Automated enzymatic assays for the determination of intestinal permeability probes in urine. 1. Lactulose and lactose.

Lactulose is becoming the disaccharide of choice in the dual sugar assessment of passive permeability of the small intestinal mucosa. However its more widespread use is hampered by current analytical methods which are tedious and time consuming. An automated spectrophotometric technique for the assay of this sugar in urine is presented in which lactulose is linked by a series of enzyme reactions to the equimolar production of NADPH. In addition to lactulose, the procedure also gives accurate values for lactose, glucose and fructose in the urine sample. The assay has been shown to be highly specific for lactulose and lactose and was not affected by high concentrations of other sugars or other urinary constituents. Within assay and between assay precision were similar with the coefficient of variation for both sugars in the range 0.4-1.6%. The technique represents a significant improvement in time, simplicity and precision on existing methods of analysis.

Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol.

A need for a simple method for the determination of mannitol in urine has arisen because of the use of this monosaccharide in intestinal permeability tests. A rapid spectrophotometric assay for mannitol is presented based on the use of the bacterial enzyme mannitol dehydrogenase. The technique has been automated for use on the Cobas-Bio (Roche) centrifugal analyser. The assay has been shown to be highly specific for mannitol and was not affected by high concentrations of glucose, lactose or lactulose in samples. Within assay coefficient of variation was in the range 0.3 to 1.1% and 0.6 to 2.4% between assays. The technique represents a significant improvement in terms of time, simplicity and precision on existing methods.

Hypoalbuminemia in energy-malnourished rats infected with Nippostrongylus brasiliensis (Nematoda).

Rats fed a restricted amount (60% of ad libitum intake) of a well-balanced diet (protein energy:total energy ratio of 0.20) had a reduced growth rate but maintained near-normal plasma albumin concentrations. However, when such animals were infected with Nippostrongylus brasiliensis (a hookwormlike intestinal parasite), plasma albumin values fell precipitously, from a preinfection value of 34.7 g/L to 21.3 g/L on d 10 postinfection (p.i.). The hypoalbuminemia developed rapidly, without changes in body weight and despite an adequate amount of dietary protein. Similarly infected well-nourished animals showed a much less severe effect, plasma albumin values falling from 34.8 to only 31.3 g/L by d 10 p.i. Two measurements of gastrointestinal integrity, [51Cr]albumin leakage from the plasma and intestinal permeability to mono- and disaccharide sugars, suggested that the alterations in plasma albumin values could be explained on the basis of protein leakage into the gastrointestinal tract through parasite-induced lesions. Much greater changes in the indices of mucosal integrity were observed in the undernourished animals and overall it was clear that the energy-restricted animals had been far more severely affected by the parasite than their well-fed counterparts. The results are discussed with reference to the etiology of kwashiorkor in humans.

Factors affecting the integrity of the intestinal mucosa of Gambian children.

The interrelationship between diarrhea, malnutrition, and small bowel integrity was investigated prospectively in 68 Gambian infants aged 0-18 mo. Profiles of growth and morbidity were recorded for 8 mo. Each month intestinal permeability was measured by the differential uptake of orally administered lactulose (L) and mannitol (M). In well infants the mean L:M ratio was 0.42 (range 0.11-1.42). This ratio was increased slightly for underweight (60-80% wt for age) infants (mean 0.52) but considerably for those with marasmus (less than 60% wt for age) (mean 1.3, p less than 0.001), for those with acute or chronic diarrhea (mean 1.0 and 2.85, respectively; p less than 0.001), or with measles (mean 1.4, p less than 0.001). Sequential studies of ward patients with malnutrition and diarrhea showed a rapid fall in L:M ratios with resolution of diarrhea. These studies suggest that damage to the small intestine may play an important part in the development of infant malnutrition in The Gambia.

Plasma albumin concentrations and intestinal permeability in Bangladeshi children infected with Ascaris lumbricoides.

Plasma albumin concentration and intestinal permeability have been investigated in Bangladeshi children before and 9-14 d after successful treatment for ascariasis. Children infected with A. lumbricoides had lower plasma albumin concentrations than counterparts not harbouring this worm and values increased with successful treatment. Intestinal permeability tests indicated that the children had impaired gastrointestinal function and some loss of mucosal integrity; these factors had not improved 9-14 d after A. lumbricoides expulsion. The lowered nitrogen nutritional status implied by the reduced plasma albumin values in infected children, and the improvement following treatment, are in keeping with previous reports that A. lumbricoides impairs protein digestion or absorption. This may be the basis of the better growth rates of dewormed children in this area.

Protein losing enteropathy associated with Nippostrongylus brasiliensis infestation and its impact on albumin homoeostasis in rats fed two levels of dietary protein.

Alterations in plasma albumin concentration and gastrointestinal permeability have been investigated in rats infected with the nematode Nippostrongylus brasiliensis and fed adequate or low protein diets. Infection caused only minor changes in growth and food consumption of well nourished rats but resulted in significant reductions in those fed the low protein diet. Animals in both dietary groups were able to mount an immune response beyond day 10 post-infection (p.i.) which caused expulsion of the parasites, but this was less effective in rats fed the low protein food. Uninfected rats fed the low protein diet had significantly lower plasma albumin concentrations than their well nourished counterparts. Animals of both dietary groups showed a progressive reduction in plasma albumin concentration as the infection developed but values returned towards normal as the parasites were expelled. The reduction in plasma albumin concentration was closely associated with increases in gastrointestinal leakage of plasma protein but losses were far greater in the protein deficiency animals. Beyond day 10 p.i. protein loss decreased in both dietary groups and by day 21 p.i. had returned to normal in well nourished animals but not those fed the low protein diet. Intestinal permeability measured by the lactulose:mannitol ratio technique gave similar results to the protein loss data. Permeability increased as the infection progressed then fell as the worms were expelled but remained above control values in infected protein deficient animals. Overall, animals fed the low protein diet were more severely affected by the parasite than were their well fed counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary linoleic and linolenic acids on testicular development in the rat.

In two experiments a total of twelve male rats were reared from weaning for up to 63 weeks on an essential fatty acid (EFA)-deficient diet alone (2 X two animals) or supplemented with the methyl esters of linoleic acid (18:2 omega 6) (2 X two animals) or linolenic acid (18:3 omega 3) (2 X two animals). Testicular development was normal in rats given 18:2 omega 6, but in rats fed the EFA-deficient diet alone, and in those supplemented with 18:3 omega 3 the testes were reduced in size. Histologically, a degeneration of the seminiferous tubules was noted, with progressive loss of the germinal cells, and with an absence of spermatozoa in the lumina of the seminiferous tubules and epididymides. Leydig cells appeared unaffected, and were prominent. The six rats in Experiment 1 were capable of mating with females reared on commercial diets, but only the two 18:2 omega 6 supplemented animals were fertile. There was a marked reduction in the percentage of arachidonic acid (20:4 omega 6) and docosapentaenoic acid (22:5 omega 6) in the total fatty acids of the atrophic testes. There was no compensatory increase in long-chain derivatives of 18:3 omega 3 in the 18:3 omega 3 fed rats and it is concluded that linolenic acid cannot replace linoleic acid in the development of the rat testis.

Effect of dietary linoleic and linolenic acids on gestation and parturition in the rat.

Six rats fed a basal, purified diet low in essential fatty acids gave birth to a total of twenty-eight pups, of which twenty-six were born dead (range of two to six pups per rat) after a mean gestation of 24 days. A seventh rat carrying ten fetuses had a protracted process of parturition with dystocia. Six rats fed the basal diet supplemented with linoleate gave birth to a total of sixty-one pups of which fifty-seven were born alive (range of two to fourteen pups per rat) after a mean gestation of 21.8 days. Seven rats fed the basal diet supplemented with linolenate and carrying a total of sixty-three fetuses (seven to twelve per rat) had a protracted process of parturition with dystocia, and only one live and one dead pup were born (mean gestation of 22.8 days). At Caesarian operation a marked uterine inertia was noted. In four more rats where the supplement of linolenate was replaced by linoleate from the seventeenth day of gestation, parturition appeared normal and twenty-nine live and four dead pups were born. The occurrence of dystocia appeared to be related to the number of fetuses being carried to term. It is concluded that, in the rat, linolenate satisfies the requirement for the growth of mother rat and of the fetuses but is inadequate for the normal process of parturition, probably through inadequate production of the prostaglandins involved. There appears to be an obligatory requirement for linoleate at parturition.

Plasma lipids and lipoproteins of some members of the order Perissodactyla.

1. The plasma lipoproteins of various members of the order Perissodactyla have been examined by electrophoresis and analytical ultracentrifugation. 2. In the Equidae, high density (alpha) lipoprotein was the major component (80-90%) and low density (beta) lipoprotein (10-20%) the minor component. 3. In the Tapiridae represented by the Malayan tapir (Tapirus indicus), high density and low density lipoproteins were present in approximately equal amounts. 4. In the Rhinocerotidae, the high density lipoprotein characteristic of the Equidae and Tapiridae was absent, and the plasma lipoproteins consisted of a complex group having beta mobility on electrophoresis and a flotation pattern usually associated with low density lipoprotein. 5. The fatty acid composition of plasma lipids was remarkably similar in all members of the Perissodactyla examined, with very high percentages of linoleic acid (greater than 70%) being found in the cholesteryl esters.