Protein-energy malnutrition

ABSTRACT. Background: Studies have investigated the consequences of intrauterine malnutrition on birth weight and overall survival but not on wound healing. This study aims to assess the influence of in utero malnutrition on wound healing of newborn rats. Methods: Pregnant Wistar rats were divided into 2 groups. Study rats were given 50% of the food intake of controls throughout pregnancy in a pair-fed manner. The body weight and length of the newborns were measured. Newborns were breast-fed until day 21, when a laparotomy was performed. The effect of the laparotomy was assessed by measure of the wound strength and collagen deposition at postoperative day (POD) 7 (n = 15) and POD 21 (n = 15). Results: The body weight and length of newborns of malnourished mothers were significantly smaller at birth compared with controls (respectively, 4.5 ± 0.1 g vs 5.8 ± 0.1 g, p = .0003 and 4.6 ± 0.1 cm vs 5.2 ± 0.1 cm, p = .0003). Maximum, rupture, and tensile strength of malnourished newborns were smaller than controls on POD 7 (0.281 ± 0.031 vs 0.470 ± 0.031, p = .0061, 0.112 ± 0.06 kgf vs 0.173 ± 0.08 kgf, p = .0495 and 0.019 ± 0.002 kgf/mm^sup 2^ vs 0.024 ± 0.003 kgf/mm^sup 2^, p = .050, respectively). On POD 21, only tensile strength remained lower (0.044 ± 0.003 kgf/mm^sup 2^ vs 0.058 ± 0.003 kgf/mm^sup 2^, p = .0477). Type I collagen deposition of malnourished newborns was similar to controls on POD 7 (57.69 ± 10.06 vs 48.34 ± 15.65, p = .3187) and on POD 21 (75.6 ± 7.21 vs 80.0 ± 9.92, p = .4212). Conclusions: In utero malnutrition decreases the abdominal wound strength of newborn rats but not the collagen deposition, suggesting that breast-feeding nutrition is effective in recovering the collagen deposition but not overall wound strength. (Journal of Parenteral and Enteral Nutrition 28:241-245, 2004)

Prevention of malnutrition in children is one of the most difficult challenges to healthcare professionals around the world. Severe protein-energy malnutrition is common not only in developing countries but also in developed ones.1 Maternal dietary restriction, decrease in time of maternal lactation and/or disease-related malnutrition, are among the main factors affecting the child's nutritional status.

Development of modern neonatal intensive care units has permitted the survival of premature infants and has increased the interest in factors that influence the healing process in low-birth-weight newborns. These infants are frequently candidates for surgery because of necrotizing enterocolitis or congenital malformation.2

The effect of mother malnutrition during pregnancy on birth weight is well known. Numerous studies in animals have shown that when protein or energy intake of the mother is restricted during pregnancy, the offspring are smaller than they would otherwise have been.3 Such undernutrition may affect the growth of organ systems differently. In early life, malnutrition tends to produce small but normally proportioned offspring, whereas malnutrition at latter stages of development causes selective restriction of organ growth and function.3 The consequences of in utero malnutrition on the healing process remain unknown, and we are aware of no studies investigating the relationship between intrauterine malnutrition and the healing process. This study aims to assess the influence of in utero malnutrition on wound healing of newborn rats from nourished and malnourished mothers.

MATERIAL AND METHODS

Animals

Fifty virgin female Wistar rats weighting 273.3 ± 21.23 g were housed at 210C on a 12-hour light-dark cycle. They were mated with healthy males at a 1:1 proportion, and the day in which vaginal plugs were observed was taken as day 1 of gestation. Thereafter, the pregnant rats were housed individually, and control rats were allowed free access to rat chow, measured daily. Study rats were given 50% of the mean daily food intake of controls in a pair-fed manner throughout pregnancy. The rat chow had a protein content of 22%. Water was allowed ad libitum throughout the study in both groups. Every other day, body weight was recorded. Maternal serum albumin was measured at delivery.

Spontaneous delivery of pups took place on day 22 of pregnancy. Newborns were allowed breast-feeding until day 21, when they were weaned. Newborns' body weights and lengths were measured at birth and every 7 days until killing. Dams were allowed free access to rat chow during lactation, and their body weights were recorded every other day.

Study Design

On day 21 after delivery, 60 newborn rats, 30 from each group (control and study rats), underwent a measured 3 cm transverse laparotomy under ether anesthesia. Wounds were closed with continuous suture of 4-0 to poliglactin (Ethicon, Sao Paulo, Brazil) placed at 5 mm intervals. The 7-0 poliglactin (Ethicon) was used in a continuous manner to close the skin. At the end of surgery, blood was collected by intracardiac puncture in order to measure serum albumin. all animals had free access to rat chow and water after weaning, and their body weights and lengths were measured every 7 days until killing.

