other two women had no
history of pre-eclampsia.
At the time of booking the mean systolic blood pressure
(SBP), mean diastolic blood pressure (DBP), and mean
arterial pressure (MAP) were significantly higher in women
who later developed gestational hypertension than in those
who remained unaffected. Although the mean SBP, DBP,
and MAP also tended to be higher at study entry in
those who developed pre-eclampsia, this difference was not
significant.
Overall, the median spot urinary ACR measured between
17 and 20 weeks of gestation was 28 mg/mmol (IQR 16
46 mg/mmol). Women who subsequently developed preeclampsia had a significantly higher initial spot urinary
ACR (median 50 mg/mmol; IQR 3390 mg/mmol) in comparison with women suffering gestational hypertension
(median 27 mg/mmol; IQR 835 mg/mmol; P = 0.035)
and unaffected women (28 mg/mmol; IQR 1646 mg/
mmol; P = 0.036). Mothers of infants with SGA also had a
significantly higher median spot urinary ACR between 17
and 20 weeks of gestation than others (median 55 versus
28 mg/mmol; IQR 3276 versus 1645 mg/mmol;
P = 0.008) (Figure 1). However, no significant correlation
was found between spot urinary ACR and birthweight
(r = 0.087; P = 0.160) or gestational age at the time of
delivery (coefficient = )0.083; P = 0.179). There was no
difference in ACR between women with a history of renal
or urinary tract disease, chronic hypertension, or diabetes
in comparison with those who did not. As expected, babies

of mothers with pre-eclampsia were smaller and delivered

earlier (see Table 1).
The sensitivity and specificity of spot urinary ACR were
calculated from the ROC (Figures 2 and 3). The optimum
spot urinary ACR to predict pre-eclampsia was 35.5 mg/
mmol, which had a test sensitivity of 83.3%, specificity of
61.2%, PPV of 63.0%, and NPV of 78.6%. The area under
the curve (AUC) was 0.753 (95% CI 0.5900.916;
P = 0.034). The relative risk of developing pre-eclampsia in
those with a urinary ACR 35.5 mg/mmol was 7.8 fold
higher than in those who had a urinary ACR of <35.5 mg/
mmol. Based on ROC, the optimum urinary ACR for predicting SGA was 34.5 mg/mmol, with a test sensitivity of
77.8%, specificity of 59.2%, PPV of 65.6%, and NPV of
72.7%. AUC was 0.762 (95% CI 0.6250.898; P = 0.008).

Discussion
Pre-eclampsia remains a leading cause of maternal and fetal
mortality and morbidity.1 In the last few years some of the
molecular mechanisms underlying pre-eclampsia have been
clarified. Studies have shown that alterations in the regulation and signalling of angiogenic pathways that contribute to
the inadequate cytotrophoblast invasion are seen in preeclampsia. High levels of anti-angiogenic factors (soluble
fms-like tyrosine kinase-1 and soluble endoglin) and low
levels of pro-angiogenic factors (VEGF, PIGF) may subsequently contribute to widespread maternal endothelial dysfunction and the clinical syndrome of pre-eclampsia.10,20,29,30
Endothelial dysfunction has been demonstrated as early as
22 weeks of gestation,12 and the level of anti-angiogenic
factors start rising as early as 17 weeks of gestation.10 It could
be expected that microalbuminuria, a marker of endothelial
dysfunction, might also be apparent by this time, although perhaps at a level undetectable by immunochemical
methods. However, HPLC is more sensitive than any of the

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Baweja et al.

