W.J. Hensley

A rapid gas chromatographic method for the quantitation of underivatised individual free fatty acids in plasma

A rapid method for quantitating individual free fatty acids (FFA) in plasma has been developed. An internal standard is added to plasma, the FFA are extracted into an organic solvent, concentrated and injected into a gas chromatograph. For routine estimations, extraction and assay of a single sample takes about 30 min and up to 30 samples can be assayed in a day. Seven FFA (myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic acids), are routinely quantitated. This method can be used to monitor individual FFA levels, to determine the percentage composition of individual FFA in plasma or to investigate metabolic disorders involving fatty acids, for example Refsums disease.

Thiamine Deficiency - A Neglected Problem of Infants and Mothers - Possible Relationships to Sudden Infant Death Syndrome

Summary: i) An unexpectedly high incidence of biochemical thiamine deficiency (erythrocyte transketolase) was found in groups of mothers and infants, selected for apparent health from a westernized Caucasian community in Australia.

The estimation of plasma valproate by gas-liquid chromatography.

A gas chromatographic method for the plasma assay of the anticonvulsant, sodium valproate, is described. Derivatization is not necessary. 200 microliter plasma are required for a single estimation. The method involves a chloroform extraction of valproate and the internal standard, cyclopentane carboxylic acid, from acidified plasma. Gas-liquid chromatography using the stationary phase 10% SP-216-PS gives complete separation of valproate and the internal standard in eight minutes. The limit of detection is 20 mumol valproate/1 plasma (equivalent to 40 pmol on column). This is well below the lower therapeutic plasma level. The between-run precision of the method indicates a variation for each sample within+/-3% of its mean value.

Postprandial changes in apolipoprotein(a) concentration of triglyceride-rich lipoproteins can be reproduced by in vitro incubation: implications for underlying mechanism

We investigated the reciprocal changes in apolipoprotein(a) concentration in the lipoprotein of d > 1.006 and the triglyceride-rich lipoprotein (TRL) fractions of plasma which occur in vivo following fat ingestion. Twenty fasting subjects were studied before and 4 h after a fat-rich meal. In 75% of cases, in vitro incubation of the postprandial (4-h) TRL with autologous fasting (0-h) lipoproteins of d > 1.006 resulted in further substantial (> 5% total) reciprocal changes in apo(a) concentration of the 2 fractions. The increase in re-isolated postprandial TRL apo(a) was 25.1% +/- 5.1% of the total apo(a), compared to the insignificant increase (0.1% +/- 0.1%) in re-isolated fasting TRL. Most of the further increase in 4-h TRL apo(a) (94% +/- 4%) could be achieved by incubation with the corresponding 4-h chylomicron fraction (CM) alone. The re-isolated 4-h TRL apo(a) concentration correlated positively with 4-h plasma TG concentration (r = 0.65, P < 0.01) and other indices of postprandial lipaemia. In vitro incubation of pooled serum lipoproteins of d > 1.006 with serial dilutions of nascent CM obtained from chylous ascitic fluid revealed that the reciprocal changes in apo(a) concentration exhibit a curvilinear relationship with the concentration of CM triglyceride which plateaued round 7 mmol/l in this instance. We conclude that the reciprocal changes in apo(a) concentration between TRL and lipoproteins of d > 1.006 which occur in the postprandial phase are quantitatively significant and largely represent a redistribution process rather than de novo synthesis because they can be reproduced by in vitro incubation.

Automated measurement of urinary proteins

We have investigated the automation of the trichloroacetic acid turbidometric method for the estimation of urinary proteins using a standard centrifugal analyser. Conditions of temperature, trichloroacetic acid concentration, sample and reagent volumes have been examined. The method requires 10 microliter of urine sample and results have been shown to be linear to 2.0 g per litre with undiluted specimens; higher levels of protein require dilution of the urine samples to obtain greater accuracy. The coefficient of variation at a level of 0.5 g per litre is 3.5% and at 1.0 g per litre is 1.3%; recoveries ranged from 98.9 to 108%. The estimation of urinary protein in 29 samples may be carried out in 5 min.

