(introduction...)

Objective
Experimental details
Summary of main results
Conclusions and comments

Jin Soon Ju, W. I. Hwang, and T.G. Ryu
Department of Nutrition and Biochemistry, Korea University, College of Medicine, Seoul, Republic of Korea

Objective

The purpose of this study was to define the changes in protein digestibility and protein requirements that occur in pre-school children infested with intestinal parasites, mainly Ascaris lumbricoides.

Experimental details

1. Subjects
Six boys and four girls, 52 to 70 months old, were selected among pre-schoolers from agricultural villages in the northwestern part of South Korea, about 48 km from Seoul. Their characteristics are given in table 1. They were healthy and normal, except for the intestinal parasites, based on medical history, physical examination, and laboratory analyses of blood and urine specimens, including transaminase and alkaline phosphatase activities.

2. Study Environrnent
The tests were done in a field metabolic unit during the fall. Outdoor temperature ranged from 3 to 25 C, and indoor temperature from 18 to 24 C.

3. Physical Activity
The activities of these children were normal for their age.

4. Duration of the Study
The study iasted ten weeks, divided into three periods: a. The first feeding period (BfV) was before treatment with a vermicide and lasted four weeks. b. The second period lasted two weeks, with a free-choice diet comparable to what the subjects ate at home. Ten mg/kg of Combantrin was given on the first day of this period. c. The third period (AV), after the vermicide treatment, lasted four weeks.

TABLE 1. Characteristics of Subjects

							Energy
							Intake		Approximate requimments ***
Subject	Sex	Age (years- months)	Height (cm) m³/hr)	Weight (kg)	SA* (m² )	BMR * (kcal/ m³/hr)	BfV **	AV	Estimated	FAO/ WHO
							(kcal/kg/day)
1	M	5-10	114.6	22.92	0.83	54.8	91.5	102.7	81.0	70.1
2	M	5- 9	11 5.6	20.5	0.80	55.4	86.9	102.7	88.2	74.2
3	M	5 - 9	107.5	1 7.67	0.72	59.9	101.8	100.0	102.5	80.3
Subjects 4 and 5 dropped
6	M	4- 4	107.3	18.64	0.73	60.7	92.9	102.5	96.9	75.7
7	M	4- 7	97.3	15.08	0.63	63.2	100.4	104.3	107.7	81 7
8	M	4- 7	96.3	14.83	0.62	64.2	101.2	106.8	109.5	82.6
9	F	5- 9	108.8	17.79	0.72	60.7	100.7	993	100,2	76.0
10	F	4- 8	95.8	15.92	0.63	61.2	103,7	99.6	98.8	80.1
11	F	4- 7	108.4	18.74	0.74	58.5	98.6	100.2	94,3	75,7
12	F	4 7	98.1	14.44	0.62	56.4	99.4	98.9	98.8	83.6

* Surface area (SA) and BMR were measured on the last day of the eighth experimental week. SA w0.425 x H0.725 x 71.84.
** BfV - before vermicide; AV ** after vermicide.
*** Approximate energy requirements: estimeted - BMR (kcal/day) x 1.7/body weight (BW); FAD/WHO = 1973 recommendations.

5. Dietary Intakes
Because of a generally low intake of animal protein, a daily supplemental feeding consisting of 150 ml of whole cow's milk was added to each subject's ordinary daily diet for one month before initiation of the study. The experimental diets were devised to provide approximately 100 kcal/kg/day and four levels of dietary protein (1.50, 1.75, 2.00, and 2.25 g/kg/day: diets A, B, C, and D, respectively), based on the local diet (see table 2). The four levels of dietary protein were fed in four consecutive weeks, both before and after treatment with the vermicide (BfV and AV, respectively). Three isoenergetic and isonitrogenous meals and light snacks were provided each day. Additional food intake, mainly rice, was allowed on request, especially in the AV period. A record was kept of each subject's food intake. Vitamin and mineral supplements were given once a day to meet requirements. Table 3 gives the estimated essential amino acid content and the chemical score of the experimental diets.