immunochemical methods, and could be used to estimate a

low level of excreted albumin in urine.
To our knowledge, this is the first study that has measured spot urinary ACR in a pregnant population by
HPLC. We found that the spot urinary ACR at 17
20 weeks of gestation was significantly higher in women
who subsequently developed pre-eclampsia and/or SGA,
than in those who did not. Figure 1 shows that although
the ACRs for the pre-eclamptic and SGA groups were significantly higher than average, some still fell within the
normal range, and therefore this may well limit the usefulness of this test in clinical practice. Comparing the 110
subsequently unaffected patients whose ACR overlapped
with those destined to develop pre-eclampsia revealed no
marked differences in blood pressure, age, parity, or gestational stage. Other factors that might have been relevant
but were not assessed included the womans level of activity
before the urine was collected and the time of day, either of
which could have affected the ACR. Although a 24-hour
collection of urine is the gold standard for quantifying
urinary albumin, a spot ACR was used in this study because
it was more likely to reflect clinical practice for a screening
test, and because an attempted 24-hour collection would
very likely have been futile and erroneous.19,31 Some but not
all past studies have shown an excellent correlation between
a spot urinary ACR and albumin excretion in a 24-hour
urine sample in normal pregnancy and pre-eclampsia.3236
Many previous studies have measured microalbuminuria
in an attempt to predict pre-eclampsia in early pregnancy,
postulating that the stage of gross proteinuria is preceded
by the stage of microalbuminuria. Using regression models,
Bar et al.16 identified the presence of microalbuminuria at
2830 weeks of gestation to be predictive of subsequent
pre-eclampsia, with an odds ratio of 2.1 (95% CI 1.26
3.53). Shaarawy found that microalbuminuria at 1012
weeks of gestation had 50% sensitivity, 58% specificity,
50% PPV, and a 91% NPV for the later development of
pre-eclampsia.18 Poon found that, although the logarithmic
conversion of urinary ACR at 1113 weeks of gestation
helped predict pre-eclampsia, it did not provide additional
value above clinical maternal variables.37 It has also been
shown that in hypertensive women, the presence of microalbuminuria at 2830 weeks of gestation has a high specificity and positive predictive value when predicting
pre-eclampsia.38 Similarly, its presence even before pregnancy in pregnant women with type-1 diabetes is a strong
predictive marker.39 It is likely that the diversity of results
from these studies indicates that other risk factors apart
from microalbuminuria can predict the establishment of
pre-eclampsia, particularly when severe or presenting early
in gestation.
A number of studies have used HPLC for urinary albumin measurement in non-pregnant populations, and have

1130

Figure 1. Graph detailing median and IQRs of pre-eclampsia,

unaffected, and SGA groups. Individual values for particular subsets are
as indicated.

Figure 2. Receiver operating curve to predict pre-eclampsia.

Figure 3. Receiver operating curve to predict SGA.

2011 The Authors BJOG An International Journal of Obstetrics and Gynaecology 2011 RCOG

Proteinuria in early pregnancy study

demonstrated that the level of albumin detected in the

urine by HPLC is significantly greater compared with
conventional assays. However, this fact has not been
verified in pregnant women. Previous studies from nonpregnant populations have reported urinary albumin excretion to be between two and four times higher when
measured by HPLC, than by one of the immunochemical
methods.2123,40,41 The median value of spot urinary ACR
(28 mg/mmol; IQR 1646 mg/mmol) in uncomplicated
pregnancy was over 20 times higher in our study compared with other studies using one of the conventional
methods.4244 This suggests that the level of immunounreactive albumin excreted in pregnancy is much greater than
in the non-pregnant woman, but the reason for this is
unknown. It can be conjectured to be the result of incomplete processing by the lysosomal pathway, as in diabetes
(although in our study, women with diabetes or hypertension did not have higher levels than the non-diabetic
women). Women with a raised ACR, measured by HPLC
rather than by immunochemical measurements, may have
a higher cardiovascular mortality risk.45,46
The major limitation of our study was that the number
of affected cases was small. The findings should therefore be
interpreted with caution. In addition, we were unable to
calculate customised birthweight centiles, as the necessary
maternal and fetal data were not collected during the study.
This prevented us from defining intrauterine growth restriction (IUGR) as an end point in the study. Furthermore, the
limited numbers precluded a regression analysis to investigate factors that contributed to changes in urinary ACR and
pre-eclampsia. Finally, as the HPLC assay is relatively
expensive, our results do not suggest that it is likely to be
useful as a screening test, although this can only be established by a much larger study. Additional studies are now
required to explore the biochemical nature and underlying
molecular mechanisms of the excreted albumin in pregnancy, to confirm the normal range of ACR by HPLC, and
to corroborate the predictive potential of this technique.

Conclusion
When urinary albumin is measured by HPLC, spot urinary
ACR values are higher in uncomplicated pregnancy in
comparison with conventional methods. A spot urinary
ACR value of 35.5 mg/mmol (measured by HPLC
between 17 and 20 weeks of gestation) predicted future preeclampsia with a sensitivity and specificity of 83.3% and
61.2%, respectively. Additional studies and a costbenefit
analysis are required to confirm these findings before
recommending this test for screening purposes. The molecular nature of the excreted albumin fractions and the
underlying mechanisms of proteinuria in early pregnancy
also need to be explored.

Disclosure of interests
None.

Contribution to authorship
SB analysed the data and wrote the article. AK was
involved in planning strategies, recruitment, and sample
collection. SR was involved in conducting patient visits and
in the collection of data. RM was involved in planning
strategies and in the enrolment of the patients. LPM was
involved in planning strategies, recruitment, analysis of
data and writing the article.

Details of ethics approval

The study was approved by the Melbourne Health Research
and Ethics Committee on 16 June 2004; ref. no. 2004.075.

Funding
Internal sources.

Acknowledgements
We thank the patients for their participation in the study
and the nursing staff of Sunshine Hospital for their cooperation. j

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