To assess wound healing following the laparotomy, the infant pups were killed by overdose with ether inhalation on either postoperative day (POD) 7 (n = 15) or POD 21 (n = 15). Each rat was weighed and had its length measured. The abdominal wall was removed en block of 3 cm × 2 cm and divided into 2 parts: the larger one (2.5 cm × 2.0 cm) was prepared for the wound strength tests, whereas the smaller portion (0.5 × 2.0 cm) was sent for collagen deposition measurement.

Wound Strength Tests

The larger part of the abdominal wall was prepared by removing the adjacent skin and the sutures, according to the technique proposed by Knolmayer et al.4 The fresh pieces were tested in a computerized traction device (Instron, London, UK), using a 10 kg cargo cell with 50 g of sensibility and a velocity of 100,000 mm/min.

The parameters evaluated were: maximum strength (kgf), defined as the maximum strength supported by the tissue before it begins to rupture; rupture strength (kgf/mm^sup 2^), the force required to break a wound with no regards to its dimensions; and tensile strength (kgf/mm^sup 2^), measured in terms of load applied per unit of cross section area.

Collagen Deposition Measurement

The smaller fragments were stained with Sirius Red after 5 µm sections and were examined under polarized light in a microscopy with 200× augmentation. The images thus obtained were captured and digitalized by a Sony camera, and were analyzed by the Image Pro Plus program (Midia Cibernetica, Sao Paulo, Brazil). Type I collagen (mature) appeared with reddish color, whereas type III collagen (immature) showed greenish color.5 The percentages of type I and type III collagen deposition were calculated for each rat, and an index was created, named collagen maturation index, which was defined as the ratio between the percentages of type I over type III collagen, where results > 1 were considered as predominance of mature collagen.

Statistical Analysis and Ethical Committee Approval

The data at each point were presented as the mean ± SEM. Differences in mean values between the 2 groups of mothers and rats were evaluated by Student's t test and multilevel test, respectively.6

The Ethical Committee for Animal Studies of the Federal University of Parana, Curitiba, Brazil, approved the study protocol.

RESULTS

Initial maternal body weight was similar in the two groups of pregnant rats (272.30 ± 33.56 g vs 272.18 ± 26.24 g for controls and study rats respectively, p = .9953). However, throughout pregnancy, the weight gain of control rats was constantly greater than that of the study group, which lost weight on the first and second weeks and had a little recovery by the third. The mean weight gain of control pregnant rats was greater than that of study ones (91.8 ± 8.61 us 12.1 ± 3.24 g, respectively,p

Rats born of mothers that had a restricted diet had their weights reduced by 22.5% of controls at birth and a reduction of 9% by day 7 (4.5 ± 0.1 g vs 5.8 ± 0.1 g, p = .0003 and 10.8 ± 0.2 g vs 11.9 ± 0.3 g, p = .0313, respectively). At weaning, both groups had similar body weights (29.3 ± 1.0 g vs 32.5 ± 1.1 g for malnourished and well-nourished groups, respectively, p = .0605). Body length was reduced in study rats at birth compared with controls, but at weaning the difference was no longer significant (4.6 ±0.1 cm us 5.2 ± 0.2 cm, p = .0003 and 29.5 ± 1.0 cm us 32.5 ± 1.1 cm, p = .6506, respectively). By POD 7, body weight was greater in control rats compared with the study group. Thus, body length gain was greater in the study group than in controls (p = .0410).

Wound Strength Tests

Results of the wound strength tests are shown in Tables I and II. The maximum, rupture, and tensile strength of the wounds of the in utero malnourished newborns were lower than controls on POD 7. On POD 21, only tensile strength remained lower in the study rats compared with controls (0.044 ± 0.003 kgf/mm^sup 2^ us 0.058 ± 0.003 kgi/mm^sup 2^, respectively, p = .0477). The maximum and the tensile strength increased from the POD 7 to POD 21 in both groups. The mean maximum strength gain was 0.511 ± 0.09 kgf (p = .0003) in the control rats and 0.626 ± 0.08 kgf (p

Collagen Deposition Measurement

Type I collagen deposition increased from POD 7 to POD 21 in both groups (p = .0344 and p = .0018 for study and control rats respectively). However, Type I collagen deposition of newborns from malnourished mothers was similar to that of controls on POD 7 (57.69% ± 8.06 vs 48.34% ± 6.65, respectively, p = .3187) and on POD 21 (75.6% ± 7.21 vs 80.0% ± 9.92, respectively, p = .4212). Collagen maturation index was higher in malnourished rats than in controls on POD 7 (2.396 ± 0.56 vs 0.991 ± 0.069, respectively, p = .0442), but was similar to control on POD 21 (4.69 ± 2.86 vs 10.606 ± 17.471 respectively, p = .2576).