6. Indicators and Measurements

1. Complete urine and faecal collections were made daily throughout the two experimental feeding periods. Total nitrogen (micro-Kjeldahl), urea nitrogen (Folin-Wu), and creatinine (diacetyl-monoxime) were measured in the urine collected during the last four days of each week with a given diet. Nitrogen was also measured in faeces pooled during the same four days and in aliquots of the diets.
2. Fats (Soxhlet) and ash (combustion) in food and faeces were measured. Carbohydrates were calculated by subtracting protein, fat, ash, and moisture from total weights of food and faeces. Their energy contents were calculated using 4, 4, and 9 kcal per gram of carbohydrate, protein, and fat, respectively.
3. Nude body weight was recorded daily after the subject had voided the first morning urine before breakfast.
4. Venous blood samples were drawn before breakfast on the first and last days of the study. Haemoglobin, haematocrit, serum glucose, urea nitrogen, creatinine, SOOT, alkaline phosphatase, total proteins, albumin, and globulins were measured.
5. Stool examinations for the quantitative determination of helminth eggs were performed each of the first three days in both experimental periods (BfV and AV). The parasites expelled on the first three days after treatment were identified and counted.
6. The basal metabolic rate (BMR) was measured on day 14 of the AV period by the Douglas bag spirometer method.
7. The faecal obligatory nitrogen losses were estimated from the Y intercept of the regression equation of faecal nitrogen (Y) on nitrogen intake (X) (table 4). Digestibilities of protein and energy were expressed as the percentage of intake that was absorbed.

TABLE 2. Food Ingredients in the Daily Diet of a 17-kg Child (g/day)

Foods	Dietary Periods
	A	B	C	D
Rice	270	270	270	270
Wheat flour	-	13	-	-
Biscuits	-	30	-	20
Potato	80	80	80	30
Soybean curd	-	22	20	30
Soybean paste	-	25	-	9
Soy sauce	6	2.5	5	1
Radish	160	80	140	110
Spinach	-	25	-	30
Onion	5	10	10	10
Carrots	5	10	10	10
Seaweed	-	-	-	3
Candy	60
Sugar	-	12	70	46
Jam	50	30	-	10
Apple	-	-	80	100
Pear	75	100	100	100
Orange juice	100	100	100	100
Oil	1	2	1	1
Beef	10	2.5	-	15
Sausage	-	-	15
Dried small fish	-	2	1	1
Egg	-	-	-	30
Energy, kcal	1,660-	1,660	1,660	1,660
Protein, total g	26.2	34.1	29.5	38.3
g/kg	(1.5)	12.0)	(1.75)	12.25)
animal	3.6	4.3	2.6	7.5
Fibre,g	3.1	5.0	3.7	4.2

TABLE 3. Essential Amino Acid Composition of Experimental Diets (mg/g Protein)

		Diet A		Diet B		Diet C		Diet D
Amino acid	FAO/WHO (S)	mg	(A/S) %	mg	(B/S) %	mg	(C/S) %	mg	(D/S) %
Isoleucine	40	45.5	114	45.3	113	47.2	118	46.0	115
Leucine	70	83.8	120	82.1	118	82.1	118	81.5	116
Lysine	55	49.1 *	89	50.8	92	49.9 *	91	51.9	94
Methionine +Cystine	35	34.2	98	32.0 *	91	33.9	97	32.5*	93
Phenylalanine +Tyrosine	60	82.0	137	80.4	134	79.7	133	79.2	132
Threonine	40	39.8	99	38.9	97	39.5	99	39.4	99
Tryptophan	10	11.6	116	12.1	121	11.7	117	12.4	124
Valine	50	57.2	114	54.9	110	55.9	112	55.8	112
Total	360		428.6		421.4		423.9		422.6

* First limiting amino acid.

An obligatory faecal nitrogen loss of 32 mg/kg/day was used to calculate "true" protein digestibility. True nitrogen balance was calculated assuming 5 mg N/kg/day for integumental and miscellaneous losses. The biological value (BV) of protein was estimated as:

BV = (intake-faecal-urinary + faecal and urinary obligatory losses) X 100/(intake-faecal + faecal obligatory loss)

where the obligatory faecal and urinary losses were assumed to be 32 and 48 mg N/kg/day, respectively. The net protein utilization (NPU) was calculated from the biological value and digestibility.

Summary of main results

1. Body Weight
Most children gained weight at a rate three times greater than the Korean standard for the corresponding age during the experimental period (an average gain of 0.99 kg in 11 weeks). This suggests that energy intakes were above requirements. There were no differences in weight gains before and after treatment.

2. Height
Most children gained 1.8 to 2.2 cm in ten weeks, which is more than the Korean standard of 1.2 cm for the corresponding age group. There were no differences before and after treatment.

3. Parasitological Observations
Table 5 gives the ova counts on each of the three days before and after treatment and the number of helminths identified in faeces during the three days after treatment.

TABLE 4. Estimation of Faecal Obligatory Nitrogen Losses

No. of subjects	Levels of nitrogen intake		Faecal nitrogen output (mg/kg/day)
	Range (mg/kg/day)	Mean (mg/kg/day)
Before vermicide
8	228 - 269	250.0 ± 4.53*	79.46 ± 5.21
9	270 - 300	286.8 ± 3.47	84.86 ± 7.03
10	308- 340	322.1 ± 3.54	94.03± 5.77
10	344 - 371	353.5 ± 2.61	98.53 + 6.13
Y = 31.64 + 0.19x (for X = 0, Y = 32)
After vermicide
9	262 - 276	269.8 ± 1.70	83.90 ± 4.23
8	282 - 311	299.3 ± 3.44	92.66 ± 4.16
9	315 - 362	346.9 ± 4.70	96.61 ± 5.65
9	356 - 397	381.1 ± 3.32	111.90 ± 6.17
Y=21.69+0.23x (for X=0, Y=22)

* Mean ± S.E.

4. Nitrogen Digestibility and Balance
Table 6 gives the "true" nitrogen digestibility and "true" nitrogen balance data, as well as the biological values and NPU. There were no differences before and after treatment. There was a poor correlation between nitrogen balance and nitrogen intake.

5. Urinary Urea and Creatinine Excretions
Table 7 gives the results obtained.

6. Absorption of Nutrients
Table 8 gives the apparent absorption of carbohydrates and fat, the "true" absorption of protein, and the estimated absorption of total dietary energy.

TABLE 5. Parasitological Findings in Stools

Subject		EPG* before vermicide			Worms excreted in 3 days after vermicide			EPG after vermicide
		Day 3	Day 2	Day 1				Day 1	Day 2	Day 3
1	Al **	8,100	11,900	15,300	Al	8	Al	Neg.***	Neg.	Neg.
	Tt **	100	100		Tt	2	Tt	Neg.	Neg.	Neg.
2	Al	28,700	11,600	18,000	Al	8	Al	Neg.	Neg.	Neg.
	Tt	1,400	400	1,200	Tt	1	Tt	100	Neg.	Neg.
3	Al	22,600	32,000	35,100	Al	14	Al	Neg.	Neg.	Neg.
	Tt	500	500	300	Tt	2	Tt	100	Neg.	Neg.
Subjects 4 and 5 dropped
6	Al	28,300	44,900	4,300	AL	8	Al	Neg.	Neg.	Neg.
	Tt	400					Tt	Neg.	Neg.	Neg.
7	Al	6,500	10,200	19,200	Al	14	Al	Neg.	Neg.	Neg.
	Tt	3					Tt	Neg.	Neg.	Neg.
8	AL	6,100	9,800	2,700	Al	2	Al	Neg.	Neg.	Neg.
9	Al	4,200	13,700	18,100	Al	6	Al	Neg.	Neg.	Neg.
10	Al	11,000	10,900	5,000	Al	2	Al	Neg.	Neg.	Neg.
	Tt	200	400		Tt	2	Tt	Neg.	Neg.	Neg.
11	Al	300	600	4,300	Al	10	Al	Neg.	Neg.	Neg.
12	Al	200	400	200	Al	1	Al	Neg.	Neg.	Neg.

* EPG = number of eggs per gram of stool.
** Al = Ascaris lumbricoides; T = Trichuris trichiura.
*** Neg. = negative.

TABLE 6. Nitrogen Balance, BV, and NPU before and after Vermicidal Treatment

Diet	No of subjects	Nitrogen intake	Faecal nitrogen (mg/kg/day)	Urinary nitrogen	Balance	True digestibility (%)	BV	NPU (kcal/kg/ day)	Energy intake
Before vermicide
A	9	259.94 *	92.80	10.8.37	53.79	76.49	69.68	53.24	103.50
		±8.21	±6.70	±6.48	±9.44	±2.67	±3.05	±2.91	±2.74
C	10	276.36	92.48	130.67	48.26	78.17	61.50	48.05	98.0
		±9.12	±6.39	±4.85	±7.95	±2.15	±1.99	±1.89	±3.13
B	10	323.60	89.44	161.98	67.18	82.49	56.97	47.04	97.56
		±7.72	±6.97	±3.84	±7.05	±1.91	±1.73	±1.98	±2.36
D	11	340.53	91.95	191.09	52.49	82.43	49.05	40.55	92.15
		±7.14	±5.78	±6.30	±6.30	±1.60	±1.82	±1.89	±1.86
Average		302.12	91.65	150.10	55.39	79.90	59.30	47.22	98
			±3.11	±3.68	±3.84	±2.08	±2.15	±2.19	± 1.4
After vermicide
A	10	270.88	8522	117.28	63.38	80.36	68.17	54.76	102.49
		±1.94	±4.01	±5.11	±5.80	±1.50	±2.28	±1.99	±0.91
C	8	303.30	97.20	140.20	60.90	78.54	61.08	48.11	101.60
		±2.94	±6.60	±4.15	±7.15	±2.09	±1.96	±2.37	±0.89
B	9	353.49	92.93	178.79	76.77	82.77	55.28	45.73	101.98
		±3.52	±3.41	±4.68	±5.33	±0.97	±1.53	±1.37	±1.06
D	8	383.13	113.35	210.58	52.97	78.75	46.06	36.19	102.39
		±4.00	±6.97	±6.98	±2.99	±1.69	±1.36	±0.91	±0.90
Average		324.90	96.37	159.66	63.85	80.11	57.65	46.20	102
			±3.03	±6.65	±3.03	±1.56	±1.78	±1.67	± 0.5

* Mean ± S.E.

TABLE 7. Daily Urinary Excretion of Urea Nitrogen and Creatinine (mg/kg/day)

Item	Diets
	A	C	B	D
Before vermicide
Ures N (UuN)	77.97 + 5.50 *	91.15 ± 4.05	112.38 ± 2.96	139.57 ± 5.37
Creatinine (UCr)	15.65 ± 0.57	15.15 ± 0.57	16.12 ± 0.54	15.20 ± 0.48
UtN **/nitrogen intake	9.42	0.47	0.50	0.56
UuN/UCr	4.98	6.00	6.98	9.18
UuN/nitrogen intake	0.39	0.33	0 35	0.41
UuN/UtN	0.72	0.70	0.70	0.73
After vermicide
Urea N (UuN)	89.55 ± 3.91	109.93 ± 3.69	152.50±5.05	169.48±6.82
Creatinine (UCr)	15.13 ± 0.43	15.71 ± 0.46	15.90 ± 0.56	16.72 ± 0.69
UtN/nitrogen intake	0.43	0.46	0.51	0.55
Uun/UCr	5.93	7.00	9.59	10.15
UuN/nitrogen intake	0.33	0.36	0.43	0.44
UuN/UtN	0.76	0.78	0.85	0.80

* Mean ± S.E,
** UtN = total urinary nitrogen.

There were no differences at the various levels of protein intake or as a result of vermicidal treatment.

7. Analyses of Blood and Serum Samples
There were no significant changes during the study and all results were within normal ranges.

Conclusions and comments

1. There were no changes in weight or height gains as a result of deworming.
2. Deworming did not change the absorption of nitrogen, cabohydrates, and fats, or the total dietary energy.
3. Nitrogen balance and the protein biological value and NPU were not affected by the parasites.
4. The results coincide with our studies in adults, indicating that ascariasis and trichuriasis of the severity reported in our investigations do not interfere with the intake, absorption, and retention of protein.

TABLE 8. Absorption of Nutrients (Percentage of Intake)

Items
Energy,	BfV	94.7 ± 0.40*	95.5 ± 0.28	95.4 ± 0.45	93.8 ± 1.50
	AV	95.0 ± 0.40	94.8 ± 0.56	95.0 ± 0.36	94.3 ± 0.54
CHO,	BfV	98.9 ± 0.23	99.3 ± 0.23	99.0 ± 0.28	97.0 ± 1.74
	AV	98.9 ± 0.27	99.6 ± 0.29	99.7 ± 0.35	99.7 ± 0.21
Fat,	BfV	93.3 ± 1.02	94.5 ± 0.40	95.4 ± 0.55	92.9 ± 0.74
	AV	96.5 ± 0.25	93.5 ± 0.48	96.6 ± 0.39	93.6 ± 0.55
Protein,**	BfV	76.5 ± 2.67	78.2 ± 2.15	82.5 ± 1.91	82.4 ± 1.60
	AV	80.4 ± 1.50	78.5 ± 2.09	82.8 ± 2.09	78.8 ± 1.69

* Mean ± S.E.
** "True" absorption after accounting for estimates of obligatory faecal nitrogen.