2.3.1 Urine Analysis
Urine from adult offspring at two years of age was collected from the bladder at post- mortem and albumin (g.l-1) and creatinine (µmol.l-1) were measured using a Randox Rx Imola autoanalyser and quantitative colorimetric assay kits (Randox Laboratories, Co Antrim, UK).
2.3.1.1 Albumin
Albumin levels were determined using a Randox albumin kit. Albumin has the ability to bind to bromocresol green in a quantitative manner. The albumin-bromocresol green complex can then be detected as it absorbs maximally at 578 nm.
2.3.1.2 Creatinine
Creatinine levels were determined enzymatically using a Randox creatinine kit. Creatinine deaminase converts creatinine and H2O to N-Methylhydantoin and ammonia. Glutamate
dehydrogenase catalyses ammonia and -ketoglutarate in the presence of NADPH to form glutamate and NAD+. The absorbance decrease (at 340 nm) due to NADPH reduction is proportional to the concentration of creatinine.
2.3.2 Plasma Analysis: Randox Imola
All two-year old offspring plasma samples from Study 1 were defrosted and analysed for sodium, potassium and chloride using a Randox Rx Imola autoanalyser and quantitative colorimetric assay kits (Randox Laboratories, Co Antrim, UK). All maternal plasma samples from Study 2 were defrosted and analysed for glucose, non-esterified fatty acids (NEFA), triglycerides, albumin, total protein, urea, lactate and D-3-hydroxybutarate. Fetal plasma
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samples from Study 2 were defrosted and analysed for glucose and lactate only. All concentrations were measured in mmol.l-1 unless stated otherwise.
2.3.2.1 Sodium
Sodium concentrations were determined enzymatically using a Randox sodium kit. O- nitrophenol- -D-galactopyranoside (OPNG) is added to the plasma. -galactosidase hydrolyses sodium + OPNG to form o-nitrophenol, which is yellow. The absorbance of the yellow product is measured at 405 nm.
2.3.2.2 Potassium
Potassium was determined enzymatically using a Randox potassium kit. Pyruvate kinase reacts with phosphoenolpyruvate to yield pyruvate. The pyruvate reacts with NADH in the presence of lactate dehydrogenase to form lactate and NAD. This causes a decrease in absorbance at 340 nm which is proportional to the concentration of potassium.
2.3.2.3 Chloride
Chloride concentrations were determined using a Randox chloride kit, using a thiocyanate method. Mercurious thiocynate reacts with the chloride ions to form mercury perchlorate and thiocyanate. The thiocyanate forms a red complex with ferric chloride and the
absorbance of the red dye is measured at 500 nm.
2.3.2.4 Glucose
Glucose concentrations were determined using a Randox glucose kit, which uses the hexokinase method. The action of the hexokinase enzyme results in D-glucose being phosphorylated with an ATP molecule to form glucose-6-phosphate. Glucose-6-phosphate
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dehydrogenase catalyses G6P in the presence of NADP, to form 6-phosphogluconate and NADPH. The absorbance of NADPH is measured at 340 nm.
2.3.2.5 NEFA
NEFA concentrations were determined using a Randox NEFA kit. The enzyme acyl-CoA synthetase is added to plasma and allows NEFA to form thiol esters of coenzyme A (acyl- CoA) in the presence of adenosine triphosphate (ATP), magnesium cations and CoA. Hydrogen peroxide is produced when Acyl-CoA is oxidised by acyl CoA oxidase. Peroxidase enzymes then catalyse 3-methylN-(hydroxyethylanaline) with 4-aminoantipyrine in the presence of hydrogen peroxide to form a purple coloured adduct which is maximally absorbed at 550 nm.
2.3.2.6 Triglycerides
Triglyceride concentrations were determined using a Randox triglyceride kit, which utilises the GPO-PAP method. The triglycerides undergo enzymatic hydrolysis using lipases. Peroxidase then catalyses hydrogen peroxide, 4-aminophenazone and 4-chlorophenol to produce quinoneimine, which is maximally absorbed at 540 nm.
2.3.2.7 Albumin
Albumin levels were determined using a Randox albumin kit, see 2.3.1.1.