DISCUSSION

In the present study, we have shown that in utero malnutrition affects the healing process after a laparotomy in weaned rats. It is interesting to note that this adverse effect of malnutrition was observed in the pups even after their body weights have been recovered during lactation. These results suggest that although growth is a biologic priority, it is not accompanied by a normal response to injury.

Pregnant rats of the study group, which received only 50% of mean daily food intake of controls, had significant weight loss in the first and second weeks of gestation, with little recovery by end of the pregnancy. The weight gain during the final weeks of pregnancy can be explained by metabolic alterations in the third week added to fetal and placenta growth after the 15th day of gestation.7 In the first week of lactation, there was significant weight gain (20%) in malnourished darns. Boyle et al8 observed that rats submitted to a restrictive diet followed by recovery had a superior weight gain compared with the control group, probably because of a reduction of metabolic activity.

Serum albumin was also lower in malnourished mothers compared with controls at delivery. Serum albumin was included in the study protocol in order to further confirm the presence of malnutrition. Other studies in the literature have not measured serum albumin and defined malnutrition in terms of body weight loss only. Our results show that serum albumin correlates with malnutrition in pregnant rats and that it may be a marker to include in studies investigating malnutrition during pregnancy.

Birth weights and body weights on POD 7 were reduced in offspring born of mothers with a restricted diet. However, body weights at weaning were similar to controls, suggesting that breast-feeding was effective in inducing weight gain. In an interesting study analyzing the breast milk composition in rats, Harris et al9 studied the breast milk protein concentration in malnourished and well-nourished dams and reported no significant difference between the 2 groups. They have shown that newborn rats received adequate nourishment and grow normally independent of the nutritional status of their dams, confirmed in our study.

After the laparotomy the growth pattern changed. Body weights of control rats on POD 7 were significantly higher than those of the study group killed on the same day. However, their body length was similar, showing that they grew faster than controls. These results suggest a preference for cell proliferation instead of fat accumulation. Therefore, to grow and not to gain weight is the biologic priority in malnourished growing rats subjected to an operative trauma. These findings have not been previously published, and the reason why this happens remains unknown. We can speculate that the metabolic burden represented by the operative trauma blocks the weight gain, but not growth.

Rupture strength in malnourished rats on POD 7 was lower than that of the well-nourished group. On POD 21, after a longer time of diet recovery, the rupture strength was equivalent in both groups. Similarly, Irvin10 demonstrated an 85% loss in the rupture strength on POD 7 in adult rats under a protein-restricted diet for 8 weeks compared with the control group. In the same study, however, rats receiving a protein-restricted diet for 3 weeks only had rupture strength similar to that of well-nourished rats. These results suggest that the duration and type of restriction are determinants to the loss of wound strength.

Maximum strength was 58.7% higher in the control group on POD 7 compared with in utero malnourished rats. In contrast, on POD 21 the maximum strength was similar in both groups. Atkinson et al11 compared the maximum strength on POD 6 in rats receiving a protein-restricted diet (5.5% protein) every other day for 8 weeks, compared with controls receiving a 23.4% protein chow. They showed on POD 6, that protein restriction was associated with a decrease in maximum strength compared with controls. They mentioned in their study that the maximum strength is affected by malnutrition only in the initial phases of healing, and is equal to the maximum strength in normally nourished rats on POD 21. This finding is similar to what was observed in the present study with growing rats.

Tensile strength tests reflect wound resistance. In our study, tensile strength was higher in well-nourished rats on both POD 7 and 21 compared with malnourished study rats. Kobak et al12 compared the tensile strength in protein-depleted (1.9% protein for 3 months) adults rats compared with well-nourished controls. Control animals were found to possess strength approximately 3 times that which was observed in the protein-deficient group on POD 5. Subsequently, the healing proceeded at an almost parallel rate. They suggested the existence of an initial lag phase in wound healing and fibroplasia in proteindepleted rats, which disappeared over the subsequent weeks.

The collagen deposition was included in the present study because it has been reported to reflect the healing process.13 However, type I (mature) collagen deposition was similar in both groups on POD 7 and 21, although it increased in both groups. Because of these unexpected results, an index was created, the "collagen maturation index," which consists of percentage of mature collagen divided by the percentage of immature collagen. A value >1 for this index reflects a predominance of mature vs immature collagen.