An exhaustive tree-searching algorithm for high-resolution computer-assisted nucleotide sequence analysis

A new computer search strategy has been devised for high-resolution nucleotide sequence analysis. The strategy differs from those used by earlier sequence analysing programs in that it is exhaustive and capable of detecting all possible homologies and other types of relationships between or within sequences irrespective of the pattern of matches and mismatches encountered. The implementation of this strategy into a working algorithm is described.

Comparability of lipoprotein measurements, total: HDL cholesterol ratio and other coronary risk functions within and between laboratories in Australia

&NA; We assessed the intralaboratory imprecision and interlaboratory comparability of lipoprotein measurements and related cardiovascular risk functions such as the total to high density lipoprotein cholesterol (TC:HDL) ratio. Analysis of 5 separate plasma pools was carried out in 4 laboratories which regularly perform lipoprotein testing. We also performed a retrospective audit on RCPA‐AACB Quality Assurance Programme data from 134 laboratories participating in the Special Lipid Programme in 1991. Intralaboratory imprecision and interlaboratory comparability are reported as coefficient of variation (cv) and its 95% confidence limit. For the national data, we calculated the percentage of laboratories within specified ranges (±3, 5 or 10%) about the national mean (or median) for a given level of each analyte. Intralaboratory imprecision and interlaboratory comparability amongst the 4 laboratories were close to, or within recommended limits for, TC, TG and HDL, but the interlaboratory comparability of LDL and coronary risk functions exceeded these limits. On a national level, interlaboratory comparability of TC:HDL was within ±5% for only 43% of laboratories, and even fewer (26%) were within this range at higher values of the ratio. We conclude that it is not possible to recommend the use of coronary risk functions at present because of sub‐optimal interlaboratory comparability. Even if measures are introduced to overcome this problem, coronary risk functions may over‐simplify coronary vascular disease risk in a variety of clinical situations.

Should nucleotide sequence analyzing computer algorithms always extend homologies by extending homologies

Most computer algorithms used for comparing or aligning nucleotide sequences rely on the premise that the best way to extend a homology between the two sequences is to select a match rather than a mismatch. We have tested this assumption and found that it is not always valid.

The effects of diphenylhydantoin on the relationship between high-density lipoprotein cholesterol and several biochemical assays.

Correlations have been calculated between high-density lipoprotein cholesterol and total plasma cholesterol, albumin, gamma-glutamyl transpeptidase, aspartate aminotransferase, alkaline phosphatase, triglyceride, urea, creatinine and uric acid for diphenylhydantoin (DPH) users and for subjects attending a multiphasic health screening centre. For women DPH users, high-density lipoprotein levels correlated significantly with gamma glutamyl transpeptidase, cholesterol and alkaline phosphatase. These correlations were significantly different from those found for male DPH users and from subjects attending the health screening centre. In male DPH users, high-density lipoprotein cholesterol correlates negatively with urea and uric acid levels, a relationship which is found neither in women DPH users nor in the health screening centre population.

A centrifugal analyser technique for measuring isoenzymes of acid phosphatase in column-chromatography fractions

A column-chromatographic technique for separation of acid phosphatase (ACP) isoenzymes has been combined with a rapid and sensitive automated method for detecting ACP using a Centrifichem 300. The substrate used is p-nitrophenyl phosphate (PNPP), in a citrate buffer of pH 6.5. At this pH the ACP activity is not significantly less than optimal, while the extinction coefficient of PNPP is high enough to detect changes in OD using a direct computer analysis of the Centrifichem OD readings. Three main peaks of isoenzyme activity are found; the second peak, containing isoenzyme 2, is the major fraction found in prostatic tissue and serum from patients with prostatic cancer. Serum from patients with normal values for total ACP was used to determine a normal range for isoenzyme 2. The value of this test is the sensitivity with which small amounts of isoenzyme 2 can be detected, increasing the possibility of detecting small elevations in prostatic ACP long before high total ACP indicates that prostatic cancer has metastasized.