2.3.2.8 Total protein
Total protein concentration was determined using a Randox total protein kit, and was measured in g.l-1. The kit uses the Biuret method: under alkali conditions peptides form a violet-coloured complex with copper ions. The intensity of the colour, and therefore the absorption at 540 nm, is directly proportional to the concentration of protein.
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2.3.2.9 Urea
Urea concentration was determined using a Randox urea kit, utilising a kinetic method. The urea is hydrolysed in the presence of water and the enzyme urease to form ammonia and CO2. Glutamate dehydrogenase catalyses the ammonia to react with a-oxoglutaric acid and
NADH2, to form glutamic acid and NAD+. The absorbance decrease (at 340 nm) due to
NADH2 reduction is proportional to the concentration of urea.
2.3.2.10 Lactate
Lactate concentration was determined using a Randox L-Lactate kit. The kit uses an enzymatic method, where lactate oxidase catalyses the oxidation of L-lactate to form pyruvate and H2O2. The H2O2 is catalysed with a peroxidase to form a purple dye. The
intensity of the dye (absorbed at 550 nm) is proportional to the lactate content.
2.3.2.11 D-3-hydroxbutyrate
D-3-hydroxybutyrate concentration was determined using a Randox ranbut
(hydroxybutyrate) kit. This is a kinetic enzymatic method. The enzyme 3-Hydroxybutyrate dehydrogenase oxidises D-3-hydroxybutyrate to form acetoacetate. Concurrently NAD+ is reduced to NADH and the associated change of absorbance at 340 nm can be directly correlated with the D-3-hydroxybutyrate concentration.
2.3.2.12 Quality control
Experimental samples were run in duplicate, and each run contained the same two quality control (QC) samples. Inter-assay variation was calculated from the variance from the QC samples, and intra-assay variation from the variance of the duplicate experimental samples. Intra-assay variance ranged from 0.35 3.52, and inter-assay from 3.23 7.07.
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2.3.3 Plasma Analysis: Amino acids
Amino acids were isolated from maternal and fetal plasma and derivatised using the
E) A A K P M UK
protocol.
2.3.3.1 Amino acid extraction
F
into a preparation vial, and then passed very slowly thro E)
N
and sodium hydroxide) was drawn up into the tip and then expelled along with the sorbent
F
sequentially added to derivatise the amino acids and the resulting organic layer was recovered. The organic layer containing the amino acids was dried down using nitrogen,
-octane:chloroform (4:1 v/v). Where necessary the amino acids were stored at -20°C for up to 12 hours whilst other samples were prepared.
Amino acid standards were prepa E)
A
samples, to check for consistency.
2.3.3.2 Gas Chromatography Mass Spectrometry
One µl of each amino acid sample was injected in splitless mode (split closed for 10s) using an AS3000 autosampler (Thermo, Manchester, UK). The injector of the Trace GC Ultra gas chromatograph (Thermo) was kept at 250°C, with a starting oven temperature of 90°C which was increased to 320°C at the rate of 20°C min-1 (transfer line from the oven to mass
56
spectrometer was 300°C). Helium (8 psi) was used as the carrier gas to elute the amino acids from the ZB-AAA column (10 m x 0.25 mm ID).
The DSQ II mass spectrometer (Thermo) was operated in selected ion mode recording ions 84, 101, 114, 116, 130, 144, 146, 155, 156, 158, 172, 180, 184, 243 and 244 with a dwell time of 0.03s. To calibrate the data the peak areas for the amino acids in the standards and samples were adjusted for variation in the peak area of the internal standard.
2.3.4 Amniotic Fluid: Amino Acids
Dr Dongfang Li carried out this analysis on behalf of the study. In brief, 980 µl of the plasma sample and 20 µl internal standard norleucine were added to a conical centrifuge tube containing 60 mg of solid 5-sulphosalicylic acid (SSA), immediately mixed and allowed to stand for 1 hour at 4°C. Next the mixture was centrifuged for 15 minutes at 4°C in a high speed centrifuge to spin down the precipitate, and the supernatant was removed and filtered through a 0.2 µm filter. Standard solutions of amino acids were prepared in the same way. Finally, 20 µl of treated standards or sample were injected to a Biochrom 20 amino acid analyser (Pharmacia LKB, Biochrom Ltd, Cambridge, UK) and peak integration was performed using EZChrom Elite software. This method successfully measured 19 out of the 20 amino acids.