The collagen maturation index was greater in malnourished rats compared with controls on POD 7. This result is the opposite of what was observed by the strength test, when well-nourished rats had higher levels compared with controls. Because the healing process is a biologic priority, the collagen deposition seems to be maintained independently of nutritional status, consistent with the results of Emery and Sanderson.14 These authors submitted rats to a chow restriction (50% of control rats diet) for 7 days followed by a laparotomy. They showed that the collagen content of the wound on POD 7 was unaffected by the short chow restriction.

The fastest collagen deposition in malnourished rats appeared to be compensatory, so that malnourished rats had similar wound resistance to that of the control rats, on POD 21. Mistry et al15 found significantly less collagen on POD 7 of protein depletion rats. In contrast, the collagen content on POD 14 was not significantly different from that of controls. This is probably because of initial hepatic protein reprioritization; the body redirects amino acids and other nutrients to the liver to support acute-phase protein reparation. During the second week, the wound assumes priority, and amino acids and nutrients are directed to the wound.

A positive relationship was found between the collagen maturation index and both the tensile and the rupture strength on POD 7, when the collagen maturation index was ^2 (66% of mature collagen). Values >2 did not correlate with the strength tests, suggesting that greater deposition of mature collagen does not necessarily result in greater wound resistance in this phase. The reason for these findings remains unknown. In contrast, on POD 21 the correlation was positive and there was much higher collagen maturation index with values to 9 (90.8% of mature collagen). Therefore, the significance of the measurement of wound collagen is time-dependent and may explain some of the perceived differences among various studies.15

In summary, in utero malnutrition decreases the abdominal wound strength of newborn rats but not the collagen deposition, suggesting that breast-feeding is effective in recovering the collagen deposition but not overall wound strength.

ACKNOWLEDGMENTS

We would like to thank Dr W Frederick Schwenk, MD, for his valuable help in reviewing this manuscript.

Discussant

Daniel H. Teitelbaum, MD

Mott Children's Hospital

Comments

I must admire the great work that this group has done. This paper has great relevance in areas of obstetrics and pediatrie development. There have been several papers in recent years. This report carries the implications of this lack of nutrition further. In particular, the consequences of wound healing may greatly influence the outcomes of pediatrie surgical patients, which could greatly affect the outcome of the patient.

Questions

1. Because wound healing was particularly affected early on, do you think that with proper intervention in utero, such problems of poor wound healing could be avoidable?

2. You state that collagen deposition was not different between the study groups by POD 7, but did you have measurements of collagen deposition immediately at birth to understand if there were any in utero differences between these 2 study groups? This would seem to be quite important in understanding your model and the mechanisms involved in healing strength. Please add this to your Results or Discussion.

3. If collagen deposition was not different, what mechanism contributed to the lower tensile strength in the malnourished group?

Author's Reply

1. The prevention of malnutrition in children is one of the most difficult challenges to healthcare professionals around the world. Maternal dietary restriction during pregnancy or disease-related malnutrition is among the main factors affecting the child's nutritional status. The question of whether proper in utero nutrition intervention would reverse the adverse consequences on healing is an interesting aspect that was not investigated in the present study and certainly merits further studies. It is known, however, that the reversal of in utero malnutrition is associated with improvements in anthropometrie parameters.

2. In reality, collagen deposition was greater in malnourished newborns, as assessed by the collagen maturation index (2.29 ± 0.56 us 0.99 ± 0.07, p

The healing process is a very complex one, and certainly several factors play a role in improving the tensile strength. Although breast-feeding was effective in recovering collagen deposition, this was not followed by overall wound strength. Other factors, probably important in the healing process, are being currently investigated, such as fibronectin, thrombospondin A, and other types of collagens, such as collagens II and IV.

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Izabel Cristina Meister Coelho-Lemos, MD; Antonio C. L. Campos, MD, PhD; Marlene de Almeida, MD; Sandra Lucia Schuler, MD; Jocemara Gurmini, MD; Osvaldo Malafaia, MD, PhD; and Dalton F. Andrade, PhD

From the Department of Surgery of the Federal University of Parana, Curitiba, Brazil

Received for publication March 12, 2004.

Accepted for publication April 6, 2004.

Correspondence: Antonio C. Campos, MD, University of Parana-Brazil, Department of Surgery, R. Comendador Araujo 143 Cj113, Curitiba 80420, Brazil. Electronic mail may be sent to accampos@ hc.ufpr.br